ARF1 orchestrates a Rab cascade to control endocytic recycling

Presenting author:

Petia Adarska

FU Berlin, Department of Biology, Chemistry, and Pharmacy, Thielallee 63, 14195 Berlin [DE], petia.adarska@icloud.com

Author(s):
Francesca Bottanelli, Petia Adarska

Early endosomes (EEs) are central sorting stations that orchestrate the distribution of cargo destined for the Golgi, lysosomal degradation, or recycling back to the plasma membrane (PM) following endocytosis. Although ARF and Rab GTPases are known to be recruited to EEs, how they regulate sorting and biogenesis of distinct carriers is unclear, due to the highly dynamic nature of endosomal compartments. We previously showed that endocytic recycling cargo flux between EEs and recycling endosomes (REs) is driven by an ARF1-to-Rab11 cascade that returns cargoes to the PM (DOI: 10.1038/s41556-024-01518-4). Here, we profiled the proteome of ARF1/Rab11-positive maturing REs using contact-dependent proximity proteomics (split Turbo-ID) to elucidate how ARF1 regulates endocytic recycling. We found that the ARF GEFs BIG1 and BIG2 localize to distinct nanodomains on Rab4-positive EEs marking budding sites for ARF1/Rab4-positive tubules, which undergo fission and mature into Rab11-positive REs. Chemical inhibition and chemogenetic sequestration of BIGs using the CATCHFIRE system reversibly disrupt endosomal maturation, highlighting BIG-mediated ARF1 activation is required for Rab4-to-Rab11 conversion and RE biogenesis. Overall, we propose that ARF1 introduces a regulatory pause to slow down endocytic recycling when adaptor-dependent cargoes are present, thereby ensuring that endosomal maturation proceeds only once endosomes have been cleared of specific cargoes destined for retrieval.

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Modulating membrane compressibility to study ER membrane protein insertion and topogenesis

Presenting author:

Cynthia Alsayyah

Saarland University, Medical Biochemistry and Molecular Biology, Kirrberger Straße 100, 66421 Homburg (Saar) [DE], cynthia.alsayyah@uni-saarland.de

Author(s):
Cynthia Alsayyah, Emmanuel Rodrigues, Julia Hach, Mike F. Renne, Alexander von der Malsburg, Robert Ernst

Most membrane proteins in eukaryotic cells are inserted by a variety of dedicated membrane insertases (EMC, SEC, SND, GET). It is generally assumed that the ER lipidome (low in sterols and high in unsaturated fatty-acyl chains) provides high membrane fluidity and compressibility to handle a broad spectrum of different transmembrane domains. Reconstituting membrane proteins in vitro, however, can be extremely challenging in cholesterol-rich membranes of low compressibility causing protein aggregation and sample heterogeneity. We propose a new approach to adapt membrane compressibility of (proteo)liposomes after their formation by reversibly tuning their sterol content. Both cholesterol delivery and extraction is mediated by methyl-β-cyclodextrin in a dialysis setting, making it possible to readily exchange the surrounding medium without losing any membrane material. During these processes, cholesterol delivery and removal is monitored with the solvatochromic probe C-Laurdan, which reports on lipid packing. We first employ this approach to show the cholesterol-triggered reversible oligomerization of the yeast membrane property sensor Ire1. Furthermore, we tune membrane compressibility of HEK293T-derived ER microsomes to assess the impact of cholesterol membrane protein insertion via different insertases.

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A Dynamic Equilibrium of Crista States Adapts Cristae Architecture to Metabolism Independently of MICOS.

Presenting author:

Jannis Anstatt

, , Sertürnerstraße 13, 37085 Göttingen [DE], jannis.anstatt@mpinat.mpg.de

Author(s):
Jannis Anstatt

The inner mitochondrial membrane (IMM) forms invaginations, termed cristae, that present a characteristic stacked morphology in many mammalian cell types. Depletion of known cristae-shaping proteins disrupts this stacked morphology and induces a non-stacked membrane arrangement. We demonstrate that forcing cells to rely on glycolysis rather than respiration for energy generation also induces a non-stacked cristae architecture. Stacked and non-stacked cristae exist in a dynamic equilibrium rather than as two distinct populations as mitochondria can transition between the two phenotypes on a minute timescale. Mass spectrometric analysis revealed little molecular alterations of the IMM between the two metabolic states. Together, this shows that cristae organization is more flexible and dynamic than previously appreciated and demonstrates that alteration of IMM architecture does not necessarily lead to a widespread remodelling of mitochondrial protein complexes. We show that cristae structure is influenced by mitochondrial metabolic activity, however, we find no evidence that the immediate adaptation of cristae structure is actively controlled by known cristae-shaping proteins such as the MICOS complex.

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Effects of metabolic glycoengineering on sialylation- and adhesion-related protein expression in GNE-deficient HEK293 cells

Presenting author:

Ghaith Ardromly

Martin Luther Universität Halle-Wittenberg, Medical Faculty, Institut für Physiologische Chemie, Hollystr. 1, 06114 Halle (Saale) [DE], Ghaith.Ardromly@uk-halle.de

Author(s):
Ghaith Ardromly, Kaya Bork, Rüdiger Horstkorte, Astrid Gesper

The properties of the cell membrane are defined not only by its glycolipid composition but also by membrane-associated glycoproteins and their post-translational modifications. Sialylation is one such modification that influences protein stability, cell–cell interactions and adhesion.The enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key regulator of sialic acid biosynthesis. Loss of GNE function leads to altered cellular sialylation and may affect adhesion-related processes. Mutations in GNE are associated with a rare neuromuscular disorder, known as GNE-Myopathy.

In this study, we analyzed the effects of metabolic glycoengineering on protein expression in human embryonic kidney (HEK 293) cells with and without functional GNE using western blotting. Wild-type and GNE-knockout HEK cells were treated for 24 hours with different monosaccharides to modulate sialic acid biosynthesis.

We examined several adhesion-associated proteins and multiple integrin subunits. The analysis revealed differences in protein expression between wild-type and GNE-deficient cells. Overall, these results indicate that metabolic glycoengineering can influence the expression of adhesion-related proteins in GNE-deficient cells.

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The transmembrane protein Vac7 couples vacuole stress sensing to Fab1-kinase mediated PI(3,5)P2 synthesis

Presenting author:

Annabel Arens

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], annabel.arens@uni-osnabrueck.de

Author(s):
Annabel Arens, Julia Seimert, Bianca Esch, Stefan Walter, Lars Langemeyer, Florian Fröhlich, Christian Ungermann

The signaling lipid phosphatidylinositol-3,5-bisphosphate PI(3,5)P₂ plays a major role in lysosome membrane homeostasis by regulating membrane fission and luminal ion homeostasis. During hyperosmotic stress, yeast cells depend on the vacuolar transmembrane protein Vac7 to activate the lipid kinase complex FAB1C, which phosphorylates PI(3)P to PI(3,5)P₂. However, the function of Vac7 has remained unresolved. Here, we show that Vac7 is involved in vacuolar and thus cellular sensing. Inducing hyperosmotic stress results in a redistribution of Vac7 and the Fab1 kinase complex. We identified a part in the cytosolic domain, that is crucial for signaling. Vac7 mutants lacking the conserved luminal domain, previously described as lipid transfer domain (LTD), have increased vacuolar PI(3,5)P₂ levels. To obtain further information about the function of Vac7, we analyzed the impact of a VAC7 deletion on the vacuolar proteome. Our data suggests a model in which Vac7 functions as a sensor of the vacuolar lumen to activate FAB1C and thereby maintaining vacuolar homeostasis.

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Synthesis of Betaine Lipids and Phosphate Starvation in Physcomitrium patens

Presenting author:

Diego Artigas Hernández

RPTU Rheinland-Pfälzische Technische Universität Kaiserslautern, Biology, Konrad-Adenauer-Str. 47, 67663 Kaiserslautern [DE], artigashernandez.d@rptu.de

Author(s):
Diego Artigas Hernández, Sadia S. Tamanna, Stefanie Müller-Schüssele, Morgane Michaud

Phospholipids are major components of plant membranes. Consequently, c. a third of cellular phosphate (Pi) has a structural role. Plants are forced to acclimate to various types of stress, such as nutrient deprivation, with Pi starvation being common in land plants. Therefore, response mechanisms to overcome Pi deprivation have evolved. To optimise the phosphorous-use efficiency, phospholipids can be replaced by non-phosphorous-containing lipids, a process called lipid remodelling. From green algae to non-seed land plants, betaine lipids have been reported to replace phospholipids under low Pi. Conversely, seed plants use galactolipids, main components of plastidial membranes, as the replacement strategy.

Our project focuses on investigating this change of strategy, using the bryophyte Physcomitrium patens, that is very informative from an evolutionary perspective. The aim is to characterise BTA1 (enzyme synthetising betaine lipids) at the genetic, transcriptional, protein and lipid levels. Firstly, using Confocal Laser Scanning Microscopy, we found that BTA1 is localised in the ER in P. patens. Our preliminary data show expression of BTA1 and presence of DGTS (betaine lipid produced via BTA1) under Pi starvation in P. patens wild type after 21 days of Pi depletion. Further, we will produce KO (bta1^ge) mutants. This project will reveal how bryophytes acclimate to Pi starvation and the role of phospholipids, betaine lipids and potentially galactolipids in lipid remodelling.

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Investigating the Tardigrada Lipid Landscape

Presenting author:

Pavel Barahtjan

EPFL Lausanne, , Station 19, 1015 Lausanne [CH], pavel.barahtjan@epfl.ch

Author(s):
Pavel Barahtjan, Nika Goršek, Jean Andrea Maillat, Giovanni D'Angelo

Lipid localization and composition vary between organisms, and these differences profoundly affect physiology and adaptation to environmental factors. Tardigrades are extremotolerant invertebrates known to survive harsh environmental conditions such as desiccation, high radiation, high osmolarity, and oxygen depletion. Their survival mechanisms have mainly been attributed to proteins, with few studies investigating the role of lipids. Given the fast-paced nature of lipid and membrane remodelling, we hypothesize that rapid lipid/membrane remodelling is a major driver of extremotolerance.

To systematically investigate lipid composition and adaptation throughout tardigrade life, we combine lipid biochemistry and molecular biology tools. We use high-resolution liquid chromatography–mass spectrometry to profile the total lipidome of H. exemplaris. In parallel, we are establishing a matrix-assisted laser desorption/ionization mass spectrometry imaging approach to map lipid distributions within intact animals.

We find that H. exemplaris exhibits a high abundance of highly polyunsaturated lipid species (e.g., SM, PC, PE, and TAG) with up to 12 double bonds, and a non-homogeneous lipid distribution within individual animals, suggesting spatially restricted, location-specific functions. These preliminary results provide a framework for understanding how tardigrades tune lipid and membrane composition to withstand environmental challenges.

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Glycosphingolipid profiling reveals tumor-specific remodeling and secretion in bladder cancer

Presenting author:

Inês Barbosa Moreira

Hannover Medical School (MHH), Clinical Biochemistry, Gellerstrasse 21, 30175 Hannover [DE], vicente.ines@mh-hannover.de

Author(s):
Inês Barbosa Moreira, Charlotte Rossdam, Jonas Kaynert, Julia Beimdiek, Manuel M Vicente, Jessica Schmitz, Anika Großhennig, Astrid Oberbeck, Michele E Rosero Moreno, Daniel Steinbach, Maria L Barcena, Yannick Lippka, Jan H Bräsen, Hossein Tezval, Falk F R Buettner

Glycosphingolipids (GSLs) are key organizers of the plasma membrane and are frequently remodeled in cancer. In epithelial tumors, these changes reflect profound membrane reorganization, yet tumor-associated GSL profiles and their extracellular fate remain poorly characterized. In bladder cancer (BC), such alterations can be studied in tumor tissue and membrane-derived material released into urine. Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF), we profiled GSLs in BC samples. This analysis identified nine species exclusively detected in tumor tissue. Among these, neolactotetraosylceramide (nLc4) was absent from healthy urothelium and spatially restricted to malignant regions of the bladder epithelium, supported by immunofluorescence staining. Paired tissue-urine analysis showed higher relative nLc4 levels in urine than in matched tumor tissue, suggesting selective release from tumor cell membranes. xCGE-LIF and ELISA analyses of urinary extracellular vesicles further revealed elevated nLc4 levels in BC patients that increased with tumor malignancy. Together, these findings demonstrate cancer-specific remodeling of membrane GSLs in BC and selective secretion of nLc4 into the extracellular space, with potential utility as a urinary biomarker for non-invasive BC detection. Beyond its translational relevance, this work highlights the value of membrane-focused glyco-analytics for studying lipid-driven disease processes.

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Phospholipid Dynamics in Myoblast Plasma Membranes

Presenting author:

Julia Baum

Ruhr Universität Bochum (RUB) , Biochemie II, Universitätsstraße, 44801 Bochum [DE], julia.baum@rub.de

Author(s):
Julia Baum, Annika Haak, Thomas Günther Pomorski

The asymmetric distribution of phospholipids across the plasma membrane is a defining feature of eukaryotic cells and is actively maintained by lipid transporters. During skeletal muscle development, however, this asymmetry is transiently disrupted. In particular, the externalization of phosphatidylserine plays a critical role in the fusion of mononucleated myoblasts into multinucleated myotubes. Although the mechanism of membrane fusion through phosphatidylserine exposure is well-established, the regulatory mechanisms governing the loss and restoration of lipid asymmetry remain poorly defined. In this study, we demonstrate that elevations in intracellular Ca²⁺ induce rapid phospholipid scrambling in the plasma membrane of myoblasts. Increasing cytosolic Ca²⁺ levels using an ionophore resulted in pronounced phosphatidylserine exposure at the cell surface and triggered fast, bidirectional transbilayer movement of fluorescence-labeled phospholipids. Notably, this scrambling activity was independent of phospholipid head group identity, indicating a non-selective mechanism. Together, these findings provide functional evidence for the presence of calcium-activated phospholipid scramblases in myoblasts and highlight their capacity to transiently disrupt membrane lipid asymmetry during myogenic differentiation.

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Membranolytic versus non-membranolytic mode of action of two antimicrobial latarcin peptides from spider venom

Presenting author:

Marnie Baumeister

Karlsruhe Insitute of Technology (KIT), Institute of Organic Chemistry (IOC), Fritz-Haber-Weg 6, 76131 Karlsruhe [DE], marnie.baumeister@kit.edu

Author(s):
Marnie Baumeister, Parvesh Wadhwani, Erik Strandberg, Anne S. Ulrich

Latarcins (Ltc) are a group of 7 linear antimicrobial peptides isolated from the venom of the spider Lachesana tarabaevi. Ltc1 and Ltc2a show structural similarities but display pronounced differences in their biological action. Both peptides fold into amphipathic α-helices and bear similar length (25/26 residues), high charge (+9/+10) and antimicrobial activity (MIC ≈ 2-4 µg/mL), but differ fundamentally with regard to their membranolytic effects. Oriented circular dichroism and solid-state ¹⁵N-NMR show that Ltc1 is oriented parallel on the surface of DMPC/DMPG (7:3) bilayers, whereas Ltc2a tilts into the membrane. At the same time, vesicle leakage and hemolysis show that tilted Ltc2a exhibits indiscriminate membranolytic activity towards any type of lipid bilayer [1]. Thus, it was hypothesized that a tilted amphipathic helix induces membrane perturbation, whereas a helix on the bilayer surface does not. To test this hypothesis, polar residues in Ltc1 (K10, R15, and K19) were individually or in combination replaced with hydrophobic leucine. Our results show that a single mutation of K10àL is sufficient to influence the tilt of the peptide and make Ltc1 more hydrophobic, thereby promoting aggregation and membranolytic effects. These results will be discussed to highlight different modes of action for similar antimicrobial peptides that may act via different mechanisms.

 

[ 1 ] Wadhwani P, Sekaran S, Strandberg E, Bürck J, Chugh A, Ulrich AS. Int J Mol Sci. 2021;22,10156.

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Investigation of Cryptococcus neoformans lipid flippases

Presenting author:

Pia Beer

Ruhr-Universität Bochum, Molecular Biochemistry, Universitätsstraße 150, 44801 Bochum [DE], pia.beer@ruhr-uni-bochum.de

Author(s):
Pia Beer, Sarina Veit, Thomas Günther-Pomorski

Lipid flippases of the P4-ATPase family actively translocate phospholipids towards the cytosolic side of biological membranes. This activity is essential for cellular processes such as membrane trafficking and vesicle budding. At least one of the four lipid flippases of the pathogenic fungus Cryptococcus neoformans is implicated in its pathogenicity and antifungal drug resistance. Therefore, an understanding of the regulation of these membrane transporters and of the key features of their activity is of great interest, as it may contribute to the development of targeted therapies against cryptococcal infections.

To directly investigate the transport activity of the fungal flippases we set out to reconstitute the transporters into large unilamellar vesicles of varying compositional complexity and to establish a flippase activity assay at the level of single vesicles. Individual vesicles containing fluorescent lipids as well as the fluorescently labeled flippase can be immobilized and visualized using fluorescence microscopy. This experimental platform enables direct observation of lipid transport and allows us to characterize the transport activity of flippases and their regulatory elements.

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Loss of Glucosylceramide Synthase Drives Membrane Lipid Remodeling and Molecular Alterations in Human iPSCs

Presenting author:

Julia Beimdiek

University of Augsburg, Institute of Theoretical Medicine, Proteomics, Universitätsstraße 2, 86159 Augsburg [DE], julia.beimdiek@med.uni-augsburg.de

Author(s):
Julia Beimdiek, Karsten Cirksena, Charlotte Rossdam, Astrid Oberbeck, Philippe V. Barbosa, Ana Tzvetkova, Malte Juchem, Christian Baer, Thomas Thum, Axel Schambach, Andreas Pich, Britta Brügger, Andreas Kuss, Falk F.R. Büttner

Glycosphingolipids (GSLs) are complex membrane lipids involved in fundamental cellular processes and associated with various diseases. Although GSLs are dispensable in embryonic stem cells, their diversity is crucial during embryogenesis. To assess their role in early development, we disrupted the UDP-glucose ceramide glucosyltransferase (UGCG) gene in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9. The loss of glucosylceramide synthase activity was confirmed by glycomic profiling (xCGE-LIF). UGCG‑KO hiPSCs showed normal morphology, growth, stem cell marker expression, and differentiated efficiently into meso-, endo-, and ectodermal progenitors similar to wildtype (WT). Teratoma formation further verified their pluripotency. Quantitative proteomics of teratomas revealed 295 significantly altered proteins, enriched in pathways related to transport, signal transduction, and organelle localization. Transcriptomic analyses confirmed broad gene expression changes, and lipidomics identified markedly increased sphingomyelin levels in UGCG‑KO cells. Together, our multi‑omics data highlight that loss of GSLs does not impair pluripotency but causes molecular changes that emphasize their role in pluripotent stem cell biology.

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Optimizing proximity labeling to dissect structure-detailed interactions of Bcl-2 family proteins at the mitochondrial outer membrane

Presenting author:

Juanfran Belén Aguilar

University of Valencia (UV) , Biochemistry & Molecular Biology, Av. Vicent Andrés Estellés, 19 , 46100 Burjassot (Valencia) [ES], juanfran.belen@uv.es

Author(s):
Juanfran Belén Aguilar, Juan Ortiz Mateu, Marina Rius Salvador, Ismael Mingarro Muñoz, María Jesús García Murria, Manuel Mateo Sánchez del Pino

Apoptosis is governed by dynamic protein–protein interactions at the mitochondrial outer membrane (MOM), where Bcl-2 family proteins regulate membrane permeabilization and cell death. Despite extensive biochemical characterization, how these interactions are organized in living cells remains poorly understood, particularly for transient and membrane-confined contacts. Traditional interaction-mapping methods disrupt membrane integrity and spatial information, limiting their applicability at mitochondria. Here, we optimize proximity-dependent biotinylation to study Bcl-2 family interactomes in human cell cultures using UltraID, a compact and highly active biotin ligase enabling efficient labeling within 10 minutes. To minimize artifacts associated with overexpression, we systematically optimize construct design, promoter strength, and expression levels, and validate correct mitochondrial localization and preserved protein function. Using Bcl-xL as a model, we combine UltraID with a truncation strategy to achieve domain-specific resolution of proximal proteomes, and incorporate Split-TurboID to selectively capture oligomer-dependent interactions. This work establishes a robust and spatially informed framework for mapping dynamic interaction networks of mitochondrial membrane proteins under physiological and apoptotic conditions.

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Activity profiles of membrane bound guanylate cyclase related to retinal disease-causing mutations

Presenting author:

Linda Bessert

, , Im Ofenerfeld 23c, 26127 Oldenburg [DE], linda.bessert@gmail.com

Author(s):
Linda Bessert, Malte Benje, Eva Maria Breuer, Lars-Oliver Peters, Alexander Scholten, Karl-Wilhelm Koch

Inherited retinal diseases like autosomal recessive Leber congenital amaurosis or autosomal dominant cone-rod dystrophy correlate with more than 140 mutations in the gene GUCY2D coding for photoreceptor guanylate cyclase GC-E. This membrane protein is part of the regeneration of the dark state after a light incident in the eye. Biochemical or cellular consequences of GC-E mutations are much less frequently investigated than the corresponding genotypes and clinical phenotypes. Here, we analyzed five point mutations of GUCY2D, that are associated with Leber congenital amaurosis (D279N, G928E) or are linked to cone or cone-rod dystrophy (R838H, R838P, E843Q). All mutants showed at least 50-100-fold less activity in the presence of guanylate cyclase-activating protein 1 (GCAP1) at low [Ca2+] than wildtype GC-E. Mutants D279N, E843Q and R838H revealed pronounced Ca2+-sensitive activation. Inhibition by retinal degeneration protein 3 was less efficient. Ca2+-feedback regulation of GC-E mutants operated within the physiological light–dark Ca2+-range, except R838H, which was constitutively active at higher [Ca2+] (dark state). Activation by GCAP2 or GCAP3 was low for the wildtype and largely Ca²⁺-independent, in contrast to GCAP1. These results highlight the molecular impact of the mutations, provide insight into the processes underlying the corresponding retinal diseases, and lay the foundation for the development of molecularly targeted treatment strategies.

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Remodelling of the Endoplasmic Reticulum in Mammalian Cells

Presenting author:

Sneha Bhatt

Heidelberg University, Biochemistry Centre, INF 328, 69120 Heidelberg [DE], sneha.bhatt@bzh.uni-heidelberg.de

Author(s):
Sneha Bhatt, Sebastian Schuck, Rolf Markus Schmidt, Giulia Ruffini

The endoplasmic reticulum (ER) is a dynamic organelle whose morphology can be remodelled to meet cellular demands. One trigger for ER remodelling is the accumulation of misfolded proteins, a condition referred to as ER stress. ER stress then activates signalling pathways called the Unfolded Protein Response (UPR). As part of this stress response, cells reshape the ER but the underlying molecular mechanisms remain poorly understood. We employed chemical ER stress inducers to alter ER morphology in U2-OS cells and used confocal microscopy to characterize the resulting morphological changes. The stressors tunicamycin and subtilase A induced extensive ER sheets, whereas thapsigargin and CPA brought about dense tubular formations. We correlated these morphological outcomes with the activation patterns of individual branches of the UPR. Furthermore, we are evaluating the global proteome to determine the protein players responsible for the morphological transformations. These studies will offer new insight into how the UPR orchestrates morphological adaptations under ER stress conditions and advance our understanding of organelle plasticity.

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Bicelles stabilize a compact conformation of opsin with enhanced α-helical packing

Presenting author:

Patryk Bielski

Carl von Ossietzky Universität Oldenburg, Department für Neurowissenschaften, Carl-von-Ossietzky-Straße 9-11, 26129 Oldenburg [DE], patryk.bielski@uni-oldenburg.de

Author(s):
Patryk Bielski, Karl-Wilhelm Koch, Justyna Bożek, Izabella Brand, Carsten Dosche, Valerio Marino, Daniele Dell'Orco

Bicelles form disc-like lipid bilayers surrounded by short-chain lipids and are well-suited to study purified transmembrane proteins in a native-like environment. In our study, we characterized bicelles using transmission electron microscopy, dynamic light scattering, fluorescence spectroscopy, infrared spectroscopy, and circular dichroism. Rhodopsin, a prototypical G protein-coupled receptor, was reconstituted into bicelles, increasing their diameter from 11.6 ± 0.6 nm to 14.9 ± 0.7 nm without detectable aggregation. Based on bicelle and rhodopsin concentrations, we estimated on average 4 ± 3 to 6 ± 1 bicelles per rhodopsin molecule, indicating that only 14-25% of bicelles contained protein. Spectroscopy showed that bicelle-reconstituted rhodopsin adopts a more compact, α-helix-rich fold and displays enhanced thermal stability compared with detergent-solubilized rhodopsin. When immobilized via concanavalin A, rhodopsin in bicelles bound the G protein transducin with at least 10-fold lower efficiency than in detergent but retained a 1:1 binding stoichiometry, consistent with a monolamellar bicelle orientation exposing natively folded rhodopsin.

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Structural basis of Protein O-mannosylation by Pmt4

Presenting author:

Florestan Bilsing

Biochemistry Center Heidelberg, Structural Biology, Im Neuenheimer Feld 328, 69120 Heidelberg [DE], florestan.bilsing@bzh.uni-heidelberg.de

Author(s):
Florestan Bilsing, Melanie McDowell, Gaetano D'Urso, Klemens Wild, Irmgard Sinning

Protein O-mannosylation is performed by protein O-mannosyltransferases (PMTs) in the ER lumen [1]. PMTs span the ER membrane and transfer mannose from Dol-P-Man to a serine or threonine residue [2]. This process is conserved from yeast to humans [3]. One of the best studied highly mannosylated proteins in humans is alpha-Dystroglycan (⍺-DG). Impaired mannoslyation of ⍺-DG causes severe developmental issues [4]. Humans have two PMTs (POMT1/2), while yeast has at least six active PMTs (Pmt1-6) of which only Pmt4 forms homodimers, while the others form heterodimers. Furthermore, Pmt4 is the only yeast PMT that mannosylates ⍺-DG in vitro and therefore serves as model for the human homolog [5].

We show recent progress in understanding structure and function of Pmt4. We obtained high-resolution structures of Pmt4 and its luminal MIR domains using cryo-EM and X-ray crystallography, respectively. We identified a cytosolic binding site for the substrate Dol-P-Man which is functionally important in vivo [6]. In combination with structure-based mutagenesis and affinity data we provide a framework towards understanding substrate specificity and function of the Pmt4 homodimer.

1 Koff, M., et al., Glycobiology, 2023

2 Strahl-Bolsinger, S. and W. Tanner, Eur J Biochem, 1991

3 Bausewein, D., et al., J Biol Chem, 2016

4 Lengeler, K.B., D. Tielker, and J.F. Ernst, Cell Mol Life Sci, 2008

5 Neubert, P. and S. Strahl, Curr Opin Cell Biol, 2016

6 McDowell, M.A., et al., Nat Commun, 2025

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Characterization of the methyltransferase Cho2 in yeast

Presenting author:

Hellen Bleeker

, , Johannisstraße 82, 49074 Osnabrück [DE], hbleeker@uos.de

Author(s):
Hellen Bleeker

Glycerophospholipids make up the majority of membrane lipids, providing structure and stability for cells. The glycerophospholipid phosphatidyl choline (PC) can be synthesized via either the Kennedy pathway or the CDP-DAG pathway. Synthesis by the CDP-DAG pathway occurs primarily under conditions of choline deficiency. In this case, PC is synthesized via a triple methylation step. In yeast, the first methylation step is believed to be catalyzed by Cho2, while the second and third steps are catalyzed by Opi3.
While the broad process of phosphatidyl ethanolamine methylation is understood, the regulatory mechanisms by which the CDP-DAG pathway may be repressed during choline availability remain unclear. We used lipidomics in combination with choline-lacking conditions to validate the partitioning of methylation steps from Cho2 and Opi3. To accomplish this, we used a deletion of the choline and ethanolamine transporter Hnm1 to inactivate the Kennedy pathway. Additionally, we purified Cho2 from an hnm1Δ strain. Mass spectrometry analysis revealed that previously observed phosphorylation sites disappear in the hnm1Δ. Earlier studies have shown that the CDP-DAG pathway is repressed in the presence of choline. In combination with our results, this suggests that Cho2 activation might occur upon dephosphorylation. Together, these findings lay the groundwork for further studies on the activity of Cho2, particularly with regard to its phosphorylation sites.

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Chemically tunable permeability of engineered alpha-Hemolysin in synthetic membranes

Presenting author:

Elisabeth Bobkova

MPI for Terrestrial Microbiology, Marburg, Biochemistry & Synthetic Metabolism, Karl-von-Frischstr. 10, 35043 Marburg [DE], elisabeth.bobkova@mpi-marburg.mpg.de

Author(s):
Elisabeth Bobkova, Anastasia Götz, Scott Scholz, Tobias Erb

 

Synthetic lipid membranes are indispensable tools in biotechnology, yet they remain largely passive structures that lack the rich, stimulus‑responsive capabilities of their biological counterparts. Imagine if these artificial barriers could be transformed into active, tunable materials whose permeability is programmed on demand.

To move toward this vision we have engineered the self-inserting pore α‑hemolysin for differential molecular diffusion through site‑specific functionalization. Using maleimide- reagents we introduce defined chemical groups at two distinct positions on the pore, enabling modification both before and after membrane insertion.

The impact of these modifications is assessed with a breakage‑controlled diffusion assay based on split nanoluc. The breakage-controlled assay discriminates diffusion from membrane rupture, delivering an unbiased, medium‑throughput read‑out. Using this platform we show that our mutated pores exhibit distinct permeability patterns for peptides of varying charge and structure, and visualize how maleimide functionalization can reshape these patterns.

With the potential for AI-ready data generation, our work paves the way for further optimization and customization of diffusion properties over synthetic membranes. These advances open new possibilities for spatially controlled bioreactions, synthetic cell systems, and functionalized biomaterials.

 

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Role of Pex31 in organelle adaptation to metabolic cues

Presenting author:

Johanna Bode

, , Hirseweg 69, 48163 Münster [DE], johanna.bode@uni-muenster.de

Author(s):
Marie Hugenroth, Johanna Bode, Pascal Höhne, Xuetong Zhao, Rebecca Fausten, Mike Wälte, Maria Bohnert

To access its full potential each cell needs to be able to sense nutrients in its surroundings and respond to their availability accordingly. We recently identified Pex31, a member of the Pex30 protein family, as a molecular player in metabolic adaptation in the yeast Saccharomyces cerevisiae. Through a systematic microscopy-based screen, we found that Pex31 localizes to the contact site between the nucleus and the vacuole, also called the nucleus-vacuole junction (NVJ). The NVJ is highly responsive to glucose availability: The contact site is small at glucose repletion, but its size and proteome increase under glucose starvation. Pex31 shows an atypical glucose dependence, being enriched at the NVJ at glucose-replete conditions, but absent under glucose-depleted conditions. Pex31 overexpressing blocks NVJ expansion at glucose starvation, while PEX31 deletion results in an expansion of the NVJ size and proteome at glucose repletion, indicating that Pex31 functions as a negative regulator of the NVJ. Cells lacking Pex31 also exhibit additional hallmarks of starvation at glucose replete conditions, including starvation-like alterations in lipid storage, a reorganization of the vacuole, and a decreased nuclear accumulation of the glucose repression factor Mig1. These findings suggest that Pex31 plays a critical role in coordinating metabolic processes and may be important for understanding how cells adapt to different metabolic states.

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New players in lipid droplet biology: contact sites meet organelle motility

Presenting author:

Maria Bohnert

University of Münster, Münster, Germany , Cell Dynamics and Imaging, Von-Esmarch-Str. 56, 48149 Münster [DE], bohnertm@uni-muenster.de

Author(s):
Maria Bohnert

pid droplets (LDs) are ubiquitous organelles with key roles in lipid storage and metabolism. A detailed mechanistic understanding of LD biology is crucial to tackle the implications of LD dysfunctions, which are linked to diseases including obesity, diabetes, fatty liver diseases, infectious diseases, and lipodystrophy. However, the molecular basis of the LD life cycle is still partially enigmatic.

We combine microscopy-based high-content screening approaches in yeast with mechanistic analyses to fill in the blank spots in the LD map. Our current work focusses on the following questions: (1) how do LDs communicate with other organelles? And (2) how is intracellular LD motility mediated on a molecular level? We have identified the molecular players that mediate membrane contact sites between LDs and other organelles, and the machinery that mediates actomyosin-based LD motility. We identify an unexpected molecular link between LD contact site formation and motility, suggesting a regulatory mechanism for the coordination of the different aspects of the LD life cycle.

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Metabolic control at the nuclear envelope: lipid composition as a regulator of gene silencing

Presenting author:

Sigurd Braun

Justus-Liebig Universität Giessen, Institut für Genetik, Heinrich-Buff-Ring 17, 35392 Gießen [DE], sigurd.braun@gen.bio.uni-giessen.de

Author(s):
Abubakar Muhammad, Agnisrota Mazumder, Zsuzsa Sarkadi, Sigurd Braun

The nuclear envelope (NE) acts as a specialized regulatory compartment that facilitates gene repression by recruiting factors that promote heterochromatin assembly and RNA degradation. We previously showed that the conserved inner nuclear membrane protein Lem2 interacts with the repressor complex SHREC^NuRD and the RNA surveillance complex MTREC^PAXT to coordinate heterochromatin silencing and transcript processing in fission yeast, thereby providing mechanistic insight into how the NE actively shapes gene repression (PMID 26744419, 36123402). However, Lem2-mediated silencing has also been linked to NE lipid metabolism by other studies (PMID 36799444, 30975915), suggesting that membrane composition itself may influence gene regulation.

Building on this framework, we performed a systematic genome-wide screen to identify novel factors required for heterochromatin silencing (PMID 39565189). This unbiased approach revealed several enzymes involved in lipid metabolism, including the SAM-dependent methyltransferases Cho1 and Cho2, which catalyze the terminal steps of phospholipid synthesis. Loss of Cho1 or Cho2 primarily affected silencing at subtelomeric regions, indicating that lipid metabolism preferentially impacts specific heterochromatin domains. Together, our findings suggest that nuclear membrane lipid composition represents a functional layer of gene regulation, supporting an emerging view in which NE composition integrates metabolic state with chromatin organization.

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Synthesis of fluorescent sphingolipids for the analysis of plasma membrane lipid dynamics

Presenting author:

Lasse Bredegaard

Ruhr Universität Bochum, Biochemistry II, Universitätsstrasse 150, 44801 Bochum [DE], Lasse.Bredegaard@ruhr-uni-bochum.de

Author(s):
Lasse Bredegaard, Elias Haid, Thomas Günther-Pomorski

Lipids play essential roles in cellular physiology beyond their structural function in membranes. Among these, sphingolipids are still a poorly studied lipid class, despite strong evidence implicating species such as sphingosine-1-phosphate in cell migration and actin cytoskeleton dynamics, and ceramide in the control of cell viability. A major limitation in sphingolipid research is the difficulty of tracking lipid localisation and dynamics, as many species lack suitable detectable analogues. Fluorescent lipid probes partially address the challenge, allowing for detection using microscopy and flow cytometry, however available sphingolipid variants remain limited. Here, we present a synthesis procedure for fluorescently labelled sphingolipids. The process involves reacting NBD-Hexanoic acid with the free amine at the sn-2 position of sphingolipids. The resulting products are purified via thin-layer chromatography and structurally validated using ¹H-NMR and MS. This process allows for quick and inexpensive small-scale synthesis of novel sphingolipid species suitable for cellular studies, providing a versatile tool to investigate sphingolipid dynamics in biological systems.

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TORC1-dependent sorting of PI(3,5)P₂ is required for vacuole membrane remodeling and signaling endosome formation

Presenting author:

Frederik Brinks

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], frederik.brinks@uni-osnabrueck.de

Author(s):
Frederik Brinks, Annabel Arens, Rainer Kurre, Jacob Piehler, Lars Langemeyer, Christian Ungermann

Lysosomes, as central organelles of the endolysosomal system, support cell
growth by releasing nutrients derived from hydrolytic digestion of macromolecules. Additionally,
they serve as storage organelles for ions and amino acids and must respond to
changes in osmolarity by adjusting their membrane to maintain membrane integrity. The
nutrient-sensing target of rapamycin complex 1 (TORC1) and the lipid kinase Fab1 (PIKfyve
in mammals) are key regulators of these processes on yeast vacuoles. TORC1 phosphorylates
Fab1, yet how their activities are functionally coupled is unknown. Here, we
show that yeast TORC1 is essential for the sorting of Fab1-derived phosphatidylinositol-
3,5-bisphosphate (PI(3,5)P2) from vacuoles to signaling endosomes (SEs), whose formation
depends on the CROP membrane remodeling complex. TORC1 phosphorylation activates
Fab1, presumably to maintain elevated PI(3,5)P2 levels on SEs toward cell growth. In mutants
defective in endosome–vacuole fusion, PI(3,5)P2 accumulates on endosomes adjacent
to the vacuole, indicating that its hydrolysis primarily occurs on the vacuolar membrane.
Our ndings reveal that synthesis and spatial distribution of the vacuolar signaling lipid
PI(3,5)P2 are directly coordinated by TORC1, coupling nutrient sensing to membrane remodeling
and endosomal

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Bridging the gap: Lipid trafficking between the inner and outer membranes of Agrobacterium tumefaciens

Presenting author:

Lina Brodskaia

Ruhr-Unitversität Bochum, Faculty of Biology and Biotechnology , Universitätsstraße 150, 44780 Bochum [DE], lina.brodskaia@rub.de

Author(s):
Lina Brodskaia, Franz Narberhaus, Marten Exterkate, Meriyem Aktas

Gram-negative bacteria possess a dual-membrane envelope with a phospholipid (PL) inner membrane (IM) and an outer membrane (OM), separated by the periplasm. PLs synthesized on the IM’s cytoplasmic side must be transported between the membranes during growth and membrane remodeling making intermembrane PL trafficking essential for envelope biogenesis. The molecular mechanisms of this process, however, remain poorly understood. Recent studies suggest that AsmA-like proteins form bridge-like structures that facilitate PL transport between the IM and OM by shielding hydrophobic acyl chains during their passage through the aqueous periplasm. Additionally, DedA-family proteins are believed to mediate transbilayer transport within the IM.

This study investigates the role of AsmA- and DedA-family proteins in lipid trafficking in Agrobacterium tumefaciens, a plant pathogen that relies on a complex lipid composition for its pathogenicity. To explore this, we use mutagenesis approaches to examine how the loss of these putative transporters affects membrane stress adaptation and cellular integrity. Mass spectrometry is applied to compare PL distribution in the IM and OM of wild-type and mutant strains. This analysis will help identify lipid imbalances resulting from the loss of transporters. Additionally, the proteins' activity will be assessed in vitro using model membranes to measure lipid translocation, substrate specificity, and potential cooperation in membrane dynamics.

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Triglyceride synthesis pathway and stress sensing in lipid droplet-mediated longevity

Presenting author:

Janine Cathrin Brück

Institute of Molecular Biology (IMB), , Ackermannweg, 4, 55128 Mainz [DE], J.Brueck@imb-mainz.de

Author(s):
Janine Cathrin Brück

Lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and serve as central hubs for cellular lipid homeostasis, buffering metabolic stress and lipotoxicity. Caenorhabditis elegans worms enriched in lipid droplets exhibit extended lifespans, yet how specific steps of the triacylglycerol (TAG) synthesis pathway contribute to this lipid droplet-dependent longevity remains largely unclear. Using targeted genetic interference of C. elegans homologs of key enzymes involved in the TAG synthesis pathway known from mammals and yeast including GPATs, AGPATs, Lipin, and DGATs we systematically perturb and quantify resulting changes in lipid droplet abundance and morphology by confocal fluorescence microscopy. We then examine how perturbations that alter lipid droplet number affect stress sensing and activation in the endoplasmatic reticulum (ER), using the unfolded protein response (UPR) signaling as a readout, and we test functional consequences by measuring membrane integrity. Our initial data indicate that depletion of Lipin increases the ER stress response and decreases membrane integrity during aging. By identifying which steps of the TAG synthesis pathway are key for stress protection, this work aims to identify how lipid droplet-centered lipid remodeling promote longevity in C. elegans.

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In vitro characterization of 3- Ketosphinganine reductase Tsc10 in yeast

Presenting author:

Kira Brümmer

Universität Osnabrück, Bioanalytische Chemie, Barbarastraße 13, 49076 Osnabrück [DE], kbruemmer@uni-osnabrueck.de

Author(s):
Kira Brümmer, Florian Fröhlich, Verena Wolf

In yeast, de novo sphingolipid biosynthesis occurs at the endoplasmic reticulum. The first step is catalyzed by serine palmitoyltransferase (SPT), which is part of the SPOTS complex and generates 3-ketosphinganine (3KS) from palmitoyl-CoA and serine. Then, 3KS is reduced to dihydrosphingosine (DHS) in an NADPH-dependent reaction catalyzed by Tsc10. In mammals, the Tsc10 ortholog FVT1 is implicated in skin disorders and cancer. However, despite its central role, biochemical and functional features of Tsc10 and its potential coupling to SPOTS remain unclear.

Here, Tsc10 was purified and its activity confirmed in vitro, both in solution and after reconstitution into ER-mimicking liposomes, quantified via NADPH fluorescence and lipid extraction and MS. Kinetic analysis yielded a K_m of 12 µM for 3KS. Size-exclusion chromatography and mass photometry indicated that Tsc10 exists predominantly as a monomer. Conversion of SPOTS-derived 3KS by Tsc10 was also observed in a reconstituted membrane system containing both enzymes. These results demonstrate that Tsc10 can function downstream of SPT in vitro, providing a framework to study early sphingolipid biosynthesis. Future work aims to resolve the crystal structure of Tsc10 to detail its active site and overall architecture and investigate how the membrane environment influences substrate access, with subsequent studies extending to FVT1.

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Assessing the selectivity of non-vesicular lipid transport pipelines

Presenting author:

Martin Buitrago-Arango

MPI-CBG, Membrane Chemical Biology, Pfotenhauerstraße 108, 01307 Dresden [DE], buitrago@mpi-cbg.de

Author(s):
Martin Buitrago-Arango, Kristin Böhlig, Athanasios Papangelis, Katelyn C. Cook, André Nadler

Intracellular lipid transport regulates the organelle lipid identities of the cell and determines their physicochemical properties and function. This transport is mainly orchestrated by non-vesicular routes via lipid-transfer proteins (LTPs) that mediate fast, directed, and selective lipid transport in lipid carriers that resemble the shape of either a bridge or a cup. The absence of many of these lipid transporters results is lethal. This underlines the need to systematically characterize how lipid transfer proteins selectively transport lipid-species across the between organelle membranes. Here, we genetically perturb LTPs-expression and leverage bifunctional (clickable and crosslinkable) lipid probes to visualize individual lipid species in cells and quantify their inter-organelle dynamics. Thereby, we further the role of these proteins in the directional trafficking of selective lipid species that create the unique organelle lipid‑fingerprint.

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Polytopic inner mitochondrial membrane proteins are inserted by selective cooperation with the TIM22 insertase

Presenting author:

Jakob D. Busch

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Laboratories of Prof. Dr. Nils Wiedemann and Prof. Dr. Dr. Nikolaus Pfanner, Stefan-Meier-Str. 17, 79104 Freiburg im Breisgau [DE], jakob.busch@biochemie.uni-freiburg.de

Author(s):
Jakob D. Busch, Iniyan Ganesan, Johanna R. Diehm, Nicole Zufall, Nikolaus Pfanner, Nils Wiedemann

The cellular energy metabolism of eukaryotic cells depends on ATP synthesis as well as numerous mitochondrial metabolic and biosynthetic pathways. To facilitate the metabolite exchange across the inner membrane, mitochondria import nuclear-encoded metabolite carrier and transporter proteins. The twin-pore translocase TIM22 is essential to insert these polytopic membrane proteins. However, cryo-EM structures questioned the proposed pore-dependent insertion mechanism. Here we determined the active site of the membrane insertion machinery by generation of substrate protein/TIM22 insertion intermediates analysed by double-site-specific crosslinking. The topological conformation of the carrier protein substrate intermediates arrested in a conserved membrane exposed cavity of the essential core subunit Tim22 are in agreement with AI-assisted structural modelling. To analyse the mechanism of membrane protein insertion we systematically mutated conserved intermembrane space, transmembrane and matrix domains of Tim22. The mutants had severe growth defects, were mostly respiratory deficient or not viable. Functional membrane protein insertion assays revealed a surprising variability of the membrane insertion efficiencies of substrates in specific Tim22 mutants. Thus, a subset of the essential domains of Tim22 are required for efficient membrane insertion of individual proteins by selective cooperation between specific features of substrates and the insertase.

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Decoding ether lipid roles in ciliary function

Presenting author:

Kristin Böhlig

MPI-CBG, , Pfotenhauerstraße 108, 01307 Dresden [DE], bohlig@mpi-cbg.de

Author(s):
Kristin Böhlig, Katelyn C. Cook, Martin Buitrago Arango, André Nadler

Ether lipids play critical roles in human health, as evidenced by the severe developmental and neurological abnormalities arising from their deficiency in peroxisomal disorders such as RCDP or the Zellweger spectrum disorders. Ether lipids structurally differ only slightly from regular ester-linked phospholipids however seem to play unique functional roles. To date, the underlying cell biology and mechanistic insights remained unidentified. Here, bifunctional ether lipid probes, containing a UV-activatable diazirine and clickable alkyne moiety, were used to identify ether lipid-specific localization and intracellular trafficking as well as their unique protein interactors. A pulldown assay in RPE cells revealed unique interactions of ether lipids with cilia proteins suggesting a potential functional role in primary cilia, a link completely unexplored in the current literature. In fact, symptoms of ether lipid deficiency and of ciliopathies are virtually identical. Follow-up experiments of RCDP patient-derived fibroblasts showed significantly elongated cilia compared to healthy cells. This suggests a ciliary growth defect under ether lipid deficiency which likely impairs ciliary function and potentially accounts for the documented symptoms. The direct effects of ether lipid deficiency on ciliary function (e.g. signalling, transport) are currently under investigation.

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Neutrophil-derived extracellular vesicles associate with miRNAs to potentially silence gene expression in the human pathogenic fungus Aspergillus fumigatus

Presenting author:

Erik Böhm

Leibniz Institute for Natural Product Research and Infection Biology—Hans Knöll Institute (Leibniz-HKI), RNA Biology of Fungal Infections, Tatzendpromenade 18, 07745 Jena [DE], erik.boehm@leibniz-hki.de

Author(s):
Alexander Bruch, Erik Böhm, Lukas Schrettenbrunner, Xiaoqing Pan, Matthew G. Blango

Aspergillus fumigatus is an opportunistic fungal pathogen classified as a critical priority to global health by the WHO. It causes invasive pulmonary aspergillosis associated with mortality rates of 30­-85% in >2 million patients each year, with neutropenia the major risk factor for these severe infections. Thus, definition of the antifungal neutrophil response is likely key to improved therapeutic strategies. Previous work reported that primary human neutrophils secrete extracellular vesicles (EVs) inhibiting A. fumigatus growth ex vivo; however, the molecular arsenal within these EVs remains poorly understood. We show that antifungal neutrophil EVs co-purify with diverse RNA species enriched in let-7 miRNAs. The abundantly detected miRNA let-7a-5p was predicted to target the putative methionine synthase metE in A. fumigatus. Indeed, EV application to fungal hyphae significantly reduced metE transcript levels. Mutating the predicted let-7a-5p binding site in the fungal metE gene suggested a reversion of metE expression, despite high levels of donor variation. Our work points to an EV-mediated cross-kingdom RNA interference mechanism between human immune cells and A. fumigatus, illustrating the emerging role of EVs in innate antifungal immunity. We hope that further research will elucidate how EVs are loaded with their cargo and how it is delivered across the fungal cell wall, eventually paving the way for the application of EVs as vectors for new RNA-based antifungals.

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Pex19 mediates membrane contact sites between peroxisomes and Golgi to promote insulin-like peptide secretion

Presenting author:

Margret Bülow

University of Potsdam, Institute of Nutritional Sciences, Karl-Liebknecht-Straße 24-25, 14476 Potsdam [DE], margret.buelow@uni-potsdam.de

Author(s):
Margret Bülow, Nicole Kucharowski, Marie Anna König, Darla Patricia Dancourt Ramos

Insulin is a peptide hormone secreted in Golgi-derived dense-core vesicles (DCV) from β-cells in response to nutrients. In Drosophila, three insulin-like peptides are secreted as neuropeptides from the insulin-producing cells in the brain. Peroxisomes are lipid-metabolizing organelles that form dynamic membrane contact sites with other organelles. Impaired peroxisomal metabolism has been associated with beta-cell apoptosis and impaired insulin secretion. How peroxisomes contribute to insulin and neuropeptide secretion is unknown. Here we demonstrate that peroxisomes establish nutrient-dependent membrane contacts with the Golgi apparatus in Drosophila insulin-producing cells. Secretion of insulin-like peptide 2 is impaired in mutants lacking the peroxisome assembly factor Pex19. Using click chemistry of alkyne-labeled fatty acids and lipidomics, we show that loss of peroxisomes shifts the profile of sphingolipids towards longer sphingoid bases and causes their accumulation in Golgi membranes, revealing altered lipid homeostasis. We further demonstrate that Pex19 directly interacts with Golgi-derived DCV and promotes peroxisome-Golgi membrane interactions via the fatty acyl-CoA reductase FAR2. We propose that this peroxisome-Pex19-Golgi axis is required to adjust Golgi membrane composition under starvation by channeling long-chain lipids to peroxisomes, thereby optimizing Golgi membrane properties for efficient DCV biogenesis and insulin-like peptide secretion upon refeeding.

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Ubiquitin-dependent signal amplification in lipid saturation sensing

Presenting author:

Jona Causemann

Saarland University, Medical Biochemistry and Molecular Biology, Kirrbergerstraße 100, 66421 Homburg [DE], s8jocaus@uni-saarland.de

Author(s):
Jona Causemann, Barbara Schmidt, Daniel Granz, Thorsten Mosler, Martin Jung, Ivan Dikic, Heiko Rieger, Robert Ernst

Biophysical properties of biomembranes such as fluidity and compressibility are vital for organelle function. In the endoplasmic reticulum (ER), dedicated sensor proteins monitor these properties and trigger adaptive responses to maintain homeostasis. Because membrane-derived signals are often weak and short-lived, sensors must employ effective amplification mechanisms – yet these remain difficult to dissect. Using the yeast lipid-saturation sensor Mga2 in a fully defined biochemical reconstitution system, we identify a previously unrecognized, ubiquitin-dependent mode of signal amplification. Increased membrane saturation stimulates ubiquitylation of Mga2 by the E3 ligase Rsp5. By reconstituting lipid-regulated ubiquitylation in vitro, we show that small changes in membrane composition are converted into large differences in Mga2 modification. Quantitative kinetic modeling reveals strong positive feedback, with up to 120-fold higher rates of ubiquitin addition. At the same time, we uncover a negative-feedback branch: in more unsaturated membranes, ubiquitin is diverted from the sensor toward Rsp5 itself, resulting in autoinhibition of the ligase. Together, these findings illustrate how weak membrane cues can be transformed into robust biochemical outputs by combining positive and negative feedback. They broaden the conceptual framework for ubiquitin-based signal amplification and reveal a mechanism that operates without relying on deubiquitylating enzymes.

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Ca2+ leakage triggers multilamellar membrane remodelling after endomembrane damage in macrophages

Presenting author:

Di Chen

The Francis Crick Institute, Host-Pathogen Interactions in Tuberculosis Laboratory, 1 Midland Road, The Francis Crick Institute, NW1 1AT LONDON [GB], di.chen@crick.ac.uk

Author(s):
Di Chen, Maximiliano G. Gutierrez, Antony Fearns

Intracellular organelles are either single membrane such as phagosome, double‑membrane such as autophagosome and multilamellar such as lamellar body. However, how multilamellar structures form and what is the function remain unknown.

Endomembrane can be damaged by insults such as crystals or bacterial pathogens. This damage results in content leakage that represents a danger signal for macrophages, as it orchestrates inflammatory responses if the injury is not controlled. However, how macrophages sense and respond to damage are poorly understood.

Using human macrophages, we found Ca²⁺ leakage from damaged organelles serves as a key signal that triggers extensive endomembrane remodelling and the formation of tubulo‑vesicular structures (TVS) composed of multilamellar structures and small vesicles that associated to the damaged compartment. These dynamic TVS are marked by membrane damage sensors Galectins and the autophagy hallmark ATG8/LC3. However, their multilamellar architecture clearly distinguish them from single or double membrane organelles. Notably, the complexity and degree of acidification of TVS depended on the nature of the damage trigger. Functionally, this Ca²⁺ driven membrane remodelling results in the containment of the damaged compartment and delayed cell death. Altogether, we identify Ca²⁺ leakage as a central signal that induces protective membrane remodelling to contain vesicular damage, revealing a conserved mechanism of innate immune defence in macrophages.

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Regulation of the unfolded protein response by anionic lipids

Presenting author:

Alexandra Cogan

University of Saarland, Biochemistry, PZMS, Kirrberger Str. 100, 66421 Homburg [DE], alexandra.cogan@uni-saarland.de

Author(s):
Alexandra Cogan, Toni Radanović, Jona Causemann, Cynthia Alsayyah, Robert Ernst

The endoplasmic reticulum (ER) is central to membrane biogenesis in eukaryotic cells, requiring tight coordination between lipid biosynthesis and protein folding to prevent ER stress. Both proteotoxic stress and altered ER membrane lipids can induce ER stress, activating the unfolded protein response (UPR). In yeast, the UPR enhances lipid synthesis and ER folding capacity. We previously showed that the UPR transducer IRE1 senses ER membrane compressibility through an unusual transmembrane structure, but the influence of anionic lipids remained unclear.

Here, we biochemically reconstitute IRE1 sensor constructs in liposomes with defined lipid compositions and assess their oligomeric state using Förster resonance energy transfer. We find that cholesterol promotes, while anionic lipids inhibit, IRE1 oligomerization—indicating opposing regulatory roles. These effects are absent under high salt conditions, suggesting selective regulation by anionic lipids. In contrast, the mammalian UPR transducer ATF6 is largely unresponsive to such lipid changes.

Our findings suggest that cells may fine-tune UPR sensitivity via dynamic ER membrane lipid composition, offering new insights into the regulation of ER stress responses in metabolic and neurodegenerative diseases.

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Selective lipid-protein interactions regulate the assembly of organelle membrane complexes

Presenting author:

Katelyn Cook

Max Planck Institute for Cell Biology & Genetics, , Pfotenhauerstr. 108, 01307 Dresden [DE], cook@mpi-cbg.de

Author(s):
Katelyn Cook, Karen Palacio-Rodrίguez, Kristin Böhlig, H. Mathilda Lennartz, O. Alf Honigmann, Andrej Shevchenko, Gerhard Hummer, Alexander von Appen, André Nadler

Organelle membrane identity is encoded by protein and lipid composition. For this purpose, human cells produce thousands of distinct lipid species, yet their precise functions remain largely unknown. Using bifunctional phospholipid probes representing major membrane lipid classes, we integrate lipid imaging with quantitative proteomics to create a framework for mechanistic lipid cell biology. We map time-resolved protein interactomes of individual lipid species as they are transported through the organelle system of human cells. We measure lipid-induced protein abundance changes, identify hundreds of unique candidate lipid-protein interactions and associated protein complexes. This multi-dimensional resource encompasses temporal, spatial and molecular aspects of lipid-protein interactomes, enabling the formulation of actionable hypotheses on lipid function. As a compelling example, we investigate the selective association of phosphatidylethanolamine (PE) with the integral membrane components of the nuclear pore (NDC1) and the mitochondrial intermembrane bridging (MIC60) complexes. Using super-resolution microscopy and molecular dynamics simulations, we demonstrate that PE stabilizes both NDC1 and MIC60 in highly curved membrane segments, indicating that major membrane organizing complexes are positioned by selective lipid-protein interactions. Altogether, our experimental strategy provides a blueprint for discovering and characterizing cellular functions of individual lipids.

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Multiparameter single-liposome characterization of active membrane transport by the maltose ABC-importer from E. coli

Presenting author:

Thorben Cordes

TU Dortmund, Chemistry and Chemical Biology, Otto-Hahn-Str. 4a, 44227 Dortmund [DE], thorben.cordes@tu-dortmund.de

Author(s):
Thorben Cordes

Membrane transporters are essential for cellular processes and represent key targets for pharmaceutical development due to their roles in disease, pathogenicity, and drug resistance. While structural insights into transporters have advanced rapidly, understanding their transported substrates and mechanistic principles remains challenging. Conventional assays measure substrate uptake using radiolabeled or fluorescent substrates to derive kinetic parameters. Recent fluorescence-based techniques now enable real-time observation of transport activity and conformational dynamics down to single molecules. Inspired by single-enzyme studies revealing dynamic functional states, similar approaches promise unprecedented insight into transporter kinetics and transient intermediates. However, active membrane transporters, particularly ABC transporters, pose challenges for electrophysiological methods due to slow transport rates or uncharged substrates. Here we present a fluorescence-based assay for monitoring maltodextrin transport by the E. coli ABC importer MalFGK₂-E, applicable both in bulk and at the single-liposome level. Using the assay, we quantify transport rates for various substrates and demonstrate compatibility with single-liposome imaging to correlate kinetic data with liposome size and transporter number. This integrated approach establishes a versatile platform for mechanistic studies of primary-active transporters at both ensemble and single-molecule resolution.

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Molecular mechanism of ceramide transfer by STARD11 / CERT : an in vitro study

Presenting author:

Camille Cuveillier

University of Geneva, Physiology and metabolism, 1 rue Michel Servet, 1206 Geneva [CH], camille.cuveillier@unige.ch

Author(s):
Camille Cuveillier, Mahmoud Moqadam, Nathalie Reuter, Anne-Claude Gavin

Non-vesicular lipid transport by lipid transfer proteins (LTPs) is crucial for establishing and maintaining membrane lipid composition. LTPs shield lipids from the aqueous environment and mediate their uptake and release at membrane surfaces. These opposing steps are energetically demanding, as they require moving lipids across the hydrophilic headgroup region without external energy input. Taking STARD11 lipid transfer domain as a model, we combined molecular dynamic simulation and in vitro lipid transfer assays to uncover the central role of a conserved arginine in Ω1 loop. This residue provides structure to the gate of the lipid-binding cavity via an H-bond network and fine-tune the opening of the gate by interacting with membrane phospholipid phosphate groups leading to fast ceramide uptake. Finally, using bifunctional lipids, we show that the opening of the protein modifies membrane properties by promoting lipid fatty acid snorkeling. Overall, our findings reveal a bidirectional coupling in which the conserved Ω1-loop arginine enables STARD11 to reshape local membrane structure while membrane phospholipids reciprocally stabilize gate opening, together driving efficient lipid transport.

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A “peroxisomal burst” in neuronal membrane remodeling

Presenting author:

Darla Dancourt

University of Potsdam, Institute of Nutritional Sciences, Karl-Liebknecht-Straße 24-25, 14476 Potsdam [DE], darlapatricia.dancourtramos@med.uni-duesseldorf.de

Author(s):
Darla Dancourt, Margret Bülow, Nicole Kucharowski

Neurodevelopment requires coordinated neurite growth and synapse formation, processes that depend on extensive membrane remodeling, lipid homeostasis, and inter-organelle communication. While many organelles such as the endoplasmic reticulum, mitochondria, and endosomes/lysosomes are distributed throughout neuronal compartments, we observed that peroxisomes, single-membrane organelles involved in lipid metabolism and redox regulation, are largely restricted to the soma in differentiated neurons.

 

Strikingly, we identify a narrow developmental window characterized by a “peroxisomal burst,” during which peroxisomes become highly abundant, motile, and extend into developing axons and dendrites. This transient redistribution coincides with periods of neurite outgrowth and neuronal remodeling, suggesting a previously unrecognized role for peroxisomes in neurodevelopment. We propose that peroxisomes might support membrane dynamics by supplying lipids, regulating local redox environments, and engaging in membrane contact sites with other organelles. Using genetic approaches combined with super-resolution imaging and focused ion beam scanning electron microscopy (FIB-SEM), we aim to define the mechanisms and functional consequences of peroxisomes redistribution during neuronal maturation.

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Selective protein degradation from the Golgi apparatus membrane

Presenting author:

João Diamantino

University of Duisburg-Essen, Mechanistic Cell Biology, Universitätsstraße 2, 45141 Essen [DE], joao.diamantino@uni-due.de

Author(s):
João Diamantino, Sofia Murteira, Steffen Lawo, Farnusch Kaschani, Markus Kaiser, Doris Hellerschmied

Protein homeostasis is a hallmark of cellular health. To maintain homeostasis, cells are equipped with protein quality control (PQC) machineries that target damaged or unwanted proteins for degradation. The Golgi, as a major protein sorting and processing site, is crucial for cellular homeostasis. Despite its importance, the PQC machinery operating at the Golgi in mammalian cells has remained largely elusive. Here we used a chemical biology tool to induce misfolding and ubiquitination of model substrates localized at the Golgi membrane. This tool recruits a Cullin-RING ubiquitin ligase (CRL) complex by exposure of a C-terminal degron, recognized by the CRL substrate receptor KLHDC2. Upon ubiquitination, Golgi membrane proteins are targeted for proteasomal degradation. Clearance of these proteins is critical, since their accumulation causes Golgi fragmentation. At the molecular level, the degradation of Golgi membrane proteins depends on the AAA+ ATPase p97 together with its adaptors UFD1-NPL4. and FAF2. Furthermore, we identified RHBDD2 as a Golgi-localized rhomboid pseudo-protease that binds to misfolded membrane proteins and has a potential role in regulating Golgi morphology. We hypothesize that RHBDD2 harbours a membrane thinning activity, which might play a role in membrane protein extraction at the Golgi. Our work provides molecular insight into protein degradation from the Golgi membrane, promoting a better understanding of Golgi membrane homeostasis mechanisms.

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Delivering lipids to organelles via a novel photocage

Presenting author:

Gary Domeniconi

EPFL, SV SB GR-SCHUHMACHER, BCH 5221 (Batochime UNIL), Av. François-Alphonse Forel 2, 1015 Lausanne [CH], gary.domeniconi@epfl.ch

Author(s):
Gary Domeniconi, Linda Wedemann, Antonino Asaro, Giovanni D'Angelo, Milena Schuhmacher

Lipids are not only structural elements of the membranes, but also many-faceted signalling molecules whose structural diversity, spatial distribution and rapid kinetics are driven by a dense network of specific enzymes. However, tools to investigate lipid metabolism at such levels of specificity are scarce. To address this methodological gap, we develop a chemical biology approach that uses a new type of coumarin-based photocleavable fluorescent ‘cage’ to deliver specific lipids to live cells in a temporally-controlled manner. Chemical modifications on the photocage allow the sub-cellular targeting of the caged ('masked') lipid to specific organelles or membrane proteins with a high level of precision. By using this technique to trigger a pulse-increase in the concentration of various lipid classes (fatty acids, diacylglycerols, phosphatidic acids, …) and species in a spatially controlled manner in live cells - coupled with cell biology and molecular biology assays - we are addressing with an unprecedented level of specificity the organelle- and lipid species- dependency of various intricate lipid-mediated cellular processes.

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Interaction studies of PDGFRα mutants with the viral oncoprotein E5

Presenting author:

Moritz Enghofer

KIT - Karlsruher Institut für Technologie, Institut für Organische Chemie (IOC), Fritz-Haber-Weg 6, 76131 Karlsruhe [DE], moritz.enghofer@kit.edu

Author(s):
Moritz Enghofer, Rebecca V. Ernst, Sebastian Otteni, Sara Brand, Stephan L. Grage, Parvesh Wadhwani, Sergiy Afonin, Torsten H. Walther, Anne S. Ulrich

Receptor tyrosine kinases (RTK) are integral membrane proteins which play an important role in signal transduction. During natural signaling, RTK´s are activated by an extracellular ligand and induce a signaling cascade inside the cell. However, the short viral oncoprotein E5 can activate the receptor PDGFRβ ligand-independently by direct interactions with the transmembrane domain (TMD) of the receptor. This emphasizes that these TMD’s are functional entities themselves and are crucial in the signaling process. In earlier studies, we have demonstrated that the interaction between E5 and PDGFRβ leads to a change in the membrane orientation of both TMD’s, which can be observed through ¹⁵N solid state NMR spectroscopy. To further our understanding of these kinds of interactions, we created mutants of the TMD of the related receptor PDGFRα. PDGFRα, although similar to PDGFRβ, cannot interact with E5. By replacing specific amino acids, our goal was to find a minimal mutant which interacts with E5. With this approach we were able to determine the five key residues responsible for the interaction: Lys524, Thr538, Phe523, Ile528 and Ile531. Lys524 and Thr538 are equivalent to Lys499 and Thr513 in PDGFRβ, which are postulated to be essential for an interaction with E5. However, the insertion of only these two is not sufficient for an interaction. We could prove that also Phe523, functioning as a membrane anchor, and additionally, Ile528 and Ile531 are required for receptor activation.

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Structural studies on Pinholin of phage φ21

Presenting author:

Rebecca V. Ernst

Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Biochemistry , Durlacher Allee 38, 76131 Karlsruhe [DE], rebecca.ernst@kit.edu

Author(s):
Rebecca V. Ernst, Dennis Winkler, Sergiy Afonin, Stephan L. Grage, Parvesh Wadhwani, Torsten H. Walther, Anne S. Ulrich

Pinholin S²¹68 is a small membrane protein with two transmembrane domains (TMDs) acting as master regulator of host cell lysis in the lytic cycle of phage φ21. TMD2 is responsible for pinhole formation and is assumed to be stably membrane inserted. TMD1, in contrast, is postulated to serve a regulatory function and must exit the membrane to enable pinhole activation.

To investigate membrane protein topology, we developed a biochemical protease-accessibility assay based on proteinase K cleavage. Here, solvent-exposed fragments are cleaved while membrane-inserted parts remain intact. When applied to full-length pinholin, a partial degradation was observed. To clarify its membrane topology, we further applied this assay to the individual TMD fragments, which so far did not yield conclusive results. However, using oriented solid-state nuclear magnetic resonance spectroscopy, we were able to determine the membrane topology of the TMD2 fragment. Interestingly, in membranes with increased hydrophobic thickness, we found the TMD2 fragment lying flat on the membrane, whereas a positive membrane curvature enabled a transition to a membrane-inserted state. Secondary structure analysis of the TMD1 fragment was performed using circular dichroism spectroscopy. In disagreement with the literature, TMD1 was found to form highly stabilised amyloid-like β-sheets in solution. The extent to which these observations of the TMD fragments apply to the full-length protein is yet to be determined.

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Spatial reorganization of the ceramide synthase in response to stress

Presenting author:

Rebecca Fausten

Universität Münster, Institute of Cell Dynamics and Imaging, Von-Esmarch-Straße 56, 48149 Münster [DE], rfausten@uni-muenster.de

Author(s):
Rebecca Fausten, Maria Bohnert, Florian Fröhlich

Ceramides are essential multifunctional lipids in our cells. They are central intermediates in the biosynthesis of complex sphingolipids, act as signaling molecules and dysregulation of ceramide levels is associated with multiple human diseases. Ceramides are synthesized by enzymes called ceramide synthases. In yeast, ceramide synthase consists of the three subunits Lac1, Lag1 and Lip1 that form a complex embedded in the ER membrane. Lac1 and Lag1 are both catalytically active but only the simultaneous loss of both proteins is lethal for the cells. The regulatory subunit Lip1 interacts with Lac1 and Lag1, which is essential for the enzyme's functionality. We observe a striking spatial reorganization of all three ceramide synthase subunits during nutrient and redox stress conditions. The proteins transition from a dispersed ER localization to an accumulation in defined foci that are predominantly located at the nuclear ER. Live-cell imaging enabled observation of reversible formation and dissolution of these foci when shifting cells between stressed and non-stressed conditions. All three proteins colocalize with each other. However, a microscopy-based genome-wide screening approach uncovered no further residents of these foci. This indicates that stress promotes formation of a unique, ceramide synthase-enriched ER membrane domain. We hypothesize that this spatial sequestration of the ceramide synthase affects enzyme activity for adaptation during metabolic changes and stress.

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Building a functional atlas of homologous contact site machineries through advanced interaction mapping

Presenting author:

Emma Fenech

University of Cologne, Center for Biochemistry, Joseph-Stelzmann-Straße 52, 50931 Cologne [DE], efenech@uni-koeln.de

Author(s):
Emma Fenech

The endoplasmic reticulum (ER) coordinates a multitude of diverse and essential functions. The range and robustness of each of these functions are upheld by the unique and shared capacities of homologous proteins. Such homologs are also present at ER contact sites, with the yeast family of six LAM (Lipid transfer protein Anchored at Membrane contact sites) proteins being one example. This family is composed of three pairs of homologs which are all anchored in the ER membrane and contain lipid binding and transfer domains. However, each LAM protein is unique in its molecular architecture and localization to different ER subdomains. To shed light onto what determines the localization of the different LAMs and the specific roles they perform, we used an advanced proximity-labelling approach to profile the protein landscape of the entire family. We uncovered unique candidate interactors which supported previous observations that Lam5 resides at the ER-mitochondria contact, and we demonstrated a novel role for it in sustaining mitochondrial activity. Conversely, shared putative interactors of multiple LAMs revealed how the Lam1/3 and Lam2/4 homologous pairs could associate specifically with plasma membrane lipids. Overall, our work provides new insights into the regulation and function of the LAM family members. More globally, it demonstrates how proximity labelling can identify shared and unique functions of protein homologs resident within the ER membrane contact site network.

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Elucidating the role of lipid transfer proteins for the outer membrane homeostasis in C. jejuni

Presenting author:

Liliana Fernandes da Costa

Goethe University Frankfurt, , Max-von-Laue-Str. 9, 60438 Frankfurt [DE], l.fernandesdacosta@em.uni-frankfurt.de

Author(s):
Liliana Fernandes da Costa

The Gram-negative bacterial cell envelope is uniquely characterized by an asymmetric outer lipid bilayer, where the inner leaflet consists of phospholipids and the outer leaflet contains lipopolysaccharide molecules. As a result, the outer bacterial membrane provides intrinsic resistance to large polar and lipophilic compounds such as antibiotics, detergents and bile salts. To maintain this penetration barrier function, Gram-negative bacteria have developed different strategies, with one of them being the highly conserved maintenance of lipid asymmetry (Mla) system. In the model organism Escherichia coli, the Mla system consists of the OmpC/F-associated outer membrane lipoprotein MlaA, a periplasmic shuttle protein MlaC and the ATP-binding cassette transporter complex MlaFEDB, that mediate the retrograde transport of mislocalized phospholipids from the outer leaflet of the outer membrane to the inner membrane. Intriguingly, the mlaA and mlaC genes of the Gram-negative bacterium Campylobacter jejuni share an operon with a Resistance-nodulation-cell division (RND) transporter, named Campylobacter lipid transporter A (CltA), raising the question of whether CltA might function together with MlaA and MlaC in the anterograde transport of phospholipids. This study aimed to functionally and mechanistically characterize Cj MlaAC_CltA heterologously expressed in E. coli using biochemical assays and mass spectrometry to elucidate its role in phospholipid transport.

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Morphogenesis of complex membrane structures: Endoplasmic Reticulum stacks and whorls

Presenting author:

Natalie Friemel

Heidelberg University Biochemistry Center, , Im Neuenheimer Feld 328, 69120 Heidelberg [DE], natalie.friemel@bzh.uni-heidelberg.de

Author(s):
Natalie Friemel, Sebastian Schuck

Cells dynamically remodel the morphology of their organelles to optimise organelle function. The endoplasmic reticulum (ER) membrane can be shaped into arrays of sheets that form flat stacks or spherical whorls. ER stacks and whorls can have distinctive protein compositions and may support protein maturation, drug detoxification or autophagy. The molecular machinery for the formation of these ER subdomains and their precise membrane topology remain unknown. Here, we employ subdomains induced by the ER membrane protein Hmg2 as a model for ER morphogenesis in yeast. We combine a genetic screen, spatial proteomics and cryo-electron tomography to identify factors in stack and whorl formation and define their three-dimensional architecture. We find that the transition from flat sheets to spherical ER structures depends on membrane abundance and rim stabilisation by reticulon proteins. Additionally, certain lumenal and membrane-associated ER proteins are excluded from stacks and whorls, suggesting a protein sorting mechanism during ER subdomain formation. This selectivity may be based on protein size because stacked smooth ER sheets are about 40% thinner than rough ER sheets and thus offer less lumenal space. Overall, understanding the molecular mechanisms of ER morphogenesis will help to further define the relationship between organelle form and function.

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Mapping Na+ sites in the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae

Presenting author:

Guenter Fritz

University of Hohenheim, Cellular Microbiology, Garbenstrasse 30, 70599 Stuttgart [DE], guenter.fritz@uni-hohenheim.de

Author(s):
Sebastian Herdan, Jann-Louis Hau, Janet Vonck, Guenter Fritz, Julia Steuber

In many pathogenic bacteria the exergonic oxidation of NADH with ubiquinone is coupled to the generation of an electrochemical Na⁺ gradient [1]. This reaction is catalyzed by the membrane protein complex Na⁺-NQR (Na⁺-translocating NADH:quinone oxidoreductase), which consists of six subunits NqrABCDEF and contains four flavins and two [2Fe-2S] as cofactors [2]. Na⁺-NQR is central in the metabolism of these pathogens und thus represents a target for novel antimicrobial drugs [3]. We study the Na⁺-NQR from the human pathogen Vibrio cholerae [4]. In order to elucidate the Na⁺ binding sites during Na⁺ translocation, we performed a spectroscopic and structural analysis. Subunit NqrB of Na⁺‑NQR harbors a riboflavin in the vicinity of a proposed Na⁺ translocation pathway. Binding of Na⁺ to Na⁺-NQR induces slight changes in the UV-vis absorption envelope, which were mapped to a riboflavin bound in subunit NqrB close to a proposed Na⁺ translocation pathway. Such changes in absorption are caused by a change in the dielectric field in the environment of the flavin cofactor. Similar changes were observed using Th⁺, a structural analogue to Na⁺. The cryo-EM structure of Na⁺‑NQR incubated with Th⁺ confirmed binding of the ion close to the flavin cofactor. A reaction scheme for Na⁺-NQR is proposed, which considers the sequential interactions with substrates, ions and the different conformational states [4] of Na⁺-NQR.

[1] Häse, CC & Mekalanos, JJ (1999) Proc. Natl. Acad. Sci. U S A 96, 3183–3187.

[2] Steuber, J., Vohl, G., Casutt, M.S., Vorburger, T., Diederichs, K., Fritz, G. (2014) Nature 516, 62-67.

[3] González-Montalvo MA, Sorescu JM, Yuan M, DePaolo-Boisvert J, Liang P, Juárez OX, Tuz K. (2025) Front Microbiol. 16:1690572.

[4] Hau, J.-L., Kaltwasser, S., Muras, V., Casutt, M.S., Vohl, G., Claußen, B., Steffen W., Leitner, A., Bill, E., Cutsail, G.E., DeBeer, S., Vonck, J., Steuber, J., Fritz, G. (2023) Nature Struct Mol Biol. 30:1686-1694.

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Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH

Presenting author:

Pinki Gahlot

Universität Duisburg-Essen, Fakultät für Biologie, Universitätstraße 5, 45141 Essen [DE], pinki.gahlot@uni-due.de

Author(s):
Pinki Gahlot, Bojana Kravic, Giulia Rota, Johannes van den Boom, Sophie Levantovsky, Nina Schulze, Christian Behrends, Hemmo Meyer

Lysosomes are the main degradative organelles in the cell and serve as platforms for important signalling pathways. The loss of lysosomal function due to lysosomal membrane permeabilization (LMP) can be highly deleterious for the cell. LMP can be caused by different conditions such as lipid peroxidation, lysosomotropic drugs or disease-associated changes in lipid composition. Cells respond to LMP by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy. However, it is not fully understood how the decision between repair and lysophagy of damaged lysosomes is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. These lipid-packing defects are sensed via amphipathic helices in SPG20. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

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High-resolution structures of the UapA purine transporter reveal unprecedented aspects of the elevator-type transport mechanism

Presenting author:

Christos Gatsogiannis

Center for Soft Nanoscience (SoN), University Münster, Germany., Institut for Medical Physics and Biophysics , Busso-Peus-Str. 10, 48149 Münster [DE], gatsogiannis.office@uni-muenster.de

Author(s):
Christos Gatsogiannis

UapA is an extensively studied elevator-type purine transporter from the model fungus Aspergillus nidulans . Determination of a 3.6Å inward-facing crystal structure lacking the cytoplasmic N-and C-tails, molecular dynamics (MD), and functional studies have led to speculative models of its transport mechanism and determination of substrate specificity. Here, we report full-length cryo-EM structures of UapA in new inward-facing apo- and substrate-loaded conformations at 2.05-3.5 Å in detergent and lipid nanodiscs. The structures reveal in an unprecedented level of detail the role of water molecules and lipids in substrate binding, specificity, dimerization, and activity, rationalizing accumulated functional data. Unexpectedly, the N-tail is structured and interacts with both the core and scaffold domains. This finding, combined with mutational and functional studies and MD, points out how N-tail interactions couple proper subcellular trafficking and transport activity by wrapping UapA in a conformation necessary for ER-exit and but also critical for elevator-type conformational changes associated with substrate translocation once UapA has integrated into the plasma membrane. Our study provides detailed insights into important aspects of the elevator-type transport mechanism and opens novel issues on how the evolution of extended cytosolic tails in eukaryotic transporters, apparently needed for subcellular trafficking, might have been integrated into the transport mechanism.

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Mitochondrial targeting and pore formation by Gasdermin E during apoptosis

Presenting author:

Nadine Gehle

Universität Osnabrück, Fachbereich Biologie, Cell Death Biophysics and Center for Cellular Nanoanalytics (CellNanOs), Barbarastraße 11, 49076 Osnabrueck [DE], nadine.gehle@uni-osnabrueck.de

Author(s):
Nadine Gehle, Lisa Hohorst, Raed Shalaby, Ana Garcia-Saez, Katia Cosentino

Gasdermins (GSDMs) are pore-forming proteins that execute pyroptotic cell death by creating pores in the plasma membrane (PM), leading to inflammation. Interestingly, Gasdermin E (GSDME) is activated by apoptotic caspase-3, positioning it at the interface between apoptosis and pyroptosis. Although GSDME has been implicated in permeabilizing both the PM and mitochondrial membranes, its membrane-targeting preference and functional role at the mitochondria remain unclear.

To address this, we investigated the real-time sequence of GSDME-mediated membrane permeabilization during apoptosis, with a focus on its subcellular localization dynamics. Using live-cell imaging combined with super-resolution microscopy, we correlated mitochondrial fragmentation and PM integrity with GSDME localization and the release of mitochondrial content, at nanoscale resolution.

We found that GSDME targets mitochondria prior to PM during apoptosis. GSDME localizes to both the outer and, especially, the inner mitochondrial membranes, where pore formation enhances the release of mitochondrial DNA, a known activator of the cGAS–STING inflammatory pathway. Using dual-color DNA-PAINT microscopy, we provide direct nanometer-scale visualization of GSDME pore formation at mitochondrial membranes.

Together, these findings identify mitochondria as a primary target of GSDME during apoptosis and highlight a previously underappreciated role for GSDME in mitochondrial permeabilization and inflammatory signaling.

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Mechanistic Insights into Bacterial Gasdermin Pore Formation

Presenting author:

Lucas Gewehr

Johannes Gutenberg - Universität Mainz, Department of Chemistry, Biochemistry, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz [DE], lgewehr@uni-mainz.de

Author(s):
Lucas Gewehr, Dirk Schneider

Bacterial gasdermins (bGSDMs) are pore-forming proteins that execute a form of programmed cell death in bacteria, closely mirroring mammalian pyroptosis.

Widely distributed across diverse bacterial and archaeal taxa, bGSDMs are typically encoded alongside dedicated caspase-like proteases. Full-length bGSDMs occur as monomeric water-soluble proteins. However, upon activation via proteolytic cleavage, bGSDMs release an inhibitory C-terminal peptide, allowing the N-terminal domain to oligomerize into large membrane pores that disrupt cellular integrity.

We investigate the fundamental biophysical properties of bGSDM activation and assembly, specifically targeting the kinetics of membrane insertion and a potential lipid preference. While mammalian GSDM homologs favor membrane insertion into phosphoinositides or cardiolipin, we aim to define the specific lipid environments and temporal dynamics required for bGSDM pore formation. Additionally, we are developing an innovative in vitro system to enable precise control over bGSDM activity in model membranes. This setup utilizes a modified bGSDM containing an engineered tobacco etch virus (TEV) cleavage site, which is activated by a small-molecule inducible TEV protease variant. This platform will provide a tool for induction of gasdermin-mediated membrane permeabilization in a controlled environment.

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Functional investigation of the ESCRT-III homologue Vipp1 using in situ cryogenic electron tomography

Presenting author:

Tom Goetze

Forschungszentrum Juelich GmbH, Structural Biology ER-C-3, Wilhelm-Johnen-Straße, 52425 Jülich [DE], t.goetze@fz-juelich.de

Author(s):
Tom Goetze, Ndjali Quarta, Benedikt Junglas, Dirk Schneider, Carsten Sachse

The endosomal sorting complex required for transport machinery (ESCRT-0 to -IV) is one of few

cellular systems employed to remodel membranes away from the cytoplasm. Central to this

process are assemblies of ESCRT-III proteins, which increasingly constrict the membrane until

fission is achieved. The ESCRT-III homologue Vesicle-Inducing Protein in Plastids 1 (Vipp1)

shows membrane remodeling activity in vitro and is critical for thylakoid membrane maintenance

and biogenesis in both chloroplasts and cyanobacteria. While recent structural and functional

studies revealed that Vipp1 belongs to the ESCRT-III superfamily, the cellular context of Vipp1

remains poorly understood. In this work, we use the cyanobacterium Synechocystis PCC6803 as a

model for photosynthetic organisms to investigate Vipp1 function in situ. High-intensity light

stress triggers Vipp1 localization as punctae close to the thylakoid membrane. Cryogenic electron

tomography reveals that these punctae coincide with the formation of small, stress-induced

vesicles located towards thylakoid membrane convergence zones. These vesicles are uniform in

size and consistently situated between plasma and thylakoid membrane. Based on preliminary

findings, we aim to decipher the role of Vipp1 in thylakoid membrane biogenesis and maintenance

under stress conditions.

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Lipid signature of Atp11bKO small vessel disease (SVD) rat model

Presenting author:

Garyfallia Gouna

, Centre for Regenerative Medicine, Institute for Regeneration & Repair, 4-5 Little France Drive , EH16 4UU Edinburgh [GB], ggouna@ed.ac.uk

Author(s):
Garyfallia Gouna, Kristin Böhlig, Athanasios Papangelis, Will Mungall, Rosie Shiels, Jair Gonzales Marques Junior, Joanna Wardlaw, André Nadler, Anna Williams

Lipids in mammals compose of around 50% of brain’s dry weight, from which 25% are phospholipids. Phospholipids are major components of cells, maintaining membrane structures and contributing to the cell physiology. It is increasingly apparent that many neurodegenerative diseases, including small vessel disease (SVD), exhibit aberrant lipid metabolism associated with disease severity. The earliest pathological sign of SVD is endothelial dysfunction, which has been linked to a causative deletion mutation in the phospholipid flippase ATP11B. ATP11B is thought to mediate the translocation of phospholipids, from the outer to the inner membrane leaflets. Nevertheless, how this lipid disruption contributes to SVD pathology is incompletely understood. Using the transgenic Atp11b KO rat SVD model, I discovered distinct bulk lipidomic profiles in the KO brain and cerebellar tissue compared to the wild-type littermates. Bulk lipidomic analysis can skew the highly variable, cell-specific lipidome and lack spatial information. Therefore, in collaboration with Dr.André Nadler (MPI-CBG,Germany), I employed custom-made bifunctional phospholipid probes in Atp11b KO endothelial cells in vitro, visualising for the first time the membrane phospholipid signature in our model. Our long-term collaboration with the Nadler lab allows me to transfer of this technology in Edinburgh and elucidate how phospholipid distribution contributes to SVD pathology.

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Exploring the biochemical and biophysical characteristics of archaeal membranes

Presenting author:

Kaja Grewe

University Regensburg, Microbiology & Archaea Centre, Universitätsstraße 31, 93053 Regensburg [DE], kaja.grewe@ur.de

Author(s):
Kaja Grewe, Henry Zivkovic, Shachar Gat, Robert Reichelt, Thorsten Bauersachs, Anne Bernheim, Petra Schwille, Dina Grohmann

Ether lipids found in archaeal membranes differ fundamentally in their biochemistry from ester lipids that constitute both eukaryotic and bacterial membranes. The unique membrane lipids of Archaea, especially the membrane-spanning tetraether lipids, are considered to be key for survival of hyperthermophilic archaea living in habitats with extreme physico-chemical parameters. Currently, the details of archaeal lipid biochemistry are poorly understood and no established model system for archaeal membranes exist.

In this study, we prepared lipid extracts from four hyperthermophilic archaea and established the formation of giant unilamellar vesicles (GUVs) to serve as an in vitro model membrane system. Additionally, we determined the lipid composition of the four archaeal lipid extracts, revealing the highly different lipid composition for each strain. The lipid composition influenced biophysical parameters like the membrane fluidity. Interestingly, archaeal membranes show phase transitions distinct from bacterial membranes. Finally, we discovered a tendency for archaeal lipids to assemble into tubular and multilamellar structures, suggesting the presence of complex lipid phases and domains within archaeal membranes both in vitro and in vivo. Taken together, we this study provides the first in-depth biophysical characterisation of archaeal membranes laying the groundwork to elucidate membrane-protein interactions in archaea.

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Spatial regulation of cytosolic DNA sensing by the innate immune sensor cGAS

Presenting author:

Theresia Gutmann

Max Planck Institute of Molecular Cell Biology and Genetics, , Pfotenhauerstrasse 108, 01307 Dresden [DE], gutmann@mpi-cbg.de

Author(s):
Theresia Gutmann, Isabel LuValle-Burke, David Kuster, Andrey Poznyakovskiy, Anthony A Hyman

The presence of DNA in the cytosol constitutes a potent signal that triggers inflammation. Cytosolic DNA, arising during viral infection or from intracellular membrane damage and genotoxic stress, is recognized by the major cytosolic DNA sensor cyclic GMP–AMP synthase (cGAS). How spatial organization within the cytoplasm shapes cGAS signaling remains incompletely understood.

Here, we investigate the spatial regulation of cytosolic DNA sensing through DNA–protein interactions, biomolecular condensation, and membrane association. Combining cell-free biochemical reconstitution and biophysical single-molecule approaches, we show that a viral nucleocapsid protein directly binds and sequesters DNA, thereby outcompeting cGAS for DNA engagement. This interaction suppresses cGAS activation both in solution and within biomolecular condensates and is reversible by protein phosphorylation. Building on this, we discuss how membrane proximity may further modulate cGAS–DNA interactions and spatially tune DNA-sensing responses in cells.

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Reconstitution of membrane tethering and fusion machineries on supported membranes

Presenting author:

Eduard Haar

Universität Osnabrück, Fachbereich Biologie, Abt Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], Eduard.Haar@uni-osnabrueck.de

Author(s):
Eduard Haar, Kevin Tanzusch, Lars Langemeyer, Changjiang You, Jacob Piehler, Christian Ungermann

Eukaryotic cells rely on the endolysosomal system to maintain protein turnover, organelle homeostasis, and nutrient regulation. At the yeast vacuole, the Rab GTPase Ypt7 and the heterohexameric HOPS complex coordinate membrane tethering and fusion. HOPS binds Ypt7 through its specific subunits Vps39 and Vps41, while the SNARE-binding module composed of Vps33 and Vps16 initiates SNARE assembly and zippering. Although their essential roles in SNARE assembly and membrane docking are well established, the molecular mechanisms underlying their activity remain poorly understood. To address this, we reconstitute the tethering machinery on supported membranes, enabling us to probe spatial organization. Our experiments show that HOPS binding to Ypt7-decorated bilayers induces nanoscale clustering of both proteins. This cluster formation and membrane association are reversible and HOPS clusters can tether Ypt7-loaded vesicles. With this system, we aim to define how nanoscale organization and structural dynamics of tethering factors translates into membrane tethering and fusion.

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The molecular machinery for actin-dependent lipid droplet motility

Presenting author:

Arpita Halder

University of Münster, Institute for Cell Dynamics and Imaging, stadtlohnweg 33,wohnung 80, 48161 Münster [DE], arpita.halder@uni-muenster.de

Author(s):
Xue-Tong Zhao, Arpita Halder, Duy Diep, Louis Percifull, Rebecca Fausten, Marie Hugenroth, Pascal Höhne, Beatriz Leite, Bianca Esch, Javier Collado, Jenny Keller, Stephan Wilmes, Meryem Turhan, Mike Wälte, Thomas Becker, Daniel Kümmel, Christian Schuberth, Rubén Fernández Busnadiego, Florian Florian Fröhlich, Roland Wedlich Söldner, Maria Bohnert

Organelle motility is a dynamic process that enables cells to adapt their architecture during growth, division, and metabolic change. Lipid droplets (LDs), the major cellular lipid storage organelles, undergo regulated intracellular movement and are actively inherited during cell division. However, the molecular mechanisms that connect LDs to cytoskeletal transport systems remain incompletely understood.

Using microscopy-based genome-wide screening in Saccharomyces cerevisiae, we identified Lipid Droplet Motility protein 1 (Ldm1) as a key factor required for actin-dependent LD transport. Ldm1 functions as a myosin adaptor that links LDs to the type V myosin Myo2 and the LD surface adaptor protein Ldo16. Through this coupling, Ldm1 enables efficient directed movement of LDs within the cell.

In addition to its role in LD motility, Ldo16 is involved in LD biogenesis-related processes associated with seipin and is a core component of vacuole-LD contact sites. These findings indicate that LD motility is closely connected to organelle contact site formation and LD surface organisation. Together, our work defines a molecular transport module that links LDs to the actin cytoskeleton and provides insight into how LD motility is coordinated with other LD-associated cellular processes. Ongoing work aims to explore how this transport machinery is regulated and how LD motility is coordinated with organelle contact sites in different cellular metabolic states.

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Interrogation of effector interactions at active signaling complexes by live-cell nanopatterning

Presenting author:

Steffen Tammo Harms

Osnabrück University, Biophysics, Barbarastraße 11, 49076 Osnabrück [DE], steffen.harms@uni-osnabrueck.de

Author(s):
Steffen Tammo Harms, Silke Pudewell, Jürgen Scheller, Changjiang You, Jacob Piehler

Signaling across the plasma membrane is fundamental for cellular communication and is mediated by membrane receptors. However, quantitative insights into the interactions governing the dynamic assembly of signaling complexes at the plasma membrane are scarce due to a lack of methodologies capable of capturing transient binding events in the native context. We here present a new approach for visualizing and quantifying effector interactions with cytokine receptor signaling complex in live cells. For this purpose, we have devised spatially resolved surface functionalization for selectively capturing and locally concentrating cell surface receptors into nanodot arrays (NDAs). Thus, monitoring effector recruitment into signaling complexes by TIRF microscopy was achieved. Using the prototypical cytokine receptor Gp130, which signals via the Janus family tyrosine kinase JAK1 to phosphorylate signal transducers and activators of transcription proteins STAT1 and STAT3, we demonstrate NDAs of active signaling complexes (sNDAs), including receptor phosphorylation, STAT recruitment and nuclear translocation of pSTAT. Single-molecule imaging in sNDAs enabled unambiguous detection of transient STAT binding to GP130, uncovering characteristic changes in dwell times and surprising sequence of STAT1 and STAT3 activation. Overall, NDAs provide a powerful platform for reconstructing receptor signaling in living cells with nanoscale precision, overcoming limitations of biochemical assays.

 

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Structural characterization of a novel bacterial ESCRT-III protein in E. coli

Presenting author:

Anja Heddier

Forschungszentraum Jülich GmbH, ERC-3 Structure Biology, Wilhelm-Johnen-Straße, 52425 Jülich [DE], a.heddier@fz-juelich.de

Author(s):
Anja Heddier, Benedikt Junglas, Ilona Ritter, Carsten Sachse

The structures and functions of endosomal sorting complexes required for transport (ESCRT)-III proteins in bacteria, such as PspA and Vipp1, have been intensively studied in recent years. In E. coli, an additional potential PspA-homolog, YjfJ, has been recently identified while a biochemical, structural and functional characterization is entirely lacking. Here we show that YjfJ from Escherichia coli is a bona fide member of bacterial ESCRT-III proteins. Using cryo-EM, we solved eight structures of YjfJ helical filaments to a resolution of 3.0 to 4.0 Å as well as two structures of YjfJ in presence of membranes at 4.4 and 6.0 Å. In these structures YjfJ monomers adopt the typical ESCRT-III fold as well as extensive plasticity typical for bacterial ESCRT-III proteins. Our data also revealed that apo state YjfJ polymers are very similar to apo state PspA polymers, whereas they change to Vipp1-like polymers upon lipid reconstitution. Preliminary functional data suggest that YjfJ and PspA may exhibit complementary functions in E. coli, where YjfJ may play a critical role in maintaining the membrane fluidity under cold (stress) conditions. The characterization of YjfJ will help to understand the evolution of ESCRT-III proteins in bacteria as well as membrane remodeling in E. coli.

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Fluorescence-based oligomerization and self-assembly study of two membrane-active, amphipathic antimicrobial peptides

Presenting author:

Mark G. Herbert

Karlsruhe Insitute of Technology, IBG-2, Durlacher Allee 38, 76131 Karlsruhe [DE], mark.herbert@kit.edu

Author(s):
Mark G. Herbert, Erik Strandberg, Parvesh Wadhwani, Anne S. Ulrich

Antimicrobial peptides (AMP) are promising future alternatives to conventional antimicrobial agents. Understanding their mode of action, often associated with membrane lysis, is essential for their potential application as drug candidates. The mode of action can be examined by investigating self-assembly and oligomerisation behaviour. Fluorescence-based methodologies, such as Foerster resonance energy transfer (FRET), can provide unique insights into these processes. This study investigates the oligomerisation tendency and complex stability of two structurally distinct AMPs, KIA21 and KL14, in various lipid systems. Both peptides have been extensively studied, but their self-assembly and oligomer size have not been experimentally verified, despite the critical role of these parameters in determining crucial membrane interactions and functional properties.

For the α-helical model peptide KIA21, with the heptamer repeat sequence [KIAGKIA]₃, the findings indicate the formation of antiparallel dimers. Significant intervesicular exchange is found employing a FRET-based approach, in line with recent findings from leakage studies. Both findings are dependent on the membrane spontaneous curvature.

In contrast, the KL14 peptide, consisting of repetitive KL building blocks, is found to form large β-sheet assemblies, with no or very slow peptide exchange. Self-assembly and oligomerisation behaviour are also for this peptide influenced by the lipid composition of the membranes.

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Two wrongs make a right? - How loss of Pex31 rescues stressed ∆pah1 cells

Presenting author:

Marie Hugenroth

Universität Münster, Germany, , Von-Esmarch Str. 56, 48149 Münster [DE], m_huge02@uni-muenster.de

Author(s):
Marie Hugenroth, Rebecca Fausten, Maria Bohnert

 

Cells need to balance membrane expansion with lipid storage in lipid droplets (LDs) to adapt to changing physiological parameters. The lipin Pah1 mediates the conversion of phosphatidic acid to diacylglycerol at the endoplasmic reticulum (ER) membrane, thus acting at the crossroads of phospholipid and triacylglyerol synthesis. Mutants lacking functional Pah1 have few LDs and an expanded, misshaped ER. We performed a microscopy-based genetic screen for factors implicated in the Dpah1 phenotype, and found that loss of Pex31, an ER membrane protein comprising a reticulon homology-domain and a dysferlin domain, restores LD formation, ER morphology, and cell growth. Intriguingly, LDs of Dpah1Dpex31 cells store mainly sterol esters, indicating that PEX31 deletion bypasses rather than counteracts defects in triacylglycerol storage. By abolishing sterol ester synthesis, rescue of ER morphology and growth can be genetically uncoupled from LD formation, suggesting that ER restoration occurs upstream to or independent of LD formation. The effects of PEX31 deletion depend on the presence of Pex30, a paralog of Pex31. Our results suggest a functional interplay of two structurally related proteins in modulation of ER membrane properties and lipid storage, opening a door to a better understanding of cellular lipid homeostasis.

 

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Application of optogenetic tools to characterize contact site dynamics

Presenting author:

Pascal Höhne

Münster University, Institute of Cell Dynamics and Imaging, Von-Esmarch-Straße 56, 48147 Münster [DE], phoehne1@uni-muenster.de

Author(s):
Pascal Höhne, Leonhard Breitsprecher, Rico Franzkoch, Seraphine Wegner, Maria Bohnert

Contact sites are pivotal structures for inter-organellar communication, lipid metabolism, calcium homeostasis, structural integrity of organelles and stress-related responses. Currently, they are increasingly characterized as highly dynamic structures, reacting quickly to changes in metabolic conditions such as nutrient availability. However, tools to investigate and manipulate contact site formation and physiology with a high spatiotemporal resolution remain rare.

Therefore, we took an optogenetic approach, by tagging organelle membrane proteins in yeast with the optogenetic dimer pair iLID and nano. With one half of the opto-tether system linked to one organelle and the second half on another organelle, we are able to reversibly induce tethering, similar to what we would observe at a contact site, via blue light illumination. We are currently creating and characterizing an optogenetic toolkit for the manipulation of contact sites between different organelles, tuned to match the specific requirements of the respective interfaces. By applying this novel toolkit, we have been able to investigate the interplay of resident proteins at multiple contact sites.

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Assessment of molecular and subcellular alterations in the liver of ACBD5-deficient mice by proteomics and lipidomics analysis

Presenting author:

Markus Islinger

Heidelberg University (Medizinische Fakultät Mannheim), Neuroanatomy, Ludolf-Krehl Str. 13-17, 68167 Mannheim [DE], markus.islinger@medma.uni-heidelberg.de

Author(s):
Sandra Kühl, Victor Costina, Öznur Singín, Jan-Bert van Klinken, Hans Waterham, Frederic M. Vaz, Markus Islinger

 

Acyl-CoA binding protein 5 (ACBD5) is a tail-anchored peroxisomal membrane protein, which assists in the import of very long-chain fatty acids (VLCFA) into peroxisomes for their subsequent degradation. Importantly, ACBD5 has a second cellular function as a tethering protein facilitating membrane contacts with the ER and mitochondria interacting with either VAP-proteins or PTPIP51, respectively. Both functions place ACBD5 into the center of cellular lipid homeostasis potentially impacting membrane phospholipid composition. To unravel how ACBD5 influences cellular lipid flux and metabolism, livers from ACBD5-deficient mice were separated into subcellular fractions and analyzed by mass spectrometry. At the cellular level, the lack in ACBD5 induced the expression of peroxisomal as well as mitochondrial proteins and caused VLCFA accumulation specifically in phosphatidylcholines (PC). Subcellular proteomics revealed that the disruptions in fatty acid metabolism distinctly changed the proteome composition of both organelles presumably as a compensatory effect. Remarkably, subcellular lipidomics unveiled that VLCFA did not arbitrarily accumulate in PC but preferentially in peroxisomal and to a higher extent microsomal fractions. By contrast, mitochondrial phospholipids were not enriched in VLCFA but showed changes in cardiolipin fatty acid saturation, indicating that subtle changes in fatty acid homeostasis induce complex changes in organelle proteomes and membrane lipid composition.

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How Ceramides Are Made: Structural and Functional Analysis of Yeast and Human Ceramide Synthases

Presenting author:

Janathan Michael Juarez Altuzar

University of Osnabrück, Bioanalytical Chemistry, Barbarastrasse 13, 49076 Osnabrück [DE], janathan.juarez@uni-osnabrueck.de

Author(s):
Janathan Michael Juarez Altuzar, Lena Clausmeyer, Florian Fröhlich

The ceramide synthase (CerS) catalyzes the N-acylation of long-chain base and acyl-chain to produce ceramide, a bioactive molecule, and backbone of all complex sphingolipids. Ceramide deregulation leads to neurodegenerative and metabolic disorders. In mammals, ceramides are synthesized by six different CerS (CerS1 to CerS6), and each enzyme prefers a defined length of acyl-CoA. In yeast, the CerS consists of three subunits Lac1, Lag1, and Lip1, and mostly incorporates C26 acyl-CoA. Interestingly, their Lag1p motif harboring the catalytic region is highly conserved among eukaryotes. Of note, several human CerS can reconstitute the viability of lac1Δ lag1Δ in yeast cells. However, no functional and structural characterization has been done to elucidate the specific activity of all CerS.

 

Here, we purified the yeast CerS and solved its structure by Cryo-EM with a resolution of 3.0 Å, unraveling the complex’s architecture as a dimer of Lip1 subunits bound to the catalytic subunits Lag1 and Lac1. Furthermore, we successfully purified all human CerS and, to overcome size limitations in Cryo-EM, we artificially dimerized two hCerS. In addition, we established a mass spectrometry-based, along with a high-throughput fluorometric-based in vitro activity assay. Together with these tools, we aim to dissect the precise function of each CerS. We anticipate that this study will provide the fundamental basis for developing therapeutic strategies to target specific CerS.

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The bacterial ESCRT-III member PspA maintains ethanol-stressed membranes by vesicle shedding in E. coli

Presenting author:

Benedikt Junglas

Forschungszentrum Jülich GmbH, Ernst-Ruska Centre for Microscopy and Spectroscopy with Electrons, ER-C-3/Structural Biology, Wilhelm-Johnen-Straße, 52428 Jülich [DE], b.junglas@fz-juelich.de

Author(s):
Esther Hudina, Benedikt Junglas, Ilona Ritter, Anja Heddier, Luisa Emonds-pool, Carsten Sachse

The phage shock protein A (PspA) acts as the main effector of the psp membrane repair system in E. coli. While induction effectors and cellular localization of PspA have been described previously, the exact molecular mechanism of membrane maintenance remained unclear. In this study, we show the effects of PspA deletion on cell growth, cell morphology, and maintenance of membrane potential. Combining structural data from cryo-EM with in situ cryo-electron tomography, we resolved a total of 10 atomic models with resolutions ranging from 3.0 Å to 8.6 Å and revealed a nucleotide dependent membrane remodeling mechanism. Our structural and functional analyses show how the bacterial ESCRT-III protein PspA repairs damaged membranes in E. coli. We propose that PspA, once recruited to membrane lesions, promotes short membrane protrusions and subsequent nucleotide-dependent vesicle shedding, thereby removing the damaged membrane, leaving a sealed lesion in the inner membrane.

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Clinical and Autoantibody Profiling of U1snRNP Positivity Using Line Immunoassay

Presenting author:

Lekha Priyadharshini Kamarajan

AIIMS , PATNA, Biochemistry, GIRLS HOSTEL,HOSTEL NUMBER 5, GROUND FLOOR ROOM NUMBER 8 AIIMS Patna, Patna - Aurangabad Road, Phulw, 801506 PATNA [IN], dharshinilekhapriya@gmail.com

Author(s):
Lekha Priyadharshini Kamarajan

Anti-U1 small nuclear ribonucleoprotein (U1snRNP) antibodies are an important serological marker in systemic autoimmune rheumatic diseases and are routinely detected using multiplex immunoassays. With increasing use of line immunoassay (LIA) platforms, U1snRNP reactivity is frequently identified either in isolation or with multiple autoantibodies, complicating laboratory interpretation. This observational study analysed 150 U1snRNP-positive samples detected by LIA over one year and stratified them into sole U1snRNP positivity (n = 45) and U1snRNP positivity with multiple autoantibodies (n = 105). Clinical features, ANA immunofluorescence patterns, autoantibody spectra, and diagnostic classifications were compared. Baseline demographics were comparable between groups. Samples with multiple autoantibody positivity demonstrated a broader extractable nuclear antigen profile, commonly including anti-SmD1, anti-dsDNA, anti-SSA/Ro52, and anti-SSA/Ro60, whereas isolated U1snRNP positivity showed limited additional reactivity. Increasing U1snRNP antibody intensity on LIA was associated with co-existing autoantibody positivity. These serological patterns correlated with distinct diagnostic phenotypes, with lupus predominance in the multiple-positivity group and mixed connective tissue disease in isolated U1snRNP positivity. This study highlights the importance of comprehensive autoantibody profiling and interpretation of antibody intensity in LIA-based diagnostics.

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Dual-membrane secretion pathway for Pel polysaccharide in Pseudomonas aeruginosa

Presenting author:

Alexej Kedrov

Heinrich Heine University Düsseldorf, Synthetic Membrane Systems. Biochemistry, Universitätsstrasse 1, 40225 Düsseldorf [DE], kedrov@hhu.de

Author(s):
Marius Benedens, Cristian Rosales-Hernandez, Jennifer Loschwitz, Roland Beckmann, Alexej Kedrov

The pathogen Pseudomonas aeruginosa enhances virulence and antibiotic resistance through formation of biofilms. The exopolysaccharide Pel is a major component of the biofilm matrix, essential for early biofilm development. In Gram-negative bacteria, Pel secretion requires translocation across both inner and outer membranes and is mediated by the pelABCDEFG operon, which encodes a secretion system unrelated to known polysaccharide exporters. Despite its importance, the architecture and dynamics of the trans-envelope Pel machinery have remained poorly understood.

 

Here, we present recent advances in elucidating the mechanism of Pel secretion using cryo-EM, biophysical approaches, and molecular dynamics simulations. Structures of the outer membrane export complex PelBC in nanodisc reveal a unique assembly consisting of a PelB β-barrel surrounded by a pseudo-symmetric ring of twelve PelC lipoproteins, stabilized by extensive protein–protein and protein–lipid interactions. Simulations and single-molecule conductivity measurements suggest conformational dynamics of the PelB β-barrel that enable polysaccharide translocation. Mot recently, we resolved the architecture of the inner membrane PelDEFG complex. The structure captured in an inactive state provides insight into second messenger–dependent activation and substrate routing. Together, these findings establish a structural framework for Pel secretion across the bacterial cell envelope, explaining mechanisms of biofilm formation.

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Role of Gem1 in Endoplasmic Reticulum-Mitochondrial Encounter Structure organization and activity

Presenting author:

Rasha Khaddaj

University of Berne, Institute of Biochemistry and Molecular Medicine, Bühlstrasse 28, 3012 Bern [CH], rasha.khaddaj@unibe.ch

Author(s):
Rasha Khaddaj, Eliane Zinn, Arun John Peter, Wanda Kukulski

In yeast, tethering and lipid transfer between the endoplasmic reticulum (ER) and mitochondria is mediated by the endoplasmic reticulum–mitochondria encounter structure (ERMES), composed of Mdm10, Mmm1, Mdm12, and Mdm34. Using integrative in situ structural biology, we showed that ERMES forms discrete bridge-like complexes with a 1:1:1 stoichiometry of Mmm1, Mdm12, and Mdm34. Within membrane contact sites (MCS), ~25 ERMES complexes assemble in an irregular but constrained pattern, suggesting regulatory control, although the underlying mechanisms remain poorly defined. The outer mitochondrial membrane GTPase Gem1, a conserved Miro family member, interacts with ERMES and has been proposed to regulate its organization. Here, we examine how ERMES distribution, dynamics, and lipid transfer are affected in cells either deleted for gem1 or overexpressing Gem1. Through quantitative fluorescence live imaging, we observe that the equimolar ratio of ERMES components is disrupted in gem1Δ cells. The lack of Gem1 also has consequences for ERMES lipid transfer function, which is restored by overexpressing Gem1. Furthermore, we investigate how MCS ultrastructure and the distribution of ERMES bridges are affected by either deleting or overexpressing Gem1, using correlative light and electron microscopy and cryo-electron tomography. Our findings provide new insights into the regulation of ERMES complex and its role in maintaining ER-mitochondria contact sites.

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HOCl-Derived N-Chloramines in Cell Envelopes of Bacteria and Monocytes

Presenting author:

Lisa Roxanne Knoke

Ruhr Universität Bochum Fakultät für Medizin, Microbial Biochemistry, Universitätsstraße 150, 44801 Bochum [DE], lisa.knoke@rub.de

Author(s):
Justine A. Williams, Sara Abad Herrera, Sascha Heinrich, Frank M.L. Peeters, Natalie Lupilov, Julia E. Bandow, Thomas Günther Pomorski, Lisa Roxanne Knoke

Neutrophils, specialized in phagocytosis orchestrate a coordinated attack on engulfed bacteria, including the toxic hypochlorous acid (HOCl). In host-pathogen interactions, HOCl will naturally target either the bacterial envelope or the phagolysosomal membrane. Here, we examined the occurrence of N-chloramines in the cell envelopes of bacteria and monocytes, their biological activity, and neutralization.

Using a chemical probe, we have shown that HOCl forms N-chloramines with the membrane lipids’ amino residues found in the head groups of some lipids in E. coli and monocyte membranes. Besides lipids, cell envelopes of E. coli contain lipopolysaccharides (LPS) localized in the outer membrane covering the surface. HOCl formed N-chloramines in LPS molecules of living cells as well. N-chlorinated membranes composed of phosphatidylethanolamine, the major membrane lipid in E. coli exhibited oxidative activity towards the redox-sensitive roGFP2, suggesting that envelope N-chloramines might perturbate the cellular thiol homeostasis. While stable in vitro, they are significantly less stable in living cells indicating both, active detoxification and high reactivity with cellular components. To unravel the role of membrane lipids for HOCl susceptibility, we altered E. coli´s membrane lipid composition and analyzed N-chloramine accumulation. We propose that N-chloramines in the bacterial cell envelope are involved in inflammation by modulating host immune cells and bacterial killing.

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FAM134B-mediated ER remodeling in lipid storage and distribution

Presenting author:

Matthias Koch

Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, , Theodor-Stern Kai 7, 60590 Frankfurt am Main [DE], ma.koch@med.uni-frankfurt.de

Author(s):
Matthias Koch, Adriana Covarrubias-Pinto, Alexis González, Petar Škrobo, Thorsten Mosler, Ivan Đikić

Remodeling of the endoplasmic reticulum (ER) through ER-phagy is crucial for various cellular functions. ER-phagy receptors, such as FAM134B, mediate ER-phagy by targeting portions of the ER to the autophagosomes towards lysosomal degradation. FAM134B has been previously shown to promote lipid accumulation and differentiation of adipocytes and is besides other ER-phagy receptors downregulated in metabolic dysfunction-associated steatohepatitis (MASH) a severe liver pathology defined by hepatitis and accumulation of fat (LDs) and thus lipotoxicity. By high-resolution microscopy we observed that FAM134B can localize to LDs in various cell lines (HeLa, MEFs or Huh-7) and its ablation via knockdown or knockout leads to an increase in LD size and abundance. Further, we identified several LD‑associated proteins as potential FAM134B interactors by employing a proximity proteomics approach. Our findings suggest a hitherto unknown link between ER‑phagy and cellular lipid homeostasis via the FAM134B receptor.

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Creld as a Master Regulator of ER-Mitochondria Contact Site Dynamics and Bioenergetic Homeostasis

Presenting author:

Nicole Kucharowski

University of Potsdam, Institute of Nutritional Sciences, Karl-Liebknecht-Straße 24-25, 14476 Potsdam [DE], nicole.kucharowski@med.uni-duesseldorf.de

Author(s):
Nicole Kucharowski, Darla Patricia Dancourt Ramos, Torsten Bülow, Margret Bülow

Membrane contact sites (MCS) coordinate lipid transport, redox signalling and metabolic coupling between organelles. Among them, ER-mitochondria contacts are key hubs linking membrane organization to mitochondrial energy production, yet their molecular regulation remains incompletely understood.

Here we identify the ER-resident protein Creld as a critical organizer of ER-mitochondria contact site architecture and function [1]. Using Drosophila, Xenopus and human cells, we show that Creld is required for phospholipid flux across MCS. Creld loss causes local lipid accumulation at contact sites, leading to excessive mitochondrial fusion and impaired respiratory complex I activity.

We further demonstrate that Creld-dependent MCS integrity is essential for controlled hydrogen-peroxide signalling between the ER and mitochondria, which is required for neuronal bioenergetic adaptation. Disruption of this membrane-mediated redox communication provides a mechanistic link between altered contact-site dynamics and neurodegenerative risk.

Together, our findings establish Creld as a molecular bridge integrating membrane organization, lipid homeostasis and mitochondrial function, highlighting how regulation of membrane contact sites, far beyond simple lipid barriers, is fundamental to cellular energy balance and disease prevention.

[1] Paradis M., Kucharowski N., […] Bülow M.H. Sci. Adv. 8, abo0155 (2022)

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A conserved mechanism of membrane fusion in nuclear pore complex assembly

Presenting author:

Ashutosh Kumar

Fribourg University, Fribourg, Switzerland, Department of Biology, Chemin du Musée 10, 1700 Fribourg [CH], ashutosh.kumar@unifr.ch

Author(s):
Ashutosh Kumar, Jonas Fischer, Matthias Wojtynek, Harry Baird, Kateřina Radilová, Daria Maslennikova, Kaustubh Ramachandran, Anna Becker, Arantxa Agote-Aran, Alessia Loffreda, Annemarie Kralt, Madhav Jagannathan, Gautam Dey, Ulrike Kutay, Stefano Vanni, Karsten Weis

The nuclear pore complex (NPC) forms a large channel that spans the double lipid bilayer of the nuclear envelope and is the central gateway for macromolecular transport between the nucleus and cytoplasm in eukaryotes. NPC biogenesis requires the coordinated assembly of over 500 proteins culminating in the fusion of the inner and outer nuclear membranes. The molecular mechanism of this membrane fusion step that occurs in all eukaryotes is unknown. Here, we elucidate the mechanism by which two paralogous transmembrane proteins, Brl1 and Brr6, mediate membrane fusion in S. cerevisiae. Both proteins form multimeric, ring-shaped complexes with membrane remodeling activity. Brl1 is enriched at NPC assembly sites via a nuclear export sequence and then interacts with Brr6 across the nuclear envelope lumen through conserved hydrophobic loops. Disrupting this interaction blocks fusion and halts NPC assembly. Molecular dynamics simulations suggest that the Brl1-Brr6 complex drives membrane fusion by forming a channel across bilayers that enables lipid exchange. Phylogenetic analyses reveal that Brl1/Brr6 homologues are broadly distributed across eukaryotes, and functional experiments in human cells and D. melanogaster establish CLCC1 as an NPC fusogen in metazoans. Together, our results uncover a novel, conserved mechanism for membrane fusion in eukaryotes.

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Investigating lysosomal lipid trafficking and homeostasis using coumarin-based photoactivable lipid probes and protein mass spectrometry

Presenting author:

Runmi Kundu

University of Osnabrück, Biology/Chemistry, Barbarastraße 11, 49076 Osnabrück [DE], runmikundu@gmail.com

Author(s):
Runmi Kundu, Vivien Koch, Denisa Jamecna

Lysosomes are catabolic organelles degrading and recycling macromolecules. Their dysfunction is implicated in many diseases from neurodegenerative conditions to storage disorders, manifested by lysosomal lipid accumulation. Whereas cholesterol export is well documented, the efflux of sphingosine and other sphingolipids remains understudied. In particular, the mechanisms of the co-regulated transport of sterols and sphingolipids at the lysosome are unknown.

To address this, we synthesize multifunctional analogues of sphingosine and cholesterol - lyso-pacSph and lyso-pacChol (Altuzar et al., 2023) - integrating four functional modules: a photocleavable coumarin cage, a lysosome targeting amine, a photo-crosslinkable diazirine and a clickable alkyne. Together, these features allow: 1) monitoring metabolic conversions through TLC/lipid mass spectrometry; 2) visualization of fluorescently click labelled probe for lipid imaging: and 3) protein interaction profiling using biotin click labelling in combination with pulldown/protein mass spectrometry.

We focus on STARD3, a transporter that tethers the lysosome with the ER. STARD3 shows dual specificity for cholesterol and sphingosine, making it a great model protein for studies of lysosomal lipid transport. We apply tools like proximity biotinylation and proteomics to elucidate STARD3's molecular 'instructive' environment. Our main goal is to identify regulators and effectors of lysosomal lipid trafficking in health and disease.

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The role of the amphipathic helix 0 in membrane binding of the ESCRT-III protein Vipp1

Presenting author:

Mirka Kutzner

, , Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz [DE], mikutzne@uni-mainz.de

Author(s):
Mirka Kutzner, Sourav Maity, Nadja Hellmann, Wouter Roos, Dirk Schneider

The vesicle-inducing protein in plastids (Vipp1) plays a key role in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. Vipp1 monomers, composed of seven α-helices, assemble into large homo-oligomeric ring structures in solution and form diverse polymeric assemblies in the presence of membranes. Recently, Vipp1 was identified as a member of the membrane-remodeling ESCRT-III superfamily. Binding of (at least) monomeric Vipp1 is mediated via electrostatic interactions between the positively charged α1–3 helical hairpin and negatively charged lipid surfaces. In addition, the amphipathic helix α0 is critical for membrane association and is proposed to function as a membrane anchor. While α0 has been implicated in membrane tubulation by homo-oligomeric Vipp1 polymers, its broader role in membrane binding and remodeling remains unclear. To examine the contribution of α0 to Vipp1 membrane binding, we analyzed protein variants lacking α0 and compared their behavior in different oligomeric states. Our results show that the presence of α0 enhances the membrane-binding affinity of monomeric Vipp1. Furthermore, high-speed atomic force microscopy (HS-AFM) revealed that α0 is required for the formation of spiral, sheet-like and tubular structures on membrane surfaces. These findings indicate that helix α0 is essential for the correct orientation of Vipp1 on the membrane surface, enabling the formation of higher-order assemblies.

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Vps41 functions as a molecular ruler for HOPS tethering complex-mediated membrane fusion

Presenting author:

Caroline König

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], caroline.koenig@uni-osnabrueck.de

Author(s):
Caroline König, Dmitry Shvarev, Jieqiong Gao, Eduard Haar, Nicole Susan, Kathrin Auffarth, Lars Langemeyer, Arne Moeller, Christian Ungermann

The HOPS tethering complex consists of Vps11, Vps16, Vps18, Vps33, Vps39 and Vps41 in Saccharomyces cerevisiae. For membrane tethering HOPS binds GTP-loaded Ypt7 and assembles SNAREs from opposing membranes to mediate fusion at the vacuole. Vps41 and Vps39 at opposite ends of the complex bind to the Rab7-like Ypt7 protein. Vps33 is crucial for binding to SNAREs and is structural related to members of the Sec1/Munc1 (SM) protein family. The other five subunits are shown to share a similar architecture, comprising an N-terminal β-propeller and a C-terminal α- solenoid domain.

Using cryo-electron microscopy, we identified Vps41 in HOPS being connected to the core by a long, extended α-solenoid domain (Shvarev et al., 2022). We now showed that this solenoid acts as a molecular ruler to position the Ypt7-interacting region of Vps41 relative to the core of HOPS to support function. Mutant complexes with a shortened or extended α-solenoid region in Vps41 still tethered membranes, but failed to efficiently support their fusion. In vivo, Vps41 mutants grew poorly and showed defects in vacuolar morphology, endolysosomal sorting and autophagy. When a length-compensating linker was inserted instead, these defects were rescued. This suggests that the Rab-specific Vps41 subunit requires the exact length of the α-solenoid domain but not the α-solenoid architecture for functionality, suggesting a revised model of how HOPS supports fusion.

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Role of the hypervariable domain of Ypt7 in activation and effector binding

Presenting author:

Lars Langemeyer

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], lars.langemeyer@uni-osnabrueck.de

Author(s):
Lars Langemeyer, Tunde Lawal, Christian Ungermann

Rab-GTPases function as organelle-specific identity markers, directing proteins to distinct membranes in eukaryotic cells. They are membrane-anchored via a C-terminal prenyl group attached to a highly conserved GTPase domain through a flexible, hypervariable domain. Rabs cycle between inactive (GDP- bound) and active (GTP-bound) states. In their inactive state, they associate with GDI and are soluble; upon encountering a cognate guanine nucleotide exchange factor (GEF) on a membrane, GDP is exchanged for GTP, enabling binding to effector proteins and their recruitment to target membranes.

 

While the GTPase domain is generally considered the primary determinant of specificity in Rab-GEF and Rab-effector interactions, recent studies demonstrate a role for the hypervariable domain in GEF recognition. Here, we present evidence that the hypervariable domain of the Saccharomyces cerevisiae Rab7-like GTPase, Ypt7, is crucial for efficient GEF-mediated activation and subsequent effector recruitment and function. Our data show that Rabs use a dual binding mode for their precise membrane activity, which demonstrates their versatility as determinants of organellar membrane identity.

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Mapping protein-specific lipid environments

Presenting author:

Rene Leffers

University Osnabrück, Bioanalytische Chemie, Weseler Straße 70, 48151 Münster [DE], rene.leffers@uni-osnabrueck.de

Author(s):
Rene Leffers, Bianca M. Esch, Florian Fröhlich, Stefan Walter

Biological membranes not only act as barriers but also provide dynamic platforms for integral transmembrane and peripheral proteins. There is a wide variety of membrane lipids due to differences in head groups, the length and saturation of acyl chains, and hydroxylation. However, the precise lipid composition of the nanoenvironment surrounding specific proteins, and its influence on their function and activity, remains unclear. In this study, we present a method combining three approaches to investigate whether proteins within the same organelle are embedded in distinct lipid environments. These strategies are: (i) analyzing lipids that co-purify with detergent-purified membrane proteins; (ii) analyzing lipids from lysates of strains that overexpress the target proteins; and (iii) isolating organelles using bait proteins under endogenous conditions. By using this combined approach, we analyzed the specific lipid environment of the serine palmitoyltransferase (SPT) complex in yeast, a key enzyme in sphingolipid synthesis, and observed an enrichment of lipids with shorter acyl chain lengths, suggesting locally thinner membranes. Furthermore, the SPT complex contains either Orm1 or Orm2 regulatory subunits and we observed differences in surrounding lipid saturation dependent on the subunit present. We therefore hypothesized that these membrane alterations could affect substrate recognition and binding to the protein complex.

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PEDIATRIC ACUTE LIVER FAILURE, UNDERSTANDING THE IMPAIRED INTRACELLULAR VESICULAR TRAFFICKING IN NBAS DEFICIENCY

Presenting author:

Lina Leghlam

, , Im neuenheimer Feld 669, 69120 Heidelberg [DE], linaleghlam@gmail.com

Author(s):
Lina Leghlam, Dominic Lenz, Stefan Kölker

Aim
NBAS deficiency is a rare genetic multisystem disorder characterized by fever-triggered acute liver failure, skeletal abnormalities, neurological and immune defects. NBAS is required for ER-Golgi trafficking, but disease mechanisms remain unclear. We hypothesize that defective vesicular transport causes organelle stress and impaired protein sorting, contributing to disease phenotypes. This study investigates trafficking defects in NBAS-deficient patient cells.

Methods
Patient-derived fibroblasts were analyzed and compared to controls using western blotting, immunofluorescence (IF), transmission electron microscopy, secretome analysis, and proteomics.

Results
NBAS-deficient cells showed abnormal secretion of extracellular matrix (ECM) proteins and lysosomal enzymes. Proteomic analysis revealed ER and Golgi stress, defective post-Golgi sorting, and mis-sorting of lysosomal enzymes, accompanied by compensatory upregulation of trafficking components, ER quality control, and lysosomal biogenesis. IF and TEM demonstrated ER swelling, enlarged lysosomes, and lysosomal accumulation. Fever-mimicking conditions induced Golgi fragmentation, ER stress, and impaired Golgi-to-plasma-membrane trafficking.

Conclusion
NBAS mutations disrupt ER–Golgi and post-Golgi trafficking, causing persistent organelle stress, lysosomal dysfunction, and ECM mis-secretion. Compensatory responses fail to restore homeostasis, offering a potential mechanism for NBAS disease phenotypes.

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Functional characterization of the vCLIP-engaged lipid droplet subpopulation

Presenting author:

Beatriz Leite

University of Münster, Biology, Coerdestr. 42, 48147 Münster [DE], beatrizleite2103@gmail.com

Author(s):
Beatriz Leite, Duy Diep, Maria Bohnert

The communication of lipid droplets with other organelles is a key process in cellular metabolism. This occurs mainly at contact sites, regions where distinct organelles are close enough to allow direct material exchange without relying on vesicular transport. This communication depends on physical tethering, which is achieved by specialized proteins that link the membranes of different organelles. In yeast, contact sites between lipid droplets and the vacuole (vCLIPs) are tightly regulated by the cell's nutritional status. vCLIPs increase during glucose scarcity and are crucial for lipid droplet degradation via lipophagy upon prolonged starvation. The multifunctional proteins Ldo16 and Ldo45 mediate vCLIP formation by binding to the vacuolar protein Vac8 and recruiting the phosphatidylinositol transfer protein Pdr16. In this project, we are using a combination of microscopy-based high-content screens, proteomics, and lipidomics, to characterize the functional roles of vCLIP-engaged lipid droplets across metabolic conditions, and to define the metabolically-controlled regulation of vCLIP expansion and reduction.

 

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Quantification of lipid sorting during clathrin-mediated endocytosis

Presenting author:

Hjoerdis Mathilda Lennartz

MPI CBG, , Pfotenhauerstrasse 108, 01307 Dresden [DE], hlennart@mpi-cbg.de

Author(s):
Hjoerdis Mathilda Lennartz

Clathrin-mediated endocytosis is a major transport route for proteins from the plasma membrane to the interior of the cell. While the recruitment of cargo proteins to clathrin-coated pits is well understood, it remains an open question if lipid species are also sorted by this process. To address this question, we combined super-resolution STED imaging of bifunctional lipid probes with mathematical modeling, enabling us to resolve true lipid partitioning at clathrin-coated pits. Quantification of 10 different lipid species revealed significant differences in pit partitioning, ranging from slight enrichment to moderate exclusion. Pit partitioning of lipid species strongly correlates with lipid asymmetry in the plasma membrane, and modeling suggests that the known leaflet asymmetry of lipid species is sufficient to explain the observed trend. Taken together, our findings imply that clathrin-mediated endocytosis has a minor selectivity for cytoplasmic leaflet lipids, but overall does not significantly contribute to lipid sorting compared to non-vesicular trafficking. More broadly, we believe that lipid super-resolution imaging will be a powerful approach to quantify lipid partitioning into membrane structures in cells.

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Tumor protein D54 dynamically forms biomolecular condensates gathering intracellular nanovesicles depending on cellular cholesterol levels

Presenting author:

Sébastien Leterme

Institut de Pharmacologie Moléculaire et Cellulaire, , 660 Rte des Lucioles, 06560 Valbonne [FR], leterme@ipmc.cnrs.fr

Author(s):
Sébastien Leterme, Maud Magdeleine, Antoine Reynaud, David Kovács, Ana Rita Dias Araújo, Sophie Abélanet, Christelle Boscagli, Sandra Lacas-Gervais, Bruno Antonny

Cells are constantly subjected to a fluctuating demand for lipids to feed its various cellular processes. In addition, cells are facing alternating periods of nutrient abundance and scarcity. To efficiently respond to these rapid fluctuations, cells classically store remobilizable lipids inside lipid droplets. We recently observed that the poorly characterized protein Tumor Protein D54 (TPD54) promotes the formation of a striking structure in the cell: a biomolecular condensate in which very small (30-35 nm) vesicles, previously identified as intracellular nanovesicles, are entrapped in a dense matrix of TPD54 proteins. Here, we aim to unravel the organization, the control of assembly, the cellular dynamics and the possible functions of this biomolecular condensate gathering intracellular nanovesicles and TPD54. Biochemical reconstitution and biophysical approaches demonstrated that TPD54 is indeed able to form dynamic condensates in vitro through its structural attributes. We then concentrated on the regulation of TPD54 subcellular localization by cell cholesterol content by combining pharmacological and microscopy approaches. We further investigated the possible role of TPD54 condensates as donors or acceptors in cellular lipid fluxes using fluorescent lipid probes. Altogether, our results constitute the first characterization of a new cellular structure that could be implied in the dynamic storage of readily mobilizable lipids.

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¹⁸F-Labeled imidazolium-based cholesterol analogs for specific cell targeting

Presenting author:

Marie-Christin Leusmann

University of Münster, Institute of Organic Chemistry, , Corrensstraße 40, 48149 Münster [DE], mleusman@uni-muenster.de

Author(s):
Nele Van Wyngaerden, Nadine Heiden, Christian Paul Konken, Marie-Christin Leusmann, Sven Hermann, Frank Glorius, Michael Schäfers

To investigate the dynamics and function of innate immune cells in vivo, longitudinal, whole-body imaging strategies are required. However, genetic cell modifications can limit clinical translation. To address this limitation, we evaluated imidazolium-based cholesterol derivatives (CHIMs) as a non-genetic platform for the selective functionalization of monocyte surfaces with azide groups, enabling bioorthogonal cell labeling.

Our group previously developed CHIMs, imidazolium-based cholesterol analogs, that retain native membrane integration properties while incorporating chemically reactive functional groups such as azides. Surface-exposed azides serve as effective reaction partners in strain-promoted cycloaddition reactions, which are characterized by broad compatibility with biological fluids, high reaction rates, and excellent stability of the resulting triazole conjugates. Azide-functionalized CHIM (CHIM-L) has been shown to enable efficient fluorophore-based membrane labeling in vitro. Herein, CHIM-L was functionalized with an ¹⁸F-labeled DBCO derivative to enable bioorthogonal labeling of leukocytes for in vivo tracking using PET.

Overall, we present an efficient strategy for non-genetic cell surface functionalization of monocytes using azide-modified CHIMs. This approach provides a promising platform for multiscale bioorthogonal imaging of primary innate immune cells and supports the development of clinically translatable in vivo cell-tracking methodologies.

 

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Membrane-active Peptides as Vectors for Receptor-mediated Endosomal Escape

Presenting author:

Nicolas Lindholm

Leipzig University, Institute of Biochemistry, Brüderstraße 34, 04103 Leipzig [DE], nicolas.lindholm@uni-leipzig.de

Author(s):
Nicolas Lindholm, Moritz List, Annette Gabriele Beck-Sickinger

In the past decades, a large number of membrane-active, cell-penetrating peptides of natural or synthetic origin have been discovered that are able to bypass membranes by different mechanisms. However, it remains unclear whether the transduction of these peptides solely occurs by the plasma membrane in an unspecific manner, or by uptake into the endosomal pathway and subsequent endosomal escape.¹ As the latter bears great potential for the cell-selective delivery of membrane-impermeable macromolecular compounds, we aim to identify effective endosomal escape peptides for the delivery of peptide cargoes by receptor-mediated endocytosis, using the chemerin chemokine-like receptor 1 (CMKLR1).² Solid-phase peptide synthesis and in-solution conjugation methods enable us to synthesize modular conjugates combining the membrane-active peptide with a cargo molecule and the cyclic chemerin-9 peptide, a stabilized ligand of CMKLR1 previously developed by our group.³ By using fluorescence-based readouts and biochemical reporter assays, we assess the endosomal escape performance and cytotoxicity of our peptide conjugates, proving a concept for the development of cell-selective delivery systems.⁴

1. Ruseska et al., Beilstein J. Nanotechnol. 11, 101–123 (2020).
   2. Fischer, T. F. & Beck-Sickinger, A. G., Biological Chemistry 403, 625–642 (2022)
   3. Fischer, T. F. et al., Cancers (Basel) 13, 3788 (2021)
   4. List, M. & Beck-Sickinger, A. G. J. Am. Chem. Soc. (2025), in press

 

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Expanding a double-split reporter system for exploring membrane contact sites

Presenting author:

Monalisha Mallik

University of Cologne, Center for Biochemistry, Joseph-Stelzmann-str. 52, 50931 Köln [DE], monalisha.mallik@uk-koeln.de

Author(s):
Monalisha Mallik, Emma J. Fenech

Organelle membrane contact sites (MCSs) are essential for inter-organelle communication, as they play key roles in lipid exchange, ion transfer, and signaling between organelles. To study MCS function, it is crucial to identify proteins localized at these sites. The CsFiND system (Complementation assay using Fusion of split-GFP and TurboID) was recently developed to enable simultaneous visualization and proximity labeling of MCS-localized proteins and validated in Saccharomyces cerevisiae at ER–mitochondria contacts. In this project, we extended the CsFiND system to MCSs between other pairs of organelles and observed changes in MCS behavior under different conditions. These results pave the way towards large-scale functional interactor screening and will advance CsFiND as a versatile tool for uncovering the molecular composition and regulatory mechanisms of diverse organelle contact sites.

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The membrane landscape of the lipid flippase complex Drs2/Cdc50

Presenting author:

Ekaterina Malysenko

Ruhr University Bochum, , Am Bremkamp 14, 44795 Bochum [DE], ekaterina.malyshenko@rub.de

Author(s):
Ekaterina Malysenko, Marten Exterkate, Sandro Keller, Thomas Günther Pomorski

The protein and lipid compositions of biological membranes vary depending on the cellular organelle. Even within organelle membranes, proteins and lipids can be distributed unevenly, forming membrane domains with distinct purposes. In yeast trans-Golgi membranes, the lipid flippase complex Drs2/Cdc50 transports specific lipids unidirectionally towards the cytoplasmic membrane leaflet. This creates lipid asymmetry and contributes to the formation of secretory vesicles, which move membrane proteins and lipids within the cell. Lipids are also known to regulate flippase activity, implying that lipids surrounding Drs2/Cdc50 may impact vesicle formation. Furthermore, past studies suggest that certain lipids aggregate during the formation of secretory vesicles. Combined, these points open up the possibility of a specialized, local Drs2/Cdc50 membrane environment. To analyze the membrane environment surrounding the flippase complex, we solubilized Drs2/Cdc50 with co-polymers into native nanodiscs, which allowed an analysis of co-purified lipids and proteins. By comparing the lipid profiles of Drs2/Cdc50 nanodiscs and selected membranes, we screened for distinct lipid enrichments surrounding the flippase complex, providing starting points for an investigation of lipid impact on flippase function.

 

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Engineering the outer membrane of the purple bacterium Rhodospirillum rubrum for surface display presentation of potential vaccine epitopes

Presenting author:

Daniel Markthaler

University of Stuttgart, Institute for Energy Efficiency in Production (EEP), Karlsbader Strasse 51, 70839 Gerlingen [DE], daniel.markthaler@eep.uni-stuttgart.de

Author(s):
Robin Ghosh, Daniel Markthaler

Surface display of viral epitopes fused to the beta-barrels of bacterial outer membrane proteins, is a promising strategy for low cost vaccine production.

The outer membrane (OM) of the Gram negative purple bacterium Rhodospirillum rubrum contains 11 major proteins, dominated by two OM porins, Por39 and Por41. R. rubrum has no known pathogenic potential, and the organism can be grown to high cell densities semi-aerobically, at low aeration with a cheap well-defined medium. These factors make the organism interesting for development as a surface display platform, where epitopes inserted into the loops of the OM porins can be employed as vaccines.

Our goal is to insert key ACE2 binding site epitopes of the SARS CoV2 spike protein into a predicted OM loop of Por39. However, since the sequence similarity to porins of known structure is weak to nonexistent, we have performed an extensive modelling study [1], where the putative 3D structure of Por39 was predicted using both publicly available folding algorithms I-TASSER and AlphaFold. Our final Por39 model indicates that the Por39 porin is a beta-barrel with 16 transmembrane strands and the predicted extracellular loops L4, L6 and L8 were found to be most suitable for epitope insertion. Currently, we are testing our model, both experimentally and computationally with more detailed MD simulations.

Ref.:
[1] Markthaler, D., and Ghosh, R. (2023) Comput. Struct. Biotechnol. J., 21, 2483-2494.

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Insights into the Viral Membrane Assembly Protein L2 and Vaccinia virus-induced membrane remodeling

Presenting author:

Finn McGhee

Institute for Integrated Biology of the Cell (I2BC), Virology, Avenue de la Terrasse, 1, 91190 Gif-sur-Yvette [FR], maeve.mcghee@i2bc.paris-saclay.fr

Author(s):
Finn McGhee, Pavlina Dubois, Malika Ouldali, Marcel Zimmeck, Sonia Fieulaine, Emmanuelle R. J. Quemin

Poxviruses are large, enveloped dsDNA viruses with epidemic potential. During infection, cellular membranes are remodeled during viral genome replication and viral particle assembly. However, the underlying molecular mechanisms remain poorly understood. Remodeling of the endoplasmic reticulum (ER) is key for the formation of the replication compartment surrounded by ER cisternae, and during assembly, when ER-derived vesicles rupture to form membrane precursors of immature virions called “crescents” with stabilized ends. Based on research on the prototype vaccinia virus (VACV), L2, one of the five viral membrane assembly proteins, participates in membrane remodeling during replication and assembly. To study how L2 interacts in vitro with model membranes, we expressed L2 in a detergent-containing cell-free system for purification and reconstitution in liposomes. L2-proteoliposomes were analyzed by flotation assays and cryo-electron tomography. Presence of L2 induced liposome remodeling including tethering and rupture, and L2 localized specifically to small pieces of membranes as labeled by NiNTA coupled to nanogold. By assessing the interplay of L2 with membranes in a reconstituted setup, we aim to uncover the role of L2 in membrane remodeling during VACV infection. Our work contributes to a better understanding of host-poxvirus interactions and to the identification of potential targets for antiviral design.

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GTPase activation in SynDLP, a dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803

Presenting author:

Lara Mernberger

Johannes Gutenberg-Universität Mainz (JGU), Department Chemie – Biochemie, An den Weiden 3, 55127 Mainz [DE], mernberger@uni-mainz.de

Author(s):
Lara Mernberger, Lucas Gewehr, Benedikt Junglas, Dirk Schneider, Carsten Sachse

Dynamin-like proteins (DLPs) are large GTPases involved in membrane remodeling processes, such as fusion, fission, and cell division. While eukaryotic DLPs are well studied, only few bacterial DLPs (BDLPs) have been characterized. In cyanobacteria, BDLPs potentially play an important role in the biogenesis and dynamics of the thylakoid membrane system.

SynDLP, a BDLP of the cyanobacterium Synechocystis sp. PCC 6803, preferentially binds to negatively charged membranes. It mediates liposome fusion in vitro in a nucleotide-independent manner, suggesting that membrane remodeling does not directly rely on GTP hydrolysis. Unlike other DLPs, SynDLP forms higher-order oligomers in solution without membranes or nucleotides, resulting in an expanded intermolecular, longitudinal interface, crucial for the GTPase activity of SynDLP. Furthermore, a monomeric GTPase domain (GD) construct showed the formation of transverse head-to-head dimers in the presence of a non-hydrolysable GTP-analogue, accompanied by conformational changes in the GD and its orientation relative to the BSE domain. Based on cryo-EM analyses, a novel three-helix bundle within the GD shifts outwards during dimer formation, indicating a potential regulatory function. Another subdomain, also not present in canonical GDs, interacts with the corresponding region of the opposing monomer and is essential for dimerization. Our findings establish a novel mechanism underlying DLP-mediated GTP hydrolysis and membrane remodeling.

 

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Fluorescent-based in vitro detection of phosphoinositides for kinetic analysis of kinases and phosphatases

Presenting author:

Jana Milach

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], Jana.milach@uni-osnabrueck.de

Author(s):
Jana Milach, Katharina Olschewski, Lars Langemeyer, Sabrina Chumpen Ramirez, Christian Ungermann

 

Phosphoinositides (PtsInsPs) are important for membrane identity and signaling processes. Several kinases and phosphatases are involved in converting the different forms of PtsInsPs according to the nutritional and metabolic requirements. In autophagy for example, the generation of phosphatidylinositol 3-phosphate (PtdIns3P) on the autophagosomal membrane is critical during the biogenesis of the autophagosome. However, at late stages its depletion is necessary to ensure the maturation of this organelle. Thus, the tight coordination of the phosphorylation and dephosphorylation reactions is crucial in autophagy as also in many other metabolic pathways. The knowledge on how the enzymes are regulated to ensure proper turnover is limited, in part due to difficulty to detect PtsInsPs in vitro. Here, we aim to optimize a lipid-coated silica-beads assay to monitor PtsInsPs production/consumption by specific fluorescently labelled biosensors. As a starting point for the kinetic analysis of kinase and phosphatase activities, we focus on the PtdIns3P conversion into phosphatidylinositol (PtdIns) by the phosphatase Ymr1, involved in autophagy and endocytosis, both described as pathways highly dependent on PtdInsPs turnover.

 

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Molecular architecture of lipid transfer between the two mitochondrial membranes

Presenting author:

Agnes Moe

University of Bern, Institute of Biochemistry and Molecular Medicine, Bühlstrasse 28, 3012 Bern [CH], agnes.moe@unibe.ch

Author(s):
Agnes Moe, Wanda Kukulski

Lipid biosynthesis occurs mostly in the endoplasmic reticulum and the distribution of lipid molecules between different organelle membranes is therefore vital for establishing membrane lipid composition and identity. The inner mitochondrial membrane requires tight control of its composition to maintain crucial functions, such as respiration. The mechanism of lipid transfer between the inner and outer mitochondrial membrane is largely unknown, although several proteins have been identified as mitochondrial lipid transfer proteins. The arrangement of these proteins between the inner and outer mitochondrial membrane, and how they carry out lipid transfer in vivo, are not known. We aim to understand lipid transfer between inner and outer membranes of mitochondria in Saccharomyces cerevisiae. We implement an integrative approach to study the molecular mechanism of lipid transfer and the arrangement of its protein components. In particular, cryo-electron tomography is used to obtain structural information about organization and architecture of lipid transfer components, and their influence on mitochondrial ultrastructure. Yeast genetics and live fluorescence imaging are used to obtain insights into the specific roles of individual protein components involved in lipid transfer. Furthermore, our study addresses how lipid transfer events are distributed, relative to landmarks in mitochondrial structure, and thus how lipid transfer connects to other mitochondrial functions.

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Molecular Mechanism of Ceramide Transfer by STARD11 / CERT: A Molecular Dynamics Investigation

Presenting author:

Mahmoud Moqadam

University of Bergen, Chemistry, Bregnestien 8B, 5141 Fyllingsdalen [NO], mahmoud.moqadam@uib.no

Author(s):
Mahmoud Moqadam, Camille Cuveillier, Anne-Claude Gavin, Nathalie Reuter

Non-vesicular lipid transport by lipid transfer proteins (LTPs) is crucial for regulating cellular lipid distribution and maintaining membrane composition. A defining feature of all LTPs is their ability to shield lipids within a hydrophobic cavity, reducing the energetic cost of transfer relative to the aqueous environment. A fundamental yet unresolved question is the mechanism by which LTPs coordinate membrane binding and gate opening with lipid uptake and release. Using the ceramide transporter STARD11 as a model system, we performed molecular dynamics simulations to dissect the membrane-dependent gating mechanism. We show that an arginine located in the Ω1 loop and conserved in 14 of the 15 human START domains plays a critical dual role: it maintains the structural integrity of the gate, and strongly interacts with a membrane phospholipid that promotes and sustains gate opening. This opening disrupts local membrane lipid packing and increases lipid tail snorkeling toward the open cavity. Using in vitro lipid transfer assays of the wild-type STARD11 and mutants, we show that mutating the arginine decreases the rate of ceramide uptake. Overall, our findings reveal a functional role for conserved arginine and highlight a mechanistic coupling between membrane lipid dynamics and cavity accessibility that together drives efficient lipid transport by the START domain protein.

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Characterizing the role of RNF150 in regulating post-Golgi trafficking

Presenting author:

Sofia Murteira

University of Duisburg-Essen, Mechanistic Cell Biology , Universitätsstraße 2, 45141 Essen [DE], sofia.murteiraferreira@uni-due.de

Author(s):
Sofia Murteira, João Diamantino, Farnusch Kaschani, Markus Kaiser, Doris Hellerschmied

Ubiquitination is a post-translational modification that can target membrane proteins to various intracellular trafficking pathways. This process is essential for maintaining cellular homeostasis by ensuring the correct localization of membrane proteins and regulating their turnover. Accordingly, ubiquitin E3 ligases have emerged as key regulators by binding and modifying membrane proteins. At the center of the secretory pathway, the Golgi apparatus coordinates the appropriate modification and sorting of nearly all secretory and transmembrane proteins. We identified RNF150 as a Golgi-localized E3 ligase that belongs to the protease associated (PA) - transmembrane (TM) – RING family. Several members of this family have been implicated in regulating membrane protein trafficking. However, RNF150 remains poorly understood. Here, we combined ubiquitin-specific proximity labeling and pull-down studies coupled to mass spectrometry to identify RNF150 substrates and interactors, respectively. The identified RNF150 candidate substrates and interactors point to a role of the E3 ligase in endosome-to-Golgi trafficking. In ongoing biochemical and imaging studies we are exploring the impact of RNF150-dependent ubiquitination on the function of SNARE proteins and transmembrane receptor proteins. Collectively, our current data suggest a role for RNF150 in the regulation of post-Golgi trafficking.

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Analysis of the cell wall glycolipids of Streptococcus canis

Presenting author:

Anna Nehrmann

Research Center Borstel - Leibniz Lung Center, Bioanalytische Chemie, Parkallee, 5a, 23845 Borstel [DE], anehrmann@fz-borstel.de

Author(s):
Anna Nehrmann, Miriam Katsburg, Ursula Schombel, Dominik Schwudke, Marcus Fulde, Nicolas Gisch

 

Streptococcus canis is an opportunistic pathogen observed in animals, especially dogs and cats, but occasionally also in humans. Usually, this pathogen is classified into the Lancefield group G. Recently, two strains of S. canis belonging to Lancefield group C were identified. Whole genome sequencing analysis pointed towards a horizontal gene transfer between S. canis and Streptococcus dysgalactiae. (Katsburg et al., 2023)

Besides the Lancefield assignment, little is known about cell wall components of S. canis on the molecular level. The aim of this study is to close this knowledge gap by structurally characterizing the lipoteichoic acid (LTA) and glycosylglycerolipids (GLs) from six selected S. canis strains: four group G strains and the two group C strains. To this end, we applied our expertise for the isolation and structural characterization of Gram-positive bacterial cell wall components by mass spectrometry and nuclear magnetic resonance spectroscopy. (Gisch, Auger et al., 2018)

All examined strains express two GLs, α-D-Glc-DAG and α-D-Glc-(1→2)-α-D-Glc-DAG. They also produce a type I LTA with the diGlc-DAG as membrane anchor and poly-glycerolphosphate chains that are partially substituted with alanine, but with significant variation in their chain length. The conserved structure of GLs and LTAs in S. canis is shared with other streptococci, but the strain dependent variation in chain length might be relevant for future functional studies.

 

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Regulation of the yeast endocytic pathway by Rab5 nucleotide exchange factors Muk1 and Vps9.

Presenting author:

Alexandra Nesterova

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], alexandra.nesterova@uni-osnabrueck.de

Author(s):
Alexandra Nesterova, Lars Langemeyer, Christian Ungermann, Derek Prosser

Rab-GTPases are conserved regulators of endomembranes which cycle between the active GTP- and the inactive GDP-bound state with the help of GEFs and GAPs. The Rab5-subgroup exerts its function within the endolysosomal system at early endosomes and endocytic vesicles. Although yeast is assumed to have a minimal endolysosomal system, four Rab5-homologs were identified, named Vps21, Ypt52, Ypt53, and Ypt10. Furthermore, three Rab5-GEFs (Vps9, Muk1 and Vrl1) are encoded in the yeast genome. To determine, which GEF is specific for which Rab5-variant, we reconstituted GEF-specific Rab5-activation assays on liposomes focusing on Vps9 and Muk1 and using prenylated Rabs. While we found no substrate exclusivity, the enigmatic Ypt10 was the preferred substrate of Muk1. Using biochemical approaches, we furthermore show that Muk1 intrinsically binds membranes and oligomerizes through its C-terminal domain which is also required for in vivo localization. Functional fluorophore-tagging of Vps9 and Muk1 unveiled that the two GEFs occupy different territories in vivo and therefore seemingly acts at different stages of endolysosomal progression. Overall, our findings aid untangling the functional specificity of Rab5-GEFs of yeast.

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The role of reticulon homology domain proteins in regulated remodelling of the ER membrane

Presenting author:

Klára Odehnalová

Biochemistry Center Heidelberg, AG Schuck, Im Neuenheimer Feld 328, 69120 Heidelberg [DE], klara.odehnalova@bzh.uni-heidelberg.de

Author(s):
Klára Odehnalová, Natalie Friemel, Petra Hubbe, Sebastian Schuck

A vital aspect of ER architecture is membrane curvature which is maintained by two protein families – reticulons and REEPs. These proteins share a topological feature, the reticulon homology domain (RHD), and localize all over the highly curved ER tubules and to the curved edges of flat ER sheets. It is however unknown whether their curvature-inducing activity is regulated in order to facilitate the transition between the different ER morphologies.

 

We found that during ER stress in S. cerevisiae, when the ER membrane expands and sheets proliferate, the RHD proteins co-cluster. This stress-induced clustering is diminished upon disruption of the unfolded protein response (UPR), the signalling pathway which governs cell’s response to ER stress. We discovered that Rtn1, the most abundant RHD protein in yeast, gets phosphorylated upon ER stress while its levels remain unchanged. On the other hand, the protein levels of Rtn2 and Yop1 increase but no phosphorylation takes place.

 

We hypothesise that the RHD protein clustering serves to remove them from the bulk of the ER, thereby generating large stretches of membrane with no curvature inducing proteins and facilitating the formation of ER sheets. RHD protein clustering could thus present a new mechanism of ER remodelling.

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Discovering unique roles for the human KDEL receptor homologs

Presenting author:

Leon Olimski

University of Cologne, Center for Biochemistry, Joseph-Stelzmann-Straße 52, 50931 Cologne [DE], leon.olimski@uk-koeln.de

Author(s):
Leon Olimski, Marine Huhardeaux, Alessandra Leone, Julia Etich, Oliver Semler, Frank Zaucke, Emma Fenech

Correct protein folding and the associated quality control (QC) mechanisms in the endoplasmic reticulum (ER) are essential for cells. In mammalian cells, the three KDEL receptors ensure that escaped ER-resident chaperones, which are involved in these QC processes, are recycled from the Golgi apparatus back to the ER. However, the individual endogenous interactors and the resulting functional specificities of these three seemingly redundant receptors remain poorly characterised. In this project, we use a proximity labelling-based approach in combination with loss-of-function mutants, to identify the endogenous clients and regulators of the individual KDEL receptors. Understanding these specific interactomes will help us gain insight into the unique function of each KDEL receptor, as well as their role in diseases such as osteogenesis imperfecta.

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Two-colour lipid imaging

Presenting author:

Athanasios Papangelis

Max Planck Institute of Molecular Cell Biology and Genetics, , Pfotenhauerstr. 108 , 01307 Dresden [DE], papangel@mpi-cbg.de

Author(s):
Athanasios Papangelis, Mathilda Hjoerdis Lennartz, André Nadler

 

Studying the colocalization of individual lipid species in membrane nano-domains has been challenging, if not impossible with current methods in lipid research. Recent advances in the use of bifunctional lipid probes in combination with fluorescence imaging and high-resolution mass spectroscopy has further enabled tracking and quantification of lipid localization within eukaryotic cells. Here, we employ two distinct sets of bifunctional lipid molecules for two-color lipid imaging in mammalian cells. The probes carry two structurally minimal modifications, namely a diazirine moiety as well as orthogonal handles for fluorescence labelling. Using a newly developed labeling scheme, the individual lipids can be visualized within the cell’s organelle system via fluorescence microscopy. Future use of super high-resolution microscopy will enable detailed localization in membrane nano-domains.

 

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Conformational plasticity of oncogenic TpoR dimers in the plasma membrane revealed by live-cell single-molecule FRET

Presenting author:

Bakeeran Pathmalolan

University of Osnabrueck, Department of Biopyhsics, Kardinal-von-Galen Straße 3, 49082 Osnabrück [DE], bpathmalolan@uni-osnabrueck.de

Author(s):
Bakeeran Pathmalolan, Hauke Winkelmann, Mykhailo Girych, Ilpo Vattulainen, Rainer Kurre, Jacob Piehler

Class I/II cytokine receptors play central roles in hematopoiesis and immunity, and their dysregulation is linked to diverse hematological malignancies. They transmit signals across the plasma membrane by assembling into dimeric and higher-order complexes, which activate Janus family tyrosine kinases (JAKs) associated with the cytosolic receptor domains. While transmembrane (TM) and juxtamembrane (JM) regions are known to be critical for receptor activation, how functional coupling across the membrane is achieved remains poorly understood.

We have devised live-cell single-molecule FRET (smFRET) in live cells to uncover dimerization dynamics and conformational organization of cytokine receptors in their native membrane environment. Comparison of ligand-induced dimers with mutation-driven dimers arising from oncogenic mutations in either the TM/JM region or the associated JAK pseudokinase domain uncovered an unexpected degree of conformational plasticity within receptor dimers.

Our data support a model in which frustrated cooperation between TM/JM-mediated and JAK pseudokinase–mediated interactions give rise to distinct dimer conformations with potentially different signaling activities. In line with this hypothesis, we observed differential perturbation of receptor dimers by pharmacological inhibitors.

Together, our results establish live-cell smFRET as a powerful approach to link receptor dimerization and conformational ensembles in dysregulated cytokine receptor signaling.

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Lipid Droplet - Mitochondria Contact Sites

Presenting author:

Louis Percifull

Universität Münster, , Von-Esmarch-Strasse 56, 48149 Münster [DE], louis.percifull@uni-muenster.de

Author(s):
Louis Percifull, Duy Trong Vien Diep, Maria Bohnert

Lipid droplet mitochondria contact sites are key regulators of long-term lipid storage and mobilization in mammalian cells. They have been shown to directly couple the lipid storage organelle with the place of bulk beta-oxidation, the mitochondria. However, the contact site also exists in yeast cells, which do not perform beta-oxidation in mitochondria. This raises the question which functions this contact site might have beyond fatty acid transfer.

We are analysing the yeast lipid droplet-mitochondria contact site by the use of genome-wide screening approaches. With the help of a split-Venus reporter we can visualise the contact site location and expanse. Genome-wide screens uncovered contact site residents and proteins that affect contact site formation. We are working toward resolving the lipid droplet-mitochondria tethering machinery and unravelling functions of this contact site besides fatty acid transfer for beta-oxidation. Our findings will hopefully not only broaden our understanding of the cellular contact site network in yeast, but also help in understanding overlooked features of this contact site in mammalian cells.

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Visual dysfunction caused by impaired membrane-bound Guanylate cyclase activity

Presenting author:

Lars-oliver Peters

Carl von Ossietzky Universität Oldenburg, AG Biochemie, Porsenbergstraße 12, 26121 Oldenburg [DE], lars-oliver.peters@uni-oldenburg.de

Author(s):
Lars-oliver Peters

Guanylate Cyclase E (GC-E) is localized to the disc membranes of the outer segments of human photoreceptors. Its activity is regulated by guanylate cyclase activating protein 1 (GCAP1) in a calcium-dependent manner. At high Ca²⁺ concentrations, GCAP1 inhibits GC, while at low Ca²⁺ concentrations, it activates GC to restore cyclic GMP (cGMP) levels, ensuring proper phototransduction recovery. Mutations in GUCA1A, the gene encoding GCAP1, and in GUCY2D encoding human GC-E, disrupt this regulation and cGMP synthesis. They are implicated in inherited retinal diseases such as retinitis pigmentosa (RP), leading to progressive vision impairment. We investigated the biochemical and functional impact of a newly identified GCAP1 variant, p.R93C, which was recently associated with RP. This missense variant results from a single nucleotide substitution in GUCA1A (c.277C>T), replacing a positively charged, hydrophilic arginine (R) with a neutral, more hydrophobic cysteine (C) in the EF3-hand motif. The R93C mutant showed a decrease in specific GC activity and in Ca²⁺-sensitivity, potentially leading to a disruption of phototransduction and of the recovery process to the dark-adapted state. The mutation impairs the Ca²⁺-binding and has an impact on Ca²⁺-dependent conformational changes monitored by tryptophane fluorescence spectroscopy. The R to C substitution probably disrupts electrostatic interactions and alters protein folding, because it exhibited strong tendencies to aggregation.

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Characterization of the protein environment of ER shaping proteins REEP & RTN

Presenting author:

Martijn Plug

Heidelberg University Biochemistry Center, Biochemie-Zentrum (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg [DE], martijn.plug@bzh.uni-heidelberg.de

Author(s):
Hatice Kara, Martijn Plug, Anne-lore Schlaitz

The endoplasmic reticulum (ER) is a complex membrane-bound network that spans across the entire cell. Near the nuclear envelope, the ER consists mostly of low membrane curvature domains called ER sheets, while near the cell periphery, it consists mostly of highly curved tubules. The high curvature of ER tubules and sheet edges is maintained by curvature inducing proteins, like the Receptor expression-enhancing proteins (REEPs) and Reticulons (RTNs). While family members share some redundancy in promoting membrane curvature, they likely have individual functions, too. For example, REEP5 and RTN4 are important for lipid droplet biogenesis, and REEP3 & REEP4 are important for mitotic ER morphology. Thus, we would like to uncover the individual functions of the REEPs and Reticulons.

To identify potential interaction partners of the individual REEPs and Reticulons, proximity-dependent biotinylation (TurboID) was used. REEP3-6, RTN1, 3 & 4 and a general ER membrane marker were tagged with a TurboID tag. Biotinylated proximal proteins will be enriched by streptavidin pulldown and identified by mass spectrometry, enabling the systematic detection of neighbouring proteins for each individual family member. Furthermore, given the mitosis-specific functions of REEP3 and REEP4, we will also characterize the mitosis-specific protein environment in metaphase-synchronized cells.

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Uncovering novel mediators of mitochondrial stress responses

Presenting author:

Noga Preminger

Weizmann Institute of Science, Department of Molecular Genetics, 234 Herzl St., 7610001 Rehovot [IL], noga.preminger@weizmann.ac.il

Author(s):
Noga Preminger, Felix Boos, Johannes M. Herrmann, Maya Schuldiner

Mitochondria require tightly regulated internal conditions to function properly. Disruption of this balance activates organelle-nucleus signaling pathways that coordinate stress responses and promote recovery from stress. While pathways such as retrograde signaling and the mitochondrial unfolded protein response maintain mitochondrial homeostasis, the variety of molecular players transmitting specific stress signals to the nucleus remains incompletely mapped.

To address this, we used systematic microscopy screens in Saccharomyces cerevisiae to identify mitochondrial proteins potentially involved in stress-responsive signaling. We found a subset of proteins that undergo a localization change in response to stress, and focused on those that re-localize to the nucleus. For these, we are now defining the triggers and downstream consequences of their activation to comprehend their physiological impact on gene expression and stress adaptation. Together, this work provides a framework for identifying proteins that bridge mitochondrial stress with nuclear responses, advancing our understanding of organelle communication and cellular capacity to cope with stress.

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Ameliorating the distorted ER morphology phenotype caused by the mutated ER membrane shaping protein ATLASTIN 3

Presenting author:

Johannes Rickert

, , Fritz-Reuter-Straße 5, 07745 Jena [DE], jr.rickert.johannes@gmail.com

Author(s):
Johannes Rickert, William Durso, Torsten Kroll, Christoph Kaether

ATLASTIN 3 (ATL3) is one of three mammalian ATLs, endoplasmic reticulum (ER) membrane-resident GTPases which are responsible for mediating homotypic membrane fusion of ER tubules, thus establishing the branchpoints of the ER network. The ATL3-Y192C mutant causes autosomal-dominant hereditary sensory and autonomic neuropathy type 1 (HSAN1), an axonopathy characterized by loss of distal sensory function and pain perception. Mechanistically, the mutation impairs the ATL3-mediated membrane fusion, resulting in a disrupted ER network. Reverting this ER morphology impairment might offer an approach for treating HSAN1. Hence, a human genome-wide siRNA library and a pilot compound library were employed in a high content imaging assay to screen for targets mediating ER morphology rescue under overexpression of ATL3-Y192C. The branchpoints count serves as a parameter to observe changes to ER complexity. Candidate targets are validated in a U2OS cell model system expressing a costitutive ER marker and inducible GFP-ATL3-Y192C, as well as in ATL3-Y192C patient fibroblasts. Subsequently, pathways and mechanisms linking the identified hits to modulation of the ER network will be assessed. Moreover, the impact of ATL3-Y192C on axonal ER morphology will be characterized and quantified in primary murine and iPSC-derived neurons cultured in microfluidic chambers. This will allow for studying the capacity of the selected siRNAs and compounds to modulate the neuronal ATL3-Y192C ER phenotype.

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High-throughput CGMD simulations enable proteome-wide identification of lipid scramblases in yeast

Presenting author:

Cristian Rocha Roa

Fribourg University, Fribourg, Switzerland, Department of Biology, Chemin du Musée 10, 1700 Fribourg [CH], cristian.rocharoa@unifr.ch

Author(s):
Cristian Rocha Roa, Stefano Vanni

Membrane proteins constitute nearly a quarter of the proteins encoded in most organisms, representing approximately 50% of the cell membrane mass. Therefore, lipid-protein interactions are crucial for many cellular processes, and lipid transport between organelles is essential for cellular homeostasis. It has been demonstrated that lipid transport proteins team-up with lipid scramblase proteins. This protein-protein complex is proposed to be required for processes such as membrane expansion, e.g. autophagosome biogenesis. Using coarse-grained molecular dynamics simulations of predicted structures from AlphaFold2 for the yeast S. cerevisiae, we have identified several previously unknown putative lipid scramblase proteins. As a consequence of the presence of these new putative scramblases, our results suggest that cell membranes are more dynamic than previously thought. They provide an initial framework for understanding how the cell regulates its lipid membrane and open the door to new interpretations of previous gaps in lipid-mediated processes.

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Dynamics and cofactor interactions of the AAA+ ATPase Vps4 in the ESCRT-III pathway

Presenting author:

Sarah Roman

Forschungszentrum Juelich GmbH, ER-C-3, Wilhelm-Johnen-Straße, 52428 Juelich [DE], s.roman@fz-juelich.de

Author(s):
Sarah Roman, Benedikt Junglas, Carsten Sachse

The AAA+ ATPase Vps4 is an essential component in the endosomal sorting complex required for transport (ESCRT) machinery. Vps4 mediates the ATP-dependent disassembly of ESCRT-III polymers. In this study, we used cryo-EM to resolve the structure of the ATPase-deficient mutant Vps4^E233Q, its complex with the cofactor Vta1, and a complex including the filament-forming ESCRT-III chimera Vps24-Vps2^MIM.

We determined the Vps4^E233Q hexamer with ATP at 2.8 Å resolution, the Vps4^E233Q-Vta1 complex with ATP at 3.5 Å resolution and the Vps4^E233Q-Vta1 together with the ESCRT-III substrate at 5.3 Å resolution. Structural analyses of the Vps4^E233Q hexamer and the Vps4^E233Q-Vta1 complex revealed distinct subunit dynamics, variable Vta1 binding occupancies and a stabilising effect of Vta1 on the hexamer, suggesting a link between ATPase activity and Vta1-mediated stabilisation. Additionally, we identified a loop region (residues 261 – 271) within the large ATPase cassette in two distinct conformations, potentially contributing to nucleotide stabilisation during ATP hydrolysis. Together, our data provides insights into the ATPase mechanism, interaction with cofactor Vta1 and Vps24 substrate engagement, highlighting its role in ESCRT-III disassembly.

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Analysis of the influence of lipid flippase CDC50A knock-out on the Golgi and mitochondria of C2C12 cells

Presenting author:

Fabian Ruebsamen

Department of Biochemie II, Ruhr-Universität Bochum, , Universitätsstr 150, 44801 Bochum [DE], fabian.ruebsamen@ruhr-uni-bochum.de

Author(s):
Fabian Ruebsamen, Thomas Günther-Pomorski, Annika Haak

Flippases are P4-type adenosine triphosphatases, which can translocate lipids from the exoplasmic/luminal to the cytosolic leaflet. A subset of flippases requires a heterodimeric interaction with β subunits to exit the endoplasmic reticulum and be fully functional. In mouse C2C12 myoblasts, a knock-out of the β subunit (CDC50A) inhibits the formation of multinuclear myotubes. Furthermore, a loss of aminophospholipid flippase activity has been observed, which indicates a loss of membrane lipid asymmetry. The GM130 labelled cis Golgi in the presence and absence of CDC50A was investigated microscopically for differences. The Golgi’s lateral extent into the cytosol relative to the nuclear cross-section did not exhibit any significant differences. Yet, the axial Golgi thickness was significantly (p = 0.021) increased in the absence of CDC50A ((1.407 ± 0.152) µm) compared to unaltered myoblasts ((0.973 ±0.135) µm). The mitochondrial morphology showed no significant difference in the absence and presence of CDC50A.

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Analysis of glycoRNAs in bladder cancer

Presenting author:

Sankritya Sarma

University of Augsburg, Institute of Theoretical Medicine, Proteomics, Biochemistry and Molecular Biology, Parkring 13, 85748 Garching bei München [DE], sankritya.sarma@med.uni-augsburg.de

Author(s):
Sankritya Sarma, Falk F. R. Buettner, Julia Beimdiek

Aberrant protein N- and O-glycosylation as well as a distinct profile of glycosphingolipids are hallmarks of malignant tumours. Recently, RNA molecules were discovered as novel carriers of glycans. Such glycoRNAs have been reported to be closely associated with cell membranes, raising compelling questions about their origin and recruitment, and emphasising their functional significance in cell-cell communications and immune interactions. GlycoRNAs are predominantly found to be N-linked and are frequently sialylated and fucosylated, suggesting their potential involvement in modulating oncogenic pathways. This project focusses on defining the contribution of RNA-glycosylation, in vitro and in situ, to lifestyle-associated early-onset tumorigenesis, and on distinguishing glycoRNA-specific effects from those mediated by glycoproteins. Therefore, we intend to adapt the xCGE-LIF technique, which is well established in our group to profile glycoRNA glycosylation from bladder cancer cell lines under different nutritional conditions. Additionally, we will employ knockout models targeting enzymes participating in glycoRNA synthesis to functionally assess how the lack of glycoRNAs affect the tumour-specific features of bladder cancer cells.

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Tafazzin: transacylation from a new angle

Presenting author:

Jonathan Schiller

Goethe - Universität Frankfurt a. M., Institute of Biochemistry II, Theodor-Stern-Kai 7, 60590 Frankfurt (Main) [DE], J.Schiller@med.uni-frankfurt.de

Author(s):
Jonathan Schiller, José Guadalupe Rosas Jiménez, Janet Vonck, Gerhard Hummer, Volker Zickermann

Cardiolipin (CL) is the signature lipid of the inner mitochondrial membrane (IMM) and is essential for its distinctive structure and the functionality of the OXPHOS complexes. CL is synthesized in the IMM but a remodelling process is required to increase the proportion of CL with unsaturated acyl chains. CL remodeling is catalyzed by Tafazzin, an enzyme with a broad substrate spectrum that transfers fatty acids from phospholipids to lyso-phospholipids. In biochemical assays, the enzymatic reaction of Tafazzin was shown to be independent of Coenzyme A (CoA), in contrast to related enzymes such as GPAT1 or PlsC. We recently published the 3.2 A resolution structure of Tafazzin from the aerobic yeast Y. lipolytica. Optimization of purification and data collection now yielded a high-resolution map in which we modelled an acyl-CoA molecule. Using molecular dynamics simulations, we showed that monolysocardiolipin (MLCL) can bind to tafazzin in the presence of acyl-CoA. Indeed, the thioester bond and the free hydroxyl group adopted a catalytically competent conformation close to the strictly conserved histidine residue of the active site. We hypothesize that CoA acts as a prosthetic group and temporary storage site for an acyl chain cleaved from a phospholipid, which is then transferred to MLCL. The dysfunction of Tafazzin causes Barth syndrome, a severe multisystem disorder. Our data lead us to a detailed mechanism of transacylation and a molecular understanding of Barth syndrome.

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Homeostatic control of the sphingolipid biosynthesis regulators Orm1 and Orm2 via ER retention and export.

Presenting author:

Oliver Schmidt

Medical University of Innsbruck, Innsbruck, Austria, Biocenter - Institute of Cell Biology, Innrain 80, 6020 Innsbruck [AT], oliver.schmidt@i-med.ac.at

Author(s):
Oliver Schmidt

 

ORMDL family proteins are conserved regulators of sphingolipid biosynthesis, with the main function to inhibit the rate-limiting enzyme serine-palmitoyl transferase (SPT) in the endoplasmic reticulum (ER). In humans, dysregulation of SPT activity causes a form of amyotrophic lateral sclerosis (ALS), and is genetically linked to several inflammatory diseases [1,2,]. In budding yeast the ORMDL family consists of two proteins, Orm1 and Orm2. Both are embedded in a homeostatic system that adjusts sphingolipid levels to cellular demands.
Although Orm1 and Orm2 share about 70% sequence identity, the two proteins inhibit the SPT with different potency, Orm2 being the stronger inhibitor [3]. Their regulation is different, with Orm2 being subject to ER export and proteolytic turnover via the Endosome and Golgi-associated degradation (EGAD) pathway, which is not observed for Orm1 [4]. How the specificity of ER export vs. retention of these highly homologous proteins is achieved is unknown. Here, we identified a hydrophobic sequence responsible for stably retaining Orm1 in the ER. A second, polybasic retention motif in Orm2 is controlled by phosphorylation and responsible for regulated ER export. We found this polybasic motif to be transferable onto another, constitutively exported ER protein, suggesting a more general mechanism for regulated ER to Golgi traffic.

[1] Mohassel et al., 2021

[2] Moffatt et al., 2007

[3] Körner; Schäfer et al., 2024

[4] Schmidt et al., 2019

 

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Regulation of ClC-7 by PIPs

Presenting author:

Julius Schreer

University of Münster, Institute for Biochemistry, Corrensstraße 36, 48149 Münster [DE], j_schr85@uni-muenster.de

Author(s):
Julius Schreer, Maximilian Rüttermann, Daniel Kümmel

The lysosome not only degrades intra and extracellular proteins, lipids and polysaccharides back to their respective building-block molecules but also functions as a storage organelle for sodium and calcium cations. For this, a large number of ion channels and transporters are needed to maintain luminal pH and the high concentration of cations as well as the signaling properties of the lysosome. Several of these membrane proteins are regulated by phosphatidylinositides especially phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), which is increasingly present in the lysosomal membrane. For example, the ClC-7 proton/chloride antiporter was proposed to act in the counterion pathway for the acidification of lysosome. Functional data suggest ClC-7 is deactivated by PI(3,5)P2 but structural studies so far have only shown the transporter bound to PI3P . We want to investigate how ClC-7 is deactivated by PI(3,5)P2 and which conformational changes can be seen that differentiate the active and inactive Protein. We use cryo electron microscopy to determine the structure of ClC-7 and its β-subunit OSTM1 in presence of PI(3,5)P2 diC8, a soluble variant of the phospholipid and compare our structure to the published structures of the activated protein complex.

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Understanding the regulation of endoplasmic reticulum – plasma membrane contact sites

Presenting author:

Isabelle Christine Schulte-Herbrüggen

University of Cologne, Center for Biochemistry, Joseph-Stelzmann-Straße 52, 50931 Köln [DE], isabelle.schulte-herbrueggen@uk-koeln.de

Author(s):
Isabelle Christine Schulte-Herbrüggen, Zoe Kolb, Emma J. Fenech

Membrane contact sites are areas of close apposition between membranes within cells. They have a specific lipid and protein composition and perform essential functions including lipid transport, signalling and many others. The contact between the endoplasmic reticulum (ER) and plasma membrane (PM) contains several different tether proteins, and most of these proteins play an important role in lipid metabolism and lipid homeostasis. Interestingly, we have observed changes in the expression and localization of a subset of these proteins upon the induction of ER stress in yeast. However, how this regulation is controlled is not understood. To uncover this, we are using the CsFiND (Complementation Assay using Fusion of split-GFP and TurboID) system which we have optimised for use at the ER-PM. The TurboID module will enable us to perform proximity-labelling to identify regulators of this contact. Furthermore, the generation of whole genome CsFiND libraries will provide a powerful tool to study the interactome and localization of membrane contact sites.

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The retrograde trafficking map: Determining cargo receptor relationships of yeast SNX-BAR proteins by vacuolar proteomics

Presenting author:

Julia Seimert

, , Hardinghausstraße 15, 49090 Osnabrück [DE], juseimert@uni-osnabrueck.de

Author(s):
Julia Seimert, Stefan Walter, Florian Fröhlich

The endosomal sorting system is essential for protein trafficking, degradation, and recycling, operating through interconnected pathways that maintain cellular homeostasis. It directs endocytosed plasma membrane and intracellular proteins either toward vacuolar degradation or recycling to the Golgi apparatus or plasma membrane. Defects in endosomal recycling are linked to lysosomal storage diseases and neurodegenerative disorders.

Sorting nexins are key regulators of this system, coordinating cargo selection and membrane remodeling to form carriers for retrograde transport. They are a conserved protein family defined by a Phox homology (PX) domain that binds phosphatidylinositol 3-phosphate (PI3P), enabling membrane association. In yeast, seven sorting nexins have been identified: Vps5 and Vps17, Snx4, Snx41, Snx42, Mvp1, and Ykr078w.

Here, we systematically characterize retrograde sorting pathways by generating a comprehensive trafficking map based on deletions of all sorting nexins. Using proteomics, we analyze cargo profiles in their native, untagged state. Our results reveal distinct cargo specificities for the retromer cargo recognition complex and the SNX-BAR Vps5/Vps17 subcomplex. In addition to confirming known cargo–receptor relationships, we identify novel SNX-BAR cargoes, including the uncharacterized protein Ptm1, which is transported via a strictly Mvp1-dependent pathway. These findings provide a valuable resource for the trafficking community.

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Crossing the Lipid Divide: Substrate Specificity of Bacterial Phospholipid N-Methyltransferases?

Presenting author:

Irina Shevyreva

Ruhr university Bochum, Microbial Biology, Universitätsstr. 150, 44801 Bochum [DE], irina.shevyreva@ruhr-uni-bochum.de

Author(s):
Irina Shevyreva, Devin Korsch, Maik Muskietorz, Meriyem Aktas, Franz Narberhaus, Marten Exterkate

The lipid divide describes the fundamental divergence between archaeal and bacterial/eukaryotic membranes. Archaeal membranes contain highly branched isoprenoid chains linked via ether bonds to a sn-glycerol-1-phosphate (G1P) backbone, whereas bacterial/eukaryotic lipids feature fatty acyl chains esterified to sn-glycerol-3-phosphate (G3P). Despite these structural differences in the lipids, many archaeal and bacterial enzymes remain active on non-native substrates. The archaeal cardiolipin synthase from Methanospirillum hungatei can utilize bacterial phosphatidylglycerol as a substrate (Exterkate et al., 2021); while Bacillus subtilis phosphatidylserine synthase can synthesize archaetidylserine from archaeal precursors (Morii & Koga, 2003).

This study examines the promiscuity of bacterial phospholipid N-methyltransferases (Pmts). These cytosolic enzymes gradually methylate of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). The activity of representatives from two distinct Pmt classes: the Rhodobacter (R) PmtA from Rubellimicrobium thermophilum (RtPmtA) and Sinorhizobium (S) PmtA from Agrobacterium tumefaciens (AtPmtA) is examined in vitro. Using a range of PE substrates, we demonstrate that AtPmtA and RtPmtA display different substrate specificities depending on the lipid backbone chemistry, linkage type, and hydrophobic tail composition. These findings highlight the evolutionary adaptability and the biochemical limitations of lipid-modifying machinery.

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ER and mitochondria contacts with endolysosomes facilitate endocytic lysosomal reformation

Presenting author:

Anne Spang

University of Basel, Biozentrum, Spitalstrasse 41, 4056 Basel [CH], anne.spang@unibas.ch

Author(s):
Samit Desai, Emma Martin Sanchez, Danilo Ritz, Alexander Schmidt, Anne Spang

Endocytic lysosome reformation (ELR) regenerates functional lysosomes following degradation of endocytic cargo, yet the mechanisms driving this process remain largely unknown. Here, we define the molecular machinery underlying ELR and reveal its dependence on inter-organellar contact sites. Using proximity labeling and live-cell imaging, we find that unlike autophagic lysosome reformation (ALR), ELR proceeds independently of mTOR and dynamin 2, but requires the mitochondrial fission GTPase DRP1. DRP1 localizes to endolysosomal tubules and mediates their scission, with fission sites marked by contacts with the endoplasmic reticulum (ER) and mitochondria. Disruption of DRP1 function or ER-endolysosome contact results in elongated, unfissioned tubules, indicating defective lysosome reformation. Moreover, mitochondrial activity is essential for tubule initiation, and calcium transfer from endolysosomes to mitochondria serves as a trigger for ELR onset. Our findings reveal a dual role for mitochondria in ELR: first in ELR initiation and second in DRP1 dependent tube fission at at ER-mitochondria-endolysosome contact sites, uncovering the previously unappreciated role of mitochondria in endolysosome remodelling and fission.

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NTA-CHOLESTEROL ANALOGUE FOR THE NONGENETIC LIQUID-ORDERED PHASE-SPECIFIC FUNCTIONALIZATION OF LIPID MEMBRANES WITH PROTEINS

Presenting author:

Jan Christopher Spies

Universität Münster, , Corrensstraße 36, 48149 Münster [DE], j_spie17@uni-muenster.de

Author(s):
Jan Christopher Spies, Yanjun Zheng, Tristan Wegner, Daniele Di Iorio, Marco Pierau, Frank Glorius, Seraphine Wegner

 

Imidazolium-based lipid mimetics and especially the cholesterol mimetic CHIM (cholesterol-based imidazolium salt) have been shown to be powerful tools for the modification of membranes. CHIM-NTA is a novel cholesterol analogue, which contains an NTA (nitrilotriacetic acid) headgroup. Similarly to the commercially available DGS-NTA, it integrates into lipid membranes and, when loaded with Ni²⁺, it can be used to immobilize polyhistidine-tagged proteins onto the surface of the membrane.
A series of studies was conducted to investigate the properties of CHIM-NTA. FRAP (fluorescence recovery after photobleaching) measurements revealed that CHIM-NTA and DGS-NTA have a similar effect on membrane fluidity and exhibit a similar mobility of bound proteins. However, confocal fluorescence microscopy of phase-separated giant unilamellar vesicles (GUVs) revealed that CHIM-NTA localizes in the liquid ordered (Lo) domains, opposed to the liquid unordered (Lo) domain for DGS-NTA.
Further studies revealed that CHIM-NTA readily integrates into live cell membranes and exhibits only a low toxicity. Treating cells with CHIM-NTA, Ni²⁺ and His-tagged GFP, led to staining of the cell membrane for up to 24 h and little internalization. In conclusion, CHIM-NTA provides a facile and flexible way to modify biological membranes, in particular Lo domains, with His-tagged proteins and can serve as a broadly applicable molecular tool for cell surface engineering.

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Lipid Droplet-Organelle Contacts in MUFA-mediated Longevity

Presenting author:

Sebastian Steinmüller

Institute of Molecular Biology (IMB), , Ackermannweg, 4, 55128 Mainz [DE], S.Steinmueller@imb-mainz.de

Author(s):
Sebastian Steinmüller

In the context of aging, specific lipids are potent modulators of lifespan, and lipids such as monounsaturated fatty acids (MUFAs) correlate with a long lifespan in humans. In the nematode Caenorhabditis elegans, dietary MUFAs extend lifespan by inducing lipid droplets in intestinal cells. Lipid droplets form contacts with other organelles to regulate lipid flux and energy metabolism. However, it remains unclear whether these contacts change with age, or whether they contribute to lifespan extension. We generated multi-organelle fluorophore marker strains and used colocalization as a proxy for contact to test how aging affects colocalization partners. We could verify that depletion of genes that have been reported to regulate lipid droplet contact with the endoplasmic reticulum (Seipin/seip-1), with peroxisomes (Spastin/spas-1) and with mitochondria (Miga2/miga-1) also deplete contact in our system. We find that lipid droplet-mitochondria and lipid droplet-peroxisome contact increase with age, while the contact with the endoplasmic reticulum is decreased. Interestingly, lipid droplet-mitochondria contact is increased in long-lived individuals induced by MUFAs. These findings suggest lipid droplet-organelle interactions as dynamic features of aging that may play a role longevity, providing a framework to further investigate molecular mechanisms.

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Deciphering the mechanisms of membrane association of the HOPS tethering complex

Presenting author:

Alexander Stockhammer

Universität Osnabrück, Fachbereich Biologie, Abt. Biochemie, Barbarastr. 13, 49069 Osnabrück [DE], alexander.stockhammer@uni-osnabrueck.de

Author(s):
Alexander Stockhammer, Christian Ungermann

Tethering and fusion of membranes within the cell are tightly regulated events which are controlled by a specialized molecular machinery. The hexameric tethering complex HOPS plays a key role in tethering of endolysosomal membranes and their consecutive fusion. However, key questions how HOPS functions in mammalian cells remain unresolved. While in yeast the Rab GTPase Ypt7 has been shown to be required for membrane association of HOPS, in metazoans multiple GTPases has been shown to be relevant in membrane recruitment of HOPS, namely the small GTPase ARL8B and the Rab GTPases Rab39 and Rab2. Furthermore, it has been proposed that HOPS would rather act in two subcomplexes that are recruited independently. Here, we employ the CRISPR-Cas9 system to endogenously tag different subunits of HOPS to understand their localization and dynamics in living cells using live-cell imaging. To clarify how HOPS is recruited to membranes we deploy the novel CATCHFIRE system and disrupt the endomembrane system by inhibiting PI3,5P₂ production. By doing so, we can study the association of the two membrane binding subunits of HOPS, VPS39 and VPS41, to membranes decorated with distinct GTPases within living cells. Our results shed light on the underlying mechanisms of HOPS mediated membrane fusion and bring us a step closer towards understanding organization and mechanisms of membrane trafficking within the endolysosomal system.

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Structure and function of the endosomal CORVET tethering complex

Presenting author:

Nicole Susan

Universität Osnabrück, Fachbereich Biologie, , Barbarastr. 13, 49069 Osnabrück [DE], nicole.susan@uni-osnabrueck.de

Author(s):
Nicole Susan, Dmitry Shvarev, Caroline König, Angela Perz, Arne Moeller, Christian Ungermann

 

The endolysosomal system is essential for regulating membrane proteins and nutrient uptake. The maturation of early endosomes into late endosomes involves multiple membrane fusion events. The hexameric class C core vacuole/endosome tethering (CORVET) complex plays a pivotal role in early endosome fusion by interacting with the GTP-loaded Rab5-like Vps21 on opposing membranes. CORVET shares four of its six subunits with the vacuolar HOPS tethering complex, which also contains two Rab-specific subunits at opposite ends. While significant progress has been made in understanding HOPS-mediated tethering and fusion, the mechanisms by which these related complexes specifically recognize their target membranes, and how CORVET mediates tethering, remain poorly understood. In this study, we used cryo-electron microscopy to resolve the structure of the CORVET tethering complex. Comparison with the HOPS structure revealed key differences between the two complexes. We also show a first look at the structure of the human CORVET complex. Through functional assays, we show how the membrane environment influences and enables tethering. Unlike HOPS, CORVET not only depends on its specific Rab GTPase but also requires phosphatidylinositol-3-phosphate and membrane packaging defects for efficient tethering. Our findings suggest that CORVET operates with a distinct membrane code, facilitating fusion through specialized interactions.

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The role of GOLPH3 in COPI coated vesicle cargo selection

Presenting author:

Rebecca Taylor

Max Planck Institute of Biochemistry, Cell and Virus Structure, Am Klopferspitz 18, 81475 Martinsried [DE], taylor@biochem.mpg.de

Author(s):
Rebecca Taylor

COPI coated vesicles mediate transport from the Golgi to the ER and transport within the Golgi. Distinct sets of cargo are transported between these different locations. Therefore, mechanisms of cargo selection must allow incorporation of specific cargo at different locations in the Golgi. Cargo selection is mediated by direct binding to the COPI vesicle coat or by binding to a cargo receptor that binds the coat. Transport to the ER involves direct COPI-binding via a C-terminal K(x)Kxx motif in the cytoplasmic tails of many ER residents or indirect binding of C-terminal ‘KDEL’ sequences to the KDEL receptor which binds to COPI. The recognition mechanism for intra-Golgi cargo is less well-understood. GOLPH3 is proposed to function as a cargo receptor for Golgi enzymes as it binds COPI, the cytosolic tails of some Golgi enzymes, and the Golgi-localized lipid PI4P. Here we determined the cryo-ET structure of in vitro reconstituted COPI vesicles bound to GOLPH3. GOLPH3 is present as a stoichiometric component of the coat, bound close to the membrane in a position that occludes a binding site for ER-resident cargo. Mutation of clusters of residues on the membrane-facing interface of GOLPH3 results in loss of retention of Golgi residents, and different mutations affect different residents. These results provide insight into how COPI vesicles selectively transport Golgi proteins and provide an explanation for why retention signals for Golgi residents have been hard to define.

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Why CHIMs belong in your toolbox: How imidazolium salts probe cholesterol-protein interactions

Presenting author:

Daniel Thielemann

University Münster, Institute of Organic Chemistry, Corrensstraße 36, 48149 Münster [DE], thielemann@uni-muenster.de

Author(s):
Tristan Wegner, Anna Lívia Linard Matos, Marco Pierau, Daniel Thielemann, Volker Gerke, Frank Glorius

 

The investigation of biological membranes is central to an improved understanding of their composition, cellular processes and pathogenesis. However, experiments depend on suitable analytical tools to elucidate the role of membrane components and their interactions with each other. For instance, cholesterol is structurally important and mediates a multitude of signaling and transport processes. At the same time, tools to study the dynamics of cholesterol in live cells are scarce and limited in their applicability. To address this limitation, our group developed imidazolium-based cholesterol analogues called CHIMs. These feature a steroid-derived backbone and an imidazolium headgroup with a functional group pointing toward the extracellular matrix. In vitro experiments showed that CHIMs with an azide functionality (CHIM-L) integrate into membranes, click to a fluorophore and thus efficiently label membranes.

In this project, a novel CHIM is developed as a versatile tool to investigate cholesterol-related biological processes. X-CHIM is a bifunctional analogue suitable for photoinduced proximity labeling of proteins and visualization with a clicked fluorophore. Thus, it enables the simultaneous exploration of cellular cholesterol distributions and cholesterol–protein interactions. It can unveil new components within cholesterol-rich membrane domains and identify membrane-associated proteins relevant to various cholesterol-dependent events.

 

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Mechanism of GAP tumour suppressors on endo-lysosomal membranes

Presenting author:

Sonja Titze

University of Münster, Institute of Biochemistry, Corrensstraße 36, 48149 Münster [DE], titzes@uni-muenster.de

Author(s):
Sonja Titze, Hannah Barz, René Rasche, Maximilian Rüttermann, Lisa Apken, Andrea Oeckinghaus, Daniel Kümmel

The small GTPases Ral and Rheb have been shown to support oncogenic signalling. To switch them off, the GTPase activating proteins (GAPs) RalGAP and tuberous sclerosis complex (TSC) are needed. RalGAP and TSC are multi-subunit protein complexes that catalyse GTP hydrolysis to GDP and thus represent tumour suppressor proteins. Their GAP domains are structurally related and contain a catalytic asparagine that is inserted into the GTPase nucleotide binding pocket (asparagine thumb mechanism). Like most small GTPases, Ral and Rheb are membrane localised via a C-terminal lipidation. Consequently, the recruitment of the GAPs towards membranes is crucial.

Ral is located to the plasma membrane, but also to endosomes and exocytic vesicles. We found that the RalGAP complex controls membrane trafficking and remodelling as its deficiency disturbs the secretory pathway, exocytosis and cilia formation in pancreatic acinar cells. To further understand RalGAP mechanistically, we determined the cryoEM structure and unraveled together with biochemical approaches how RalGAP functions as an obligatory tetramer.

The TSC complex on the other hand inactivates Rheb on the lysosome. This requires binding of TSC1, a subunit of the TSC complex, to PIP containing membranes. Loosing this interaction also weakens the ability to deactivate Rheb at the lysosomal membrane. Interestingly, many pathogenic mutations cluster in this region.

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Reconstitution of the Mon1Ccz1 Rab GEF on model lipid bilayer

Presenting author:

Jesse Tönjes

, , Kettelerstraße , 48147 Münster [DE], jtoenjes@uni-muenster.de

Author(s):
Jesse Tönjes, Daniel Kümmel, Lara Jorde, Arne Möller

The structure of the Rab GEF (guanine nucleotide exchange factor) Mon1Cczz1 in solution was determined using cryo-electron microscopy and helped to understand the mechanistic basis for membrane interaction of the complex. Combined with biochemical studies, a model for the Rab7 activation was developed. However, how the complex is oriented on the membrane, how the full-length Rab7 is bound, and how Rab5 binding promotes membrane recruitment and enhances catalytic activity of the complex remains elusive at a molecular level. To address these questions, we aim for the reconstitution of the endosomal and autophagosomal tetrameric complex of Mon1Ccz1 with their respective GTPases and/or with the autophagosome recruiter Atg8 on membranes for structural analysis by cryo-electron microscopy. As Rab7 has the highest affinity towards Mon1Ccz1 in the nucleotide-free state and Rab5 only interacts with Mon1Ccz1 in the GTP-bound state, getting both Rab-GTPases in their respective nucleotide loading states on one membrane represents the primary challenge for which different click techniques are needed. So far, the reconstitution of the ctYpt7/ctMC1, ctVps21b/ctMC1, and the ScAtg8/ScYpt7/ScMC1 complexes on DO/PIP+MPB liposomes was achieved. For the autophagosomal complex comprising of ScAtg8/ScYpt7/ScMC1 first cryo-electron microscopy conditions were screened and Protein decorated Liposomes were observed.

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Clickable Imidazolium-based Cholesterol Analogs for Tracking and Targeting Subcellular Cholesterol Pools

Presenting author:

Nele Van Wyngaerden

University of Münster, Institute of Organic Chemistry, , Corrensstraße 38, 48149 Münster [DE], n.vanwyngaerden@uni-muenster.de

Author(s):
Nele Van Wyngaerden, Inga Pauels, Ursula Rescher, Frank Glorius

In complex organisms, constant communication across cellular interfaces is essential to ensure proper functionality. These exchanges of signals occur mainly via assembly and disassembly of molecular domains in the plasma membrane.

Cholesterol is an important component in most biological membranes which plays a vital role in regulating membrane integrity and cellular processes. One example being that the endolysosomal cholesterol balance controls the loading of CD63 and P-selectin onto Weibel-Palade Bodies (WPBs). However, tools to probe the cholesterol dynamics in live cells are scarce and limited in their applicability. Our group has previously developed imidazolium-based cholesterol analogues called CHIMs. These can be modified with different chemically functional groups, such as azides, that points towards the extracellular matrix, while maintaining the integration properties of cholesterol. Such azide-containing CHIMs (CHIM-L) were shown to be able to efficiently label cell membranes with fluorophores in live cells.

In this project, novel CHIMs are further developed as chemical tools to investigate cholesterol-related dynamic membrane processes with the goal of further investigating the dynamics of endolysosomal cholesterol balance in various pathophysiological scenarios.

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Model membrane vesicles as a tool to study plant flippases

Presenting author:

Inja Van der Linden

, , Hustadtring 77, 44801 Bochum [DE], inja.vanderlinden@rub.de

Author(s):
Inja Van der Linden, Huriye Deniz Uzun, Thomas Günther-Pomorski

Flippases are a functionally distinct group of membrane proteins that maintain membrane asymmetry by translocating lipids from the extracellular or luminal leaflet to the cytosolic leaflet. This activity is essential for many physiological processes, including membrane trafficking and cellular signaling. In plants, flippases are implicated in fertility, development, and adaptive responses to external stressors such as temperature extremes and pathogen attack, making them attractive targets for biotechnological crop improvement. While some aspects of plant flippases have been extensively studied, many remain poorly understood, particularly their regulation and interaction with specific lipid substrates.

Model membrane systems provide a powerful tool to study flippases under controlled conditions. Protein purification and reconstitution into artificial vesicles reduce the complexity of the native cellular environment, enabling direct assessment of lipid transport under various conditions. Large unilamellar vesicles are widely used due to their ease of assembly and stability, but their small size limits direct observation and often requires labelled lipid probes. In contrast, flippase-induced shape changes of giant unilamellar vesicles, arising from a numerical leaflet lipid asymmetry, can be directly visualized by microscopy. Here, we present an optimized approach for reconstituting flippases into GUVs, enabling direct assessment of their activity using native lipid substrates.

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Visualization of phospholipid scrambling by a single cross-linked VDAC1 dimer at the single vesicle level

Presenting author:

Sarina Veit

Ruhr University Bochum , Biochemistry II, Universitätsstr. 150, 44780 Bochum [DE], sarina.veit@rub.de

Author(s):
Sarina Veit, Grace I. Dearden, Kartikeya Menon, Faria Noor, Indu Menon, Takefumi Morizumi, Oliver Ernst, Anant K. Menon, Thomas Günther Pomorski

Phospholipid scramblases facilitate ATP-independent bidirectional movement of lipids between the leaflets of a membrane bilayer. Recent work [1] identified the voltage dependent anion channel (VDAC1) as a scramblase in the outer mitochondrial membrane which plays a major role in importing phospholipids into mitochondria. VDAC1 is active as homodimer, with the lipid scrambling pathway provided by the dimer interface. We developed a fluorescence-based microscopy setup to analyze single vesicles reconstituted with fluorescently labeled lipid transporters [2]. This high-throughput approach permits parallel analysis of multiple parameters of individual proteoliposomes, including size and protein copy number, with the aim to correlate these features with lipid transport kinetics. Using artificial liposomes containing trace amounts of tail-labeled fluorescent reporter lipids, we were able to monitor the scramblase activity of reconstituted VDAC1 dimers at the single vesicle level and calculate the unitary rate for single dimers.

 

1. Jahn et al. (2023) Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase. Nature Communications 14:8115

 

2. Veit et al. (2022) A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy. ACS Applied Materials & Interfaces 14: 29659

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The interaction of antiviral molecules with membranes investigated by solid-state NMR and MD simulations

Presenting author:

Alexander Vogel

Universität Leipzig / Medical Faculty, Institute of Medical Physics and Biophysics, Härtelstr. 16-18, 04107 Leipzig [DE], alexander.vogel@medizin.uni-leipzig.de

Author(s):
Alexander Vogel, Florian Seufert, Peter Müller, Holger A. Scheidt

Albeit the mode of action for many antiviral drugs is still incompletely understood, it is common to all that they interact with the plasma membrane and/or with intracellular membranes. However, their membrane interactions are often insufficiently characterized at the molecular level. Therefore, in this work, we investigate the interaction of the antiviral drugs Molnupiravir, Ritonavir, and Nirmatrelvir which are used for treatment of COVID-19 with POPC lipid bilayers combining solid-state NMR spectroscopy and molecular dynamics (MD) simulations.

Solid-state ²H NMR spectroscopy was employed to determine the influence of the antiviral molecules on the order parameters of POPC lipids while ¹H–¹H NOESY spectra were recorded to localize the antiviral compounds within the membrane.

To aid the interpretation of the experimental data, all-atom MD simulations of 4 microsecond length each were performed. From the simulation trajectories, order parameters and intermolecular NOESY cross-relaxation rates were calculated and quantitatively compared with the experimental NMR results. Good agreement between experiment and simulation was observed for all three compounds, allowing interpretation of the NMR results.

Overall, this study demonstrates how the synergistic combination of solid-state NMR and MD simulations provides a detailed and consistent picture of antiviral drug–membrane interactions, contributing to a deeper understanding of their physicochemical behavior in lipid bilayers.

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Species-specific diacylglycerol signaling – peeking behind the mask of lipid classes

Presenting author:

Linda Wedemann

EPFL, SB SV GR-SCHUHMACHER, Av. François-Alphonse Forel 2, 1015 Lausanne [CH], linda.wedemann@epfl.ch

Author(s):
Linda Wedemann, Romain Hamelin, Florence Armand, Séverine Lorraine, Maria Pavlou, Milena Schuhmacher

Diacylglycerols (DAGs) are cellular lipids which function as second messengers in cellular signal transduction. Upon formation at the plasma membrane, DAGs recruit downstream effector proteins, which in turn activate diverse signaling cascades, ultimately leading to specific cellular outcomes. However, mammalian cells do not generate “a single DAG signal”, but dozens of structurally distinct DAG species. This chemical heterogeneity raises the question whether individual DAG species contribute distinctively to signalling pathway specificity and cellular decision-making.

The emergence of functionalized lipids has enabled the in vitro investigation of individual lipid species. Here, we employ photo-caged DAG probes, which allow the spatially controlled elevation of individual DAG species at the plasma membrane. Using these probes and following a multi-omic approach, we elucidate the species-specific effects of DAGs on cellular signaling. Combination of (phospho)proteomic, transcriptomic, and lipidomic data enables the elucidation of species-specific cellular pathway activation and cellular response patterns. Our approach provides new insight into how DAG structural diversity influences signaling networks and cellular responses.

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The role of the palmitoylation switch in activated T cells

Presenting author:

Tamina Werk

Freie Universitaet Berlin, Biochemistry, Thielallee 63, 14195 Berlin [DE], Tamina.werk@fu-berlin.de

Author(s):
Tamina Werk

S-Acylation, in particular palmitoylation, is a post-translational modification in which a fatty acid chain is covalently attached to a cysteine residue via a thioester bond. This process is catalysed by protein acyltransferases (PATs), also known as DHHC enzymes, and can be reversed by acyl-protein thioesterases (APTs). This reversible nature of palmitoylation confers a dynamic regulation, enabling it to function as a molecular switch in cellular processes, e.g. in T cell activation. However, the mechanisms underlying DHHC enzyme-mediated substrate recognition and specificity remain unknown. In my project I express and purify DHHC enzymes to investigate their interaction partners and subsequently their underlying mechanism of substrate recognition. Since DHHC enzymes are membrane proteins, their purification strategy must be carefully tailored to the specific protein. Afterwards, the interaction and binding mode are characterised by cross-linking mass spectrometry (XL-MS), while substrate specificity is assessed by a functional on-plate palmitoylation assay. In particular, a pulldown assay with DHHC20 revealed, amongst others, the beta subunit of a G protein as an interaction partner. I have investigated the interaction sites by XL-MS, which yields distance constraints that can be used for docking simulations of both proteins. I will expand this with other binding partners, to get insights on how different substrates are bound and recognised by DHHC enzymes.

 

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Investigating the subcellular localization of Ubx2 in yeast

Presenting author:

Lisa-Marie Werning

University of Cologne, Center for Biochemistry, Joseph-Stelzmann-Straße 52, 50931 Cologne [DE], lwerning@uni-koeln.de

Author(s):
Lisa-Marie Werning, Emma J. Fenech

 

For proteins to function they need to be correctly folded and localized. Failure to achieve this can lead to protein aggregation and loss of homeostasis, which trigger stress response mechanisms in different organelles. One of these mechanisms is endoplasmic reticulum (ER)-Associated Degradation (ERAD) which can degrade misfolded proteins in the ER. In mitochondria, a parallel mitochondria protein Translocation Associated Degradation (mitoTAD) pathway exists. Intriguingly, in yeast, both pathways rely on the membrane embedded protein, Ubx2. However, it is unknown whether the subcellular localization of Ubx2 is regulated depending on organellar requirements. To address this, we endogenously tagged Ubx2 and monitored it under conditions where either the ER or the mitochondria were stressed. While ER stress did not affect Ubx2, we found that mitochondrial stress did induce changes. Specifically, both the amount and localization of Ubx2 changed under these conditions. Our results therefore imply stress-dependent regulation of Ubx2. More globally, it highlights that there are undiscovered mechanisms which control the subcellular positioning of multi-localized proteins.

 

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N-Chlorotaurine – A Membrane Modifying Neutrophil-Derived Antimicrobial Oxidant?

Presenting author:

Justine Andrea Williams

Ruhr University Bochum (RUB), Microbial Biochemistry, Medical Faculty, Microbial Biochemistry, Universitätsstraße 150, 44801 Bochum [DE], justine.williams@rub.de

Author(s):
Justine Andrea Williams, Lisa Roxanne Knoke

 

During phagocytosis, neutrophils engulf pathogens and release a toxic cocktail including hypochlorous acid (HOCl). In host-pathogen interactions, taurine protects immune cells by neutralising HOCl, resulting in N-chlorotaurine (NCT) accumulation. Previous studies hypothesised that NCT targets the bacterial envelope and induces oxidative stress, however its mode of action is not fully understood. Bacteria actively take up taurine through two designated ABC transporters, TauABC and SsuABC, but whether they are important for NCT toxicity is unknown. Here, we elucidate the importance of the bacterial cell envelope and taurine import for NCT susceptibility.

Using a fluorescent chemical probe, we demonstrated that NCT forms N-chloramines with phosphatidylethanolamine (PE) in vitro. We analysed membrane permeability in response to NCT using liposomes containing self-quenching fluorophores. Although NCT modified PE, it did not permealise our model membranes. We treated living E. coli cells with NCT and applied our N-chloramine probe to analyse membrane chlorination and propidium iodide to track E. coli´s membrane integrity. To elucidate the role of taurine importers for NCT susceptibility - and hence whether NCT toxicity depends on uptake - we monitored the growth of E. coli tauB and ssuB single and double deletion mutants in the presence of NCT. We showed that the lack of TauB increased NCT resistance, suggesting that NCT acts on both the cell envelope and cytosolic components.

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Lipid Droplet Targeting Drives Substrate Access during Very Long Chain Fatty Acid Activation by Fat1

Presenting author:

Carolin Willner

University of Osnabrück, Bioanalytical Chemistry, Sutthauser Straße 110, 49080 Osnabrück [DE], carolin.willner@uni-osnabrueck.de

Author(s):
Carolin Willner, Jennifer Sapia, Carolin Körner, Florian Fröhlich, Stefano Vanni

 

Fatty acids, the most abundant building blocks for lipid synthesis, require conjugation with Coenzyme A (CoA) for their incorporation into membrane lipids. Very long-chain fatty acids (VLCFAs), the precursors of yeast ceramides, are a particularly hydrophobic class of fatty acids, yet the site and mechanism of their activation by the sole VLCFA-CoA synthetase Fat1 remains unresolved. In our work, we demonstrate that lipid droplets (LDs) serve as the site of free VLCFAs storage, as well as their place of activation. Molecular simulations and in vitro reconstitution show preferential partitioning of VLCFAs into LDs, whereas Fat1 targets the LD surface in cells via a hydrophobic N-terminal amphipathic helix. Structural predictions identify an essential hydrophobic cavity in Fat1 that connects the active site to the membrane, suggesting that VLCFAs directly shuttle from the LD core into the enzymatic pocket. Our findings reveal how the organelle targeting of a fatty acid activator evolved to adapt to the biophysical properties and consequently the subcellular localization of its highly hydrophobic substrate.

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Molecular arms race – insights into the bacterial phage shock response to holin-mediated cell lysis

Presenting author:

Dennis Winkler

Karlsruher Institut für Technologie (KIT), Biochemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe [DE], dennis.winkler@kit.edu

Author(s):
Dennis Winkler, Natalie Heissler, Patrick Sieber, Antje Löher, Torsten H. Walther, Anne S. Ulrich

Phages infect bacterial hosts in order to exploit their biosynthetic machinery for propagation. The subsequent release of viral progeny is ensured by programmed lysis of the host cell. In double-stranded DNA pages, this process is orchestrated by small membrane proteins called holins, which form large holes in the membrane at a defined time-point after infection.¹

While this process was found to be tightly regulated by the phage¹, the corresponding host response has not been addressed yet: Bacteria possess numerous membrane repair systems², which are likely to intervene and consequentially modulate lysis timing.

One of the best understood membrane repair systems in E. coli is the phage shock protein (Psp) response. Its main effector PspA can sense membrane damage and patch it via formation of protective carpets on the membrane surface.⁴

Here, we aimed to understand the effect of PspA, on holin-mediated cell lysis.

We could demonstrate that the Psp response is indeed activated by holin-stress via reporter assay. Growth analyses of a ΔpspA strain expressing the lysis effectors of phage λ revealed an inhibiting effect of PspA on holin-mediated cell lysis. Localisation studies via time-resolved fluorescence microscopy verified this effect to be a result of PspA directly acting on holin lesions.

 

1. R. Young. J Microbiol. 2014. 52: 243-258
2. A. M. Mitchell & T.J. Silhavy. Nat Rev Microbiol. 2019. 17: 417–428
3. R. Kobayashi, T. Suzuki, M. Yoshida, Mol Microbiol. 2007. 66:100-109

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Mechanical control of neurotransmission via a disordered domain of an endocytic protein

Presenting author:

Agata Witkowska

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), , Robert-Rössle-Straße 10, 13125 Berlin [DE], witkowska@fmp-berlin.de

Author(s):
Agata Witkowska, Leonie Rommel, Abdelmoneim Eshra, Gaga Kochlamazashvili, Svea Hohensee, Dmytro Puchkov, Tim Berneiser, Narasimha Swamy Telugu, Sebastian Diecke, Max Ruwolt, Fan Liu, Stefan Hallermann, Volker Haucke

Membrane mechanics is increasingly recognized as a critical regulator of membrane remodeling processes, particularly at sites of dynamic vesicle trafficking like the synapse. However, the mechanisms coordinating these processes remain incompletely understood. We demonstrate that dynamic membrane tension fluctuations directly regulate synaptic vesicle cycle dynamics, revealing a novel molecular mechanism. Using a combination of in vitro reconstitution, optical tension measurements in neurons, and neurotransmission characterization, we identified FBP17, an endocytic protein, as a presynaptic membrane tension sensor. We show that its intrinsically disordered domain undergoes a tension-dependent conformational change, triggering endocytosis, a process crucial for high-fidelity neurotransmission. The conformational change drives endocytosis and modulates neurotransmitter release, revealing how membrane tension acts as a signal, triggering protein rearrangements at the lipid-protein interface. Our findings demonstrate that membrane tension is not merely a passive biophysical property, but an active regulator of signaling, dynamically controlling membrane remodeling. We propose that this interfacial protein dynamics, linked to membrane tension, represents one of the fundamental principles governing membrane remodeling events not only at synapses, but also at other sites such as exo-endocytosis in exocrine and endocrine cells or endosomal membrane scission.

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Activity regulation mechanisms of in vitro reconstituted yeast serine palmitoyl-transferase

Presenting author:

Verena Natascha Wolf

University Osnabrueck, Bioanalytical Chemistry, Barbarastraße 11, 49076 Osnabrück [DE], verena.wolf@uni-osnabrueck.de

Author(s):
Verena Natascha Wolf, Bianca M. Esch, Carolin Körner, Florian Fröhlich

The first and rate-limiting step in sphingolipid biosynthesis to generate 3-ketodihyrosphingosine in the endoplasmic reticulum is catalyzed by the serine-palmitoyl transferase (SPT), which sits within the SPOTS complex.

Recent studies have revealed that the structure of the SPOTS complex is highly similar in mammals, plants and yeast (Saccharomyces cerevisiae). This clearly indicates the presence of conserved regulatory mechanisms across different species. However, exact mechanisms remain unclear.

Since cells have a highly adaptable lipidome we use in vitro reconstitution, to resemble a near-native environment in order to study the exact regulation mechanisms of the yeast complex. We are therefore the first, showing a successful reconstitution of the multi-subunit SPOTS complex into ER membrane mimicking liposomes.

Embedding the complex in a membrane clearly improved SPT activity. Meanwhile, Orm2 turned out to be a more potent inhibitor in comparison to Orm1. Furthermore, a strong activity reduction of an Orm free complex provides evidence that Orm proteins are required not only for regulation but also have a pivotal role to form a stable complex. To further unravel regulation mechanisms via phosphorylation and ceramide binding we will use the Ypk1 kinase and different ceramide concentrations in our activity assays. We propose that our reconstitution-based assay will help clarify how the enzyme regulation functions, providing a better understanding of misregulation in mammals.

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Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes

Presenting author:

Wenxin Zhang

Max Planck Institute of Biophysics, Mechanisms of Cellular Quality Control, Max-von-Laue-Straße 3, 60438 Frankfurt am Main [DE], wenxin.zhang@biophys.mpg.de

Author(s):
Wenxin Zhang, Delong Li, Florian Wilfling

Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. In vitro reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.

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The membrane-bound electron transport system of Methanosarcina mazei: Structural investigation of the energy-converting hydrogenase Ech

Presenting author:

Erik Zimmer

Marburg University, Center for Synthetic Microbiology, Karl-von-Frisch-Str. 14, 35043 Marburg [DE], zimmere@staff.uni-marburg.de

Author(s):
Erik Zimmer, Katrin Weidenbach, Ruth Schmitz-Streit, Jan Schuller

 

Class II methanogenic archaea conserve energy through a membrane-bound electron transport system, similar to the well-known aerobic respiratory chain. This system uses different redox cofactors and protein complexes, one of them being the energy-converting hydrogenase (Ech). The reaction catalysed by this complex is known, however, structural and mechanistic understanding and its interplay with the other complexes is lacking so far. We aim to elucidate the energy conservation mechanisms employed by the methanogenic electron transport system. Due to its simplicity compared to the aerobic respiratory chain complexes and the evolutionary conservation of similar complexes across all domains of life, we would like to infer a general theory of how biological electron transport systems work and evolved. For this, we purified the Ech complex from detergent-solubilized membranes of Methanosarcina mazei Gö1 (M. mazei) and determined its protein structure with cryo-EM single-particle analysis at 2.5 Å resolution. By comparison of conserved residues and water molecules found within the structure, to respiratory complex I and membrane-bound hydrogenases, we hypothesize on the coupling mechanisms of redox reactions and proton pumping. Next, we plan to determine catalytically relevant conformations of Ech by vitrifying the protein in copolymer-nanodiscs and native vesicles under turnover conditions.

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Unraveling Membrane Remodeling by a Self-Organized Lipid Kinase/Phosphatase System in vitro

Presenting author:

Laís do Carmo

Friedrich Miescher Laboratory, , Max-Planck-Ring 9 , 72076 Tübingen [DE], lais.docarmo@tuebingen.mpg.de

Author(s):
Laís do Carmo, Beatrice Ramm

Phosphoinositides (PI) are essential lipids in eukaryotic membrane and crucial for vesicle trafficking and membrane identity. The opposing actions of lipid kinases and phosphatases dynamically regulate PI levels, giving rise to spatial organization of membrane lipids. Recent studies showed that PI kinases and phosphatases play a critical role in membrane remodeling during infections, including those caused by Legionella pneumophila. L. pneumophila grows intracellularly and has a T4SS secretion system that releases over 300 different effector proteins into the host cell to create an endoplasmic reticulum (ER)-like niche. Recently, only two of those effectors were shown to generate dynamic patterns that remodel the host ER in vivo: the phosphatidylinositol 3-kinase MavQ and the 3-phosphatase SidP. Using supported lipid bilayers containing PI, MavQ and SidP could also self-organize in vitro into dynamic waves that condense into quasi-stationary patterns - spots, labyrinths and stripes with wavy edges, becoming a strong candidate to emerge as a minimal self-organizing system. However, the detailed molecular mechanism underlying the self-organization of the MavQ/SidP/PI system and how this causes deformation of the host ER membrane remain unclear. To adress this questions, this project aims to elucidate how the activity of individual components and their interactions with each other and the membrane cause membrane remodeling.

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Investigating a Putative Diffusion Barrier at ER–Nuclear Envelope Junctions

Presenting author:

Alexander von der Malsburg

Universität des Saarlandes, Medizinische Fakultät, Medizinische Biochemie und Molekularbiologie , Kirrberger Straße 100, 66421 Homburg [DE], alexander.malsburg@uni-saarland.de

Author(s):
Alexander von der Malsburg, Clara-Anna Wagner, Kaike Ren, Shotaro Otsuka, Robert Ernst

The endoplasmic reticulum (ER) is a vast endomembrane system physically connected to the nuclear envelope (NE) and spans the entirety of the eukaryotic cell. As a central hub for protein and lipid synthesis, the ER interacts with most organelles and strongly influences their molecular composition. ER function is dynamically adjusted to metabolic demands, while imbalances in protein load and lipid composition are monitored by dedicated sensing and signalling pathways to prevent cellular dysfunction. However, whether these adaptive changes propagate to the NE and directly affect NE function remains poorly understood.

The ER forms hourglass-shaped junctions with the outer nuclear membrane (ONM), creating a narrow constriction. We propose that this highly curved ER–NE junction acts as a selective diffusion barrier, restricting the passage of large protein complexes, aggregates, and potentially lipids between the ER and the NE, thereby shielding the NE from ER stress and preserving nuclear integrity.

To test this hypothesis, we developed an APEX2-based proximity labelling approach to define and compare ER and NE proteomes. This strategy addresses three key questions: (i) whether the ER–NE junction functions as a selective barrier; (ii) which biophysical properties underlie restricted diffusion; and (iii) how this barrier contributes to NE protection by retaining specific proteins and pathways within the ER.

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Minimal rhodamine probes for covalent protein labeling in live cells

Silja Friederike Zedlitz

Max Planck Institute of Molecular Cell Biology & Genetics, Hyman Group, Dresden, Germany, <a>zedlitz@mpi-cbg.de

Rhodamine dyes are exceptionally bright, photostable and tunable in color.[1] Paired with genetically-encoded self-labeling protein tags, like HaloTag7[2], the respective rhodamine derivatives allow selective labeling of target proteins in cultured cells. Yet, in vivo imaging of multicellular organisms with HaloTag7 remains challenging due to the limited pharmacokinetic characteristics of these fluorophore substrates.[3] Here, novel rhodamine-binding protein tags (Rho-tags)[4] present an attractive alternative because of their direct high-affinity binding to unsubstituted rhodamines. Rho-tag has been demonstrated for efficient in vivo labeling, but the non-covalent interaction remains a persistent drawback as the dye may be washed out over time. Therefore, a covalent Rho-tag (cRho-tag) modality – bearing a minimal reactive linker on the rhodamine scaffolds – was developed to address this challenge. The reactive moieties were carefully selected to ensure high labeling specificity to a residue within the binding site and favorable pharmacokinetic properties. This approach represents a promising step towards the next generation of permanent protein labeling for bioimaging.

[1] Chen, F. et al. Analysis & Sensing 2 (2022).
[2] Los, G.V. et al. ACS Chem Biol (2008).
[3] Chen, W. et al. Biocell (2022).
[4] Kompa. J. et al. bioRxiv (2025).

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Assessment and development of a preexisting pipeline for computational design of multistate proteins

Tobias Johannes Dorer

Kopenhagen, Denmark, tobias.dorer@bio.ku.dk

A proteins function relies on its dynamic behavior and the ability to program specific dynamics into de novo designed proteins is hence a momentous goal. Allostery, substrate binding and even enzymatic catalysis often depends on the specific dynamic behavior of proteins built by evolution.
While recent method development led to elaborate tools such as ProteinMPNN or RF-Diffusion for the design of proteins of arbitrary shape, the tools for the precise design of dynamic behvavior are yet to be created. Design pipelines for dynamic proteins lack elaborate inverse folding methods as well as computational methods to predict the populations of different conformations.
This thesis builds on improving the general design pipeline for the purpose of dynamic proteins. I show that the elaborate combination and modification of state-of-the-art deep learning tools promises higher success rates. I found that, while many new deep learning models are designed, the ingenious usage and the deliberate choice of existing models can have a strong influence on success. The comparison of probability distributions generated by inverse folding models holds information on the compatibility of conformations. With tied logits sampling in deep learning models, the resulting probability distribution given by the inverse folding tool can be stronger influenced by one state depending on the confidence of the model. Such principles can be used to screen and balance the sampling process for dynamic de novo proteins in the future.

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The shape of the plasma membrane regulates the nanoscale organization, function and pharmacology of transmembrane proteins

Dimitrios Stamou  

University of Copenhagen, Center for Geometrical Engineering of Cellular Systems, Copenhagen, Denmark, stamou@chem.ku.dk

Abstract not submitted yet

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Mechanical control of neurotransmission via a disordered domain of an endocytic protein

Presenting author: Agata Witkowska

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), , Robert-Rössle-Straße 10, 13125 Berlin [DE], witkowska@fmp-berlin.de

Author(s):
Agata Witkowska, Leonie Rommel, Abdelmoneim Eshra, Gaga Kochlamazashvili, Svea Hohensee, Dmytro Puchkov, Tim Berneiser, Narasimha Swamy Telugu, Sebastian Diecke, Max Ruwolt, Fan Liu, Stefan Hallermann, Volker Haucke

Membrane mechanics is increasingly recognized as a critical regulator of membrane remodeling processes, particularly at sites of dynamic vesicle trafficking like the synapse. However, the mechanisms coordinating these processes remain incompletely understood. We demonstrate that dynamic membrane tension fluctuations directly regulate synaptic vesicle cycle dynamics, revealing a novel molecular mechanism. Using a combination of in vitro reconstitution, optical tension measurements in neurons, and neurotransmission characterization, we identified FBP17, an endocytic protein, as a presynaptic membrane tension sensor. We show that its intrinsically disordered domain undergoes a tension-dependent conformational change, triggering endocytosis, a process crucial for high-fidelity neurotransmission. The conformational change drives endocytosis and modulates neurotransmitter release, revealing how membrane tension acts as a signal, triggering protein rearrangements at the lipid-protein interface. Our findings demonstrate that membrane tension is not merely a passive biophysical property, but an active regulator of signaling, dynamically controlling membrane remodeling. We propose that this interfacial protein dynamics, linked to membrane tension, represents one of the fundamental principles governing membrane remodeling events not only at synapses, but also at other sites such as exo-endocytosis in exocrine and endocrine cells or endosomal membrane scission.

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Nuclear envelope budding is a non-canonical mechanism to export large transcripts in muscle cells

S Zaganelli, JB Meehl, RG Abrisch & Gia Voeltz*

*University of Colorado, Department of Molecular, Cellular and Developmental Biology, Boulder, CO, USA, gia.voeltz@colorado.edu

The nuclear pore complex (NPC) is considered the sole route to transport molecules across the nuclear envelope (NE). In recent years, NE budding (NEB) has emerged as an alternative route for nuclear export of viral particles that are too large to pass through the NPC. Yet, the significance of this unconventional export pathway for large endogenous cargoes in mammalian cells has remained largely unexplored. Here, we use a combination of electron and fluorescence microscopy to demonstrate that NEB occurs concomitantly with the differentiation of myoblasts into myotubes. We show that NE buds are derived from the inner nuclear membrane, contain internal vesicles, and are enriched with long sarcomeric transcripts. We identify the RNA-binding protein UIF and the ESCRTIII membrane remodeling machinery function to traffic these large mRNA cargoes into NE buds for nuclear egress. Together these data reveal a non-canonical pathway for large transcript packaging and export in muscles cells.

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Orphan protein quality control at the Golgi protects organelle architecture

David Teis

Medical University of Innsbruck, Institute of Cell Biology, Innrain 80/82, 6020 Innsbruck, Austria, david.teis@i-med.ac.at

Abstract not submitted yet

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Building a functional atlas of homologous contact site machineries through advanced interaction mapping

Presenting author: Emma Fenech

University of Cologne, Center for Biochemistry, Joseph-Stelzmann-Straße 52, 50931 Cologne [DE], efenech@uni-koeln.de

Author(s):
Emma Fenech

The endoplasmic reticulum (ER) coordinates a multitude of diverse and essential functions. The range and robustness of each of these functions are upheld by the unique and shared capacities of homologous proteins. Such homologs are also present at ER contact sites, with the yeast family of six LAM (Lipid transfer protein Anchored at Membrane contact sites) proteins being one example. This family is composed of three pairs of homologs which are all anchored in the ER membrane and contain lipid binding and transfer domains. However, each LAM protein is unique in its molecular architecture and localization to different ER subdomains. To shed light onto what determines the localization of the different LAMs and the specific roles they perform, we used an advanced proximity-labelling approach to profile the protein landscape of the entire family. We uncovered unique candidate interactors which supported previous observations that Lam5 resides at the ER-mitochondria contact, and we demonstrated a novel role for it in sustaining mitochondrial activity. Conversely, shared putative interactors of multiple LAMs revealed how the Lam1/3 and Lam2/4 homologous pairs could associate specifically with plasma membrane lipids. Overall, our work provides new insights into the regulation and function of the LAM family members. More globally, it demonstrates how proximity labelling can identify shared and unique functions of protein homologs resident within the ER membrane contact site network.

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The architecture of organelle contact sites

Wanda Kukulski

University Bern, Institut für Biochemie und Molekulare Medizin, Bühlstrasse 28, 3012 Bern, Switzerland, wanda.kukulski@unibe.ch

Organelles interact through close apposition of their membranes. Such contact sites serve the exchange of lipid molecules, calcium transport and organelle biogenesis. The mechanisms by which molecules are transferred across organelle contact sites are poorly understood because little is known about the underpinning supramolecular organisation. We aim to reveal and dissect the architecture composed of proteins and apposing bilayers and thereby shed light on how functional microenvironments between the membranes of two organelles are formed. Towards this goal, we employ correlative light and electron microscopy approaches, which we complement with live fluorescence imaging, biochemical and cell biological experiments.

 

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Illuminating mitochondrial permeabilisation in apoptosis

Ana García-Sáez

Max Planck Institute of Biophysics, Membrane Dynamics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany, ana.garcia@biophys.mpg.de

Mitochondrial permeabilization is a key step in the signaling pathway of apoptosis and in the cell’s commitment to death. It affects the outer and subsequently the inner mitochondrial membranes through the opening of membrane pores via mechanisms that are not fully clear. This releases cytochrome c and SMAC into the cytosol, leading to the activation of caspases, a set of proteases that accelerate cell death and that block inflammation by inactivating intracellular innate immunity effectors. Mitochondrial inner membrane permeabilization additionally releases mtDNA into the cytosol, which in turn activates intracellular innate immunity pathways unless counterbalanced by caspases. Here, I will discuss our work using advanced microscopy methods to advance our understanding of the molecular mechanisms of mitochondrial pores in apoptosis and how they function in the regulation of cell death and of inflammatory signaling outcomes.

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Tools for measuring the compositional and biophysical paralipidome of cell membrane proteins

Ilya Levental

University of Virginia, Levental Lab of Membrane Biology, 1340 Jefferson Park Avenue, Charlottesville, VA 22903, USA, il2sy@virginia.edu

Membrane proteins constitute >30% of the mammalian proteome and 60% of all drug targets, localizing a major fraction of cellular bioactivity at membrane interfaces. Such proteins are potentially solvated by hundreds of distinct lipid subtypes with a range of physicochemical properties that can influence protein activity. Proteins have been hypothesized to selectively recruit local lipid environments, termed paralipidomes, that are chemically and physically distinct from the bulk membrane. While bulk membrane compositions and properties have been widely measured, the local lipid environments solvating membrane proteins have rarely been measured. To address this gap, we have developed tools for measuring the compositional and biophysical paralipidomes in live cells. For biophysical paralipidomes, we deployed HaloTags to covalently modify membrane proteins with environment-sensitive probes and thereby reveal the biophysical properties of various protein native paralipidomes in live cells. We use this modular approach to reveal asymmetry in local lipid packing between the inner and outer plasma membrane leaflets and how they are affected by lipid perturbations. We also observe distinct physical environments surrounding raft and non-raft protein markers, representing direct measurements of nanoscopic raft domains in live cells. For compositional paralipidomes, proteins are isolated with their native surrounding lipidomes using copolymeric nanodiscs, which are then analyzed by mass spectrometry. This approach reveals enrichment of specific lipid subtypes around GPCRs versus single-pass signaling adaptors. Thus, a modular toolbox enables straightforward measurements of local lipid nano-environments around membrane proteins and reveals layers of membrane organization in living cells.

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Biomolecular condensates as membrane sculptors

Rumania Dimova

Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany, rumiana.dimova@mpikg.mpg.de

Biomolecular condensates are important organizers of cellular biochemistry, yet their interactions with membranes remain underexplored. Because cells contain many membrane-bound compartments, encounters between condensates and membranes are unavoidable and can strongly impact cellular structure and function. Using giant unilamellar vesicles (10–100 µm) as a minimal model system, we examine how condensates adhere to, deform, and remodel lipid membranes [1–3]. We find that condensates can induce pronounced membrane shape changes [4], reorganize lipid distributions [5], initiate endocytosis-like events [6], and repair damaged membranes by sealing pores [7]. Membranes, in turn, actively regulate these interactions: lipid composition and packing strongly influence condensate affinity and wetting behavior [8]. Notably, we show that membrane affinity is not controlled by condensate hydrophobicity alone, but by the dielectric permittivity contrast between the condensate and the surrounding dilute phase [9]. Together, these results demonstrate how fundamental physicochemical principles, independent of active cellular processes or scaffolding proteins, can drive membrane remodeling and organization, and provide design rules for engineering synthetic cellular systems.

1. Mangiarotti & Dimova, Annu. Rev. Biophys. 53:319, 2024.
2. Dimova & Lipowsky, Adv. Mater. Interfaces 4:1600451, 2017.
3. Liu et al., Front. Chem. 7:213, 2019.
4. Mangiarotti et al., Nature Commun. 14:2809, 2023.
5. Mangiarotti et al., Nature Commun. 14:6081, 2023.
6. Mangiarotti, Aleksanyan et al., Adv. Sci. 11:2309864, 2024.
7. Bussi et al., Nature 623:1062, 2023.
8. Mangiarotti et al., Nature Commun. 16:2756, 2025.
9. Sabri et al., bioRxiv, doi:10.1101/2025.03.09.642144 (2025).

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Exploring the biochemical and biophysical characteristics of archaeal membranes

Presenting author: Kaja Grewe

University Regensburg, Microbiology & Archaea Centre, Universitätsstraße 31, 93053 Regensburg [DE], kaja.grewe@ur.de

Author(s):
Kaja Grewe, Henry Zivkovic, Shachar Gat, Robert Reichelt, Thorsten Bauersachs, Anne Bernheim, Petra Schwille, Dina Grohmann

Ether lipids found in archaeal membranes differ fundamentally in their biochemistry from ester lipids that constitute both eukaryotic and bacterial membranes. The unique membrane lipids of Archaea, especially the membrane-spanning tetraether lipids, are considered to be key for survival of hyperthermophilic archaea living in habitats with extreme physico-chemical parameters. Currently, the details of archaeal lipid biochemistry are poorly understood and no established model system for archaeal membranes exist.

In this study, we prepared lipid extracts from four hyperthermophilic archaea and established the formation of giant unilamellar vesicles (GUVs) to serve as an in vitro model membrane system. Additionally, we determined the lipid composition of the four archaeal lipid extracts, revealing the highly different lipid composition for each strain. The lipid composition influenced biophysical parameters like the membrane fluidity. Interestingly, archaeal membranes show phase transitions distinct from bacterial membranes. Finally, we discovered a tendency for archaeal lipids to assemble into tubular and multilamellar structures, suggesting the presence of complex lipid phases and domains within archaeal membranes both in vitro and in vivo. Taken together, we this study provides the first in-depth biophysical characterisation of archaeal membranes laying the groundwork to elucidate membrane-protein interactions in archaea.

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Bayer Pharmaceuticals PhD Award
Title to be announced

N.N.

Abstract not submitted yet

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GBM PhD Award
Title to be announced

N.N.

Abstract not submitted yet

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Feodor Lynen Lecture:
Lipid packing defects: a journey with a fuzzy but insightful concept in molecular membrane biology

Bruno Antonny

Institute of Molecular and Cellular Pharmacology, 660, route des Lucioles, Sophia Antipolis, 6560 Valbonne, France, antonny@ipmc.cnrs.fr

In this plenary presentation, I will evoke key milestones of the work of my team and of other colleagues over the past twenty years. The recognition of imperfections in the packing of membrane lipids has been the common thread of this research. Such recognition by specific protein motifs enables the spatiotemporal organisation of numerous reactions at the surface of cellular organelles.

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From lipid flip-flop to membrane nanopores and protein insertion

Gerhard Hummer

Max Planck Institute of Biophysics, Theoretical Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany, Gerhard.Hummer@biophys.mpg.de

By combining molecular dynamics (MD) simulations with artificial intelligence (AI) tools, we overcome the inherent time scale limitations in studying biomolecular processes and extract mechanistic insights in real time. These AI-guided MD simulations allowed us to capture the assembly of transmembrane helices of an ER calcium sensor with atomic resolutions, identify the transition states of the assembly process, and link them to distinct assembly states. In addition, we could extensively sample exceedingly rare lipid flip-flop events across lipid bilayer leaflets. We identified multiple distinct mechanisms of lipid flip-flop, relating them to lipid type and membrane characteristics. Notably, the spontaneous formation of transient nanopores is of particular interest, as it facilitates the translocation of lipids with large polar headgroups, as well as other polar or charged molecules.  In connection with nanoporation, I will highlight our ongoing work on protein insertion.

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Morphogenesis of complex membrane structures: Endoplasmic Reticulum stacks and whorls

Presenting author: Natalie Friemel

Heidelberg University Biochemistry Center, , Im Neuenheimer Feld 328, 69120 Heidelberg [DE], natalie.friemel@bzh.uni-heidelberg.de

Author(s):
Natalie Friemel, Sebastian Schuck

Cells dynamically remodel the morphology of their organelles to optimise organelle function. The endoplasmic reticulum (ER) membrane can be shaped into arrays of sheets that form flat stacks or spherical whorls. ER stacks and whorls can have distinctive protein compositions and may support protein maturation, drug detoxification or autophagy. The molecular machinery for the formation of these ER subdomains and their precise membrane topology remain unknown. Here, we employ subdomains induced by the ER membrane protein Hmg2 as a model for ER morphogenesis in yeast. We combine a genetic screen, spatial proteomics and cryo-electron tomography to identify factors in stack and whorl formation and define their three-dimensional architecture. We find that the transition from flat sheets to spherical ER structures depends on membrane abundance and rim stabilisation by reticulon proteins. Additionally, certain lumenal and membrane-associated ER proteins are excluded from stacks and whorls, suggesting a protein sorting mechanism during ER subdomain formation. This selectivity may be based on protein size because stacked smooth ER sheets are about 40% thinner than rough ER sheets and thus offer less lumenal space. Overall, understanding the molecular mechanisms of ER morphogenesis will help to further define the relationship between organelle form and function.

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Mechanisms of Cellular LIpid Homeostasis: From Lipid Droplets to Ferroptosis

James Olzmann

University of California, Berkeley, Departments of Molecular & Cell Biology and Nutritional Sciences & Toxicology, 1951 Oxford Street, Berkeley, CA 94720-3104, USA, olzmann@berkeley.edu

Abstract not submitted yet

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Rab GTPase phosphorylation in Parkinson’s Disease

Suzanne Pfeffer

Stanford University, Department of Biochemistry, Beckman Center, Room B413, 279 Campus Drive, Stanford, CA 94305-5307, USA, pfeffer@stanford.edu

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of Rab GTPases and activating mutations in LRRK2 are a common cause of Parkinson’s. Loss of function mutations in glucocerebrosidase (GBA1) are also linked to this disease.  We have shown that both LRRK2 pathway and GBA1 mutations may cause Parkinson's by a convergent pathway--loss of Hedgehog signaling. Remarkably, treatment of mice with a LRRK2 inhibitor restores cilia and rescues neuroprotective factor production, which is great news for patients.

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Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes

Presenting author: Wenxin Zhang

Max Planck Institute of Biophysics, Mechanisms of Cellular Quality Control, Max-von-Laue-Straße 3, 60438 Frankfurt am Main [DE], wenxin.zhang@biophys.mpg.de

Author(s):
Wenxin Zhang, Delong Li, Florian Wilfling

Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. In vitro reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.

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Protecting Membranes from Stress by Very Long-Chain Fatty Acids

Florian Fröhlich

University of Osnabrück, Bioanalytical Chemistry, Barbarastraße 11, 49076 Osnabrück, Germany, florian.froehlich@uni-osnabrueck.de

Abstract not submitted yet

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Uncovering the private lives of lipids with new chemogenetic imaging approaches

Itay Budin

University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA, ibudin@ucsd.edu

Cell membranes are composed of hundreds of different lipid species that are distributed across organelles in a highly anisotropic manner. This heterogeneity is now thought to be driven primarily by protein machinery, including cytosolic lipid transfer proteins that act at organelle contact sites and flippases/scramblases that dictate transbilayer distributions. A long-standing challenge in cell biology has been to quantitatively image lipid pools at the length scale relevant for investigating these transport processes. In this talk, I will focus on new chemical biology approaches our lab has developed to map phospholipids in cells and determine factors that control their trafficking and transbilayer distributions. A key advance has been the use of fluorogenic dyes for biorthogonal labeling reactions that allow compartment and leaflet-specific detection of phospholipid pools. These tools can be used to test the function of lipid transfer proteins and the mechanisms underlying membrane asymmetry in cells.

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Decoding membrane dynamics with quantitative and super-resolution microscopy

Francesca Bottanelli

Freie Universität Berlin, Membrane Trafficking, Thielallee 63, 14195 Berlin, Germany, francesca.bottanelli@fu-berlin.de

In the laboratory, we combine endogenous tagging with live-cell super-resolution STED microscopy to follow dynamic processes at the nanoscale and in their own unperturbed physiological cellular environment (Bottanelli et al., 2016, 2017, Wong-Dilworth et al., 2023). We have implemented a pipeline for the rapid generation of knock ins and generated a library of over 150 tagged proteins involved in membrane trafficking (Adarska et al., 2025), which can be used for dynamic nanoscale microscopy and for high sensitivity proximity biotinylation experiments (Stockhammer et al., 2024). The ability to explore cellular dynamics of an extensive collection of trafficking machinery at sub 50 nm resolution in an unperturbed cellular environment is revealing novel and unexplored sorting mechanisms. Our primary biological questions focus on the mechanisms underlying endosomal organelle biogenesis (Stockhmmer, Adarska et al., 2024) and on membrane reorganization processes that support immune function (Rodilla-Ramirez et al., 2025).

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Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH

Presenting author: Pinki Gahlot

Universität Duisburg-Essen, Fakultät für Biologie, Universitätstraße 5, 45141 Essen [DE], pinki.gahlot@uni-due.de

Author(s):
Pinki Gahlot, Bojana Kravic, Giulia Rota, Johannes van den Boom, Sophie Levantovsky, Nina Schulze, Christian Behrends, Hemmo Meyer

Lysosomes are the main degradative organelles in the cell and serve as platforms for important signalling pathways. The loss of lysosomal function due to lysosomal membrane permeabilization (LMP) can be highly deleterious for the cell. LMP can be caused by different conditions such as lipid peroxidation, lysosomotropic drugs or disease-associated changes in lipid composition. Cells respond to LMP by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy. However, it is not fully understood how the decision between repair and lysophagy of damaged lysosomes is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. These lipid-packing defects are sensed via amphipathic helices in SPG20. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

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Structural characterization of a novel bacterial ESCRT-III protein in E. coli

Presenting author: Anja Heddier

Forschungszentraum Jülich GmbH, ERC-3 Structure Biology, Wilhelm-Johnen-Straße, 52425 Jülich [DE], a.heddier@fz-juelich.de

Author(s):
Anja Heddier, Benedikt Junglas, Ilona Ritter, Carsten Sachse

The structures and functions of endosomal sorting complexes required for transport (ESCRT)-III proteins in bacteria, such as PspA and Vipp1, have been intensively studied in recent years. In E. coli, an additional potential PspA-homolog, YjfJ, has been recently identified while a biochemical, structural and functional characterization is entirely lacking. Here we show that YjfJ from Escherichia coli is a bona fide member of bacterial ESCRT-III proteins. Using cryo-EM, we solved eight structures of YjfJ helical filaments to a resolution of 3.0 to 4.0 Å as well as two structures of YjfJ in presence of membranes at 4.4 and 6.0 Å. In these structures YjfJ monomers adopt the typical ESCRT-III fold as well as extensive plasticity typical for bacterial ESCRT-III proteins. Our data also revealed that apo state YjfJ polymers are very similar to apo state PspA polymers, whereas they change to Vipp1-like polymers upon lipid reconstitution. Preliminary functional data suggest that YjfJ and PspA may exhibit complementary functions in E. coli, where YjfJ may play a critical role in maintaining the membrane fluidity under cold (stress) conditions. The characterization of YjfJ will help to understand the evolution of ESCRT-III proteins in bacteria as well as membrane remodeling in E. coli.

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Otto Meyerhof Prize
IRGQ-Mediated Autophagy in MHC-I Quality Control Drives Tumor Immune Evasion

Lina Herhaus^1,9,10*, Uxía Gestal-Mato^1,9, Vinay V. Eapen^2,3, Igor Mačinković^1,4, Henry J. Bailey^1,5, Cristian Prieto-Garcia¹, Mohit Misra^1,5, Anne-Claire Jacomin¹, Aparna Viswanathan Ammanath¹, Ivan Bagarić¹, Jolina Michaelis¹, Joshua Vollrath^1,5,6, Ramachandra M. Bhaskara^1,5, Georg Bündgen⁷, Adriana Covarrubias-Pinto¹, Koraljka Husnjak¹, Jonathan Zöller⁶, Ajami Gikandi², Sara Ribičić¹, Tobias Bopp⁷, Gerbrand J. van der Heden van Noort⁸, Julian D. Langer⁶, Andreas Weigert⁴, J. Wade Harper³, Joseph D. Mancias², Ivan Dikic ^1,5,6,*

1 Institute of Biochemistry II, Goethe University Frankfurt, Medical Faculty, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
2 Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Institutes of Medicine, 450 Brookline Avenue, Boston, MA 02215
3 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115
4 Institute of Biochemistry I, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
5 Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Riedberg Campus, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany
6 Max Planck Institute of Biophysics, Goethe University Frankfurt, Riedberg Campus, 60438 Frankfurt am Main, Germany
7 Institute for Immunology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany
8 Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
9 These authors contributed equally
10 current address: HZI - Helmholtz Centre for Infection Research, Cellular Immune Signals, Inhoffenstraße 7, 38124 Braunschweig, Germany
*Shared senior authorship, correspondence should be addressed to: dikic@biochem2.uni-frankfurt.de; lina.herhaus@gmail.com

The autophagy-lysosome system directs the degradation of a wide variety of cargo and is also involved in tumor progression. Here we show that immunity-related GTPase family Q protein (IRGQ), an uncharacterized protein to date, acts in the quality control of MHC-I molecules. IRGQ directs misfolded MHC-I towards lysosomal degradation through its unique binding mode to GABARAPL2 and LC3B. In the absence of IRGQ, free MHC-I heavy chains do not only accumulate in the cell, but are also transported to the cell surface, thereby promoting an immune response. Mice and human patients suffering from hepatocellular carcinoma show improved survival rates with reduced IRGQ levels, due to increased reactivity of CD8+ T cells towards IRGQ knock-out tumor cells. Thus, we reveal IRGQ as a novel regulator of MHC-I quality control, mediating tumor immune evasion.

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Otto Warburg Medal
Targeting, Tethering, and Talking: Membranes Beyond Barriers

Maya Schuldiner

Weizmann Institute of Science, Dept. of Molecular Genetics, Meyer Bldg. Room 122, 7610001 Rehovot, Israel, maya.schuldiner@weizmann.ac.il

Organelles are the fundamental building blocks of eukaryotic life, providing specialized environments for biochemical reactions, signaling, and cellular homeostasis. Despite decades of research, many of the proteins that shape organelle structure, regulate their function, and maintain their dynamic balance remain uncharacterized. Functional genomics offers a transformative approach to this challenge by systematically linking genes to organelle biology through unbiased, large-scale analyses. By combining genetic perturbations with imaging, proteomics, and metabolic profiling, functional genomics enables the discovery of novel organelle factors and the delineation of pathways underlying their biogenesis, communication, and turnover. In my talk, I will discuss how we have utilized high-content screens to uncover new concepts in organelle function, reshaping our ability to chart the molecular landscape of organelles and providing critical insights into their integration within the cell and their impact on health and disease.

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Molecular architecture of lipid metabolism: insights through the lens of mass spectrometry

Maria Fedorova

Dresden University of Technology, Center for Membrane Biochemistry and Lipid Research, Fetscherstraße 74, 01307 Dresden, Germany, maria.fedorova@tu-dresden.de

Abstract not submitted yet

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High-throughput CGMD simulations enable proteome-wide identification of lipid scramblases in yeast

Presenting author: Cristian Rocha Roa

Fribourg University, Fribourg, Switzerland, Department of Biology, Chemin du Musée 10, 1700 Fribourg [CH], cristian.rocharoa@unifr.ch

Author(s):
Cristian Rocha Roa, Stefano Vanni

Membrane proteins constitute nearly a quarter of the proteins encoded in most organisms, representing approximately 50% of the cell membrane mass. Therefore, lipid-protein interactions are crucial for many cellular processes, and lipid transport between organelles is essential for cellular homeostasis. It has been demonstrated that lipid transport proteins team-up with lipid scramblase proteins. This protein-protein complex is proposed to be required for processes such as membrane expansion, e.g. autophagosome biogenesis. Using coarse-grained molecular dynamics simulations of predicted structures from AlphaFold2 for the yeast S. cerevisiae, we have identified several previously unknown putative lipid scramblase proteins. As a consequence of the presence of these new putative scramblases, our results suggest that cell membranes are more dynamic than previously thought. They provide an initial framework for understanding how the cell regulates its lipid membrane and open the door to new interpretations of previous gaps in lipid-mediated processes.

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Biophysical mechanisms of cholesterol synthesis and storage

Rachid Thiam

ENS-PSL, Laboratoire de physique de l’ENS, 24, rue Lhomond, 75231 Paris, France, thiam@phys.ens.fr

Cells store excess cholesterol as esters within lipid droplets, yet these lipids can form highly ordered phases. How such an order is accommodated within dynamic organelles remains unclear. We show that triglycerides are key to maintaining fluidity, allowing cholesterol ester incorporation and influencing the phase behavior of lipid droplet organelles. Instead of protein scaffolds alone, the balance and sequence of neutral lipid deposition, set at the endoplasmic reticulum, determine whether droplets stay fluid, enter mixed states, or become locked in liquid-crystalline phases. These ordered states prevent droplet ripening and reorganize the protein landscape at the organelles. In my presentation, I will share our recent advances in understanding the mechanisms of cholesterol synthesis and storage.

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Developing super-resolution microscopy to reveal sub-cellular complexity

Jörg Bewersdorf

Yale School of Medicine, Cell Biology, 333 Cedar Street, New Haven, CT 06520-8002, USA, joerg.bewersdorf@yale.edu

Abstract not submitted yet

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Time for Lipid Cell Biology -
Combining lipid imaging and proteomics for mechanistic discovery

André Nadler

Max Planck Institute of Molecular Cell Biology and Genetics, Membrane Chemical Biology, Pfotenhauerstraße 108, 01307 Dresden, Germany, nadler@mpi-cbg.de

Abstract not submitted yet

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Molecular mechanism of ceramide transfer by STARD11 / CERT

Molecular mechanism of ceramide transfer by STARD11 / CERT : an in vitro study

Presenting author: Camille Cuveillier

University of Geneva, Physiology and metabolism, 1 rue Michel Servet, 1206 Geneva [CH], camille.cuveillier@unige.ch

Author(s):
Camille Cuveillier, Mahmoud Moqadam, Nathalie Reuter, Anne-Claude Gavin

Non-vesicular lipid transport by lipid transfer proteins (LTPs) is crucial for establishing and maintaining membrane lipid composition. LTPs shield lipids from the aqueous environment and mediate their uptake and release at membrane surfaces. These opposing steps are energetically demanding, as they require moving lipids across the hydrophilic headgroup region without external energy input. Taking STARD11 lipid transfer domain as a model, we combined molecular dynamic simulation and in vitro lipid transfer assays to uncover the central role of a conserved arginine in Ω1 loop. This residue provides structure to the gate of the lipid-binding cavity via an H-bond network and fine-tune the opening of the gate by interacting with membrane phospholipid phosphate groups leading to fast ceramide uptake. Finally, using bifunctional lipids, we show that the opening of the protein modifies membrane properties by promoting lipid fatty acid snorkeling. Overall, our findings reveal a bidirectional coupling in which the conserved Ω1-loop arginine enables STARD11 to reshape local membrane structure while membrane phospholipids reciprocally stabilize gate opening, together driving efficient lipid transport.
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Molecular Mechanism of Ceramide Transfer by STARD11 / CERT: A Molecular Dynamics Investigation

Presenting author: Mahmoud Moqadam

University of Bergen, Chemistry, Bregnestien 8B, 5141 Fyllingsdalen [NO], mahmoud.moqadam@uib.no

Author(s):
Mahmoud Moqadam, Camille Cuveillier, Anne-Claude Gavin, Nathalie Reuter

Non-vesicular lipid transport by lipid transfer proteins (LTPs) is crucial for regulating cellular lipid distribution and maintaining membrane composition. A defining feature of all LTPs is their ability to shield lipids within a hydrophobic cavity, reducing the energetic cost of transfer relative to the aqueous environment. A fundamental yet unresolved question is the mechanism by which LTPs coordinate membrane binding and gate opening with lipid uptake and release. Using the ceramide transporter STARD11 as a model system, we performed molecular dynamics simulations to dissect the membrane-dependent gating mechanism. We show that an arginine located in the Ω1 loop and conserved in 14 of the 15 human START domains plays a critical dual role: it maintains the structural integrity of the gate, and strongly interacts with a membrane phospholipid that promotes and sustains gate opening. This opening disrupts local membrane lipid packing and increases lipid tail snorkeling toward the open cavity. Using in vitro lipid transfer assays of the wild-type STARD11 and mutants, we show that mutating the arginine decreases the rate of ceramide uptake. Overall, our findings reveal a functional role for conserved arginine and highlight a mechanistic coupling between membrane lipid dynamics and cavity accessibility that together drives efficient lipid transport by the START domain protein.

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Membrane editing and proximity proteomics reveals regulators of lipid homeostasis

Jeremy Baskin

Cornell University, Dept. Of Chemistry & Chemical Biology, 122 Baker Laboratory, Ithaca, NY 14853, USA, jeremy.baskin@cornell.edu

Cellular lipid metabolism is subject to strong homeostatic regulation, but players involved in and mechanisms underlying these pathways remain mostly uncharacterized. I will describe a “Feeding–Fishing” approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics via proximity labeling (fishing) to elucidate molecular players and pathways involved in homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. By performing proximity biotinylation using a membrane-tethered TurboID alongside membrane editing to selectively deliver PA to the same membrane, we identified numerous PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Subsequent mechanistic analysis revealed that PA homeostasis in the cytosolic leaflets of the plasma membrane and lysosomes is mediated by both local PA metabolism and the action of members of the lipid transfer protein families that carry out interorganelle lipid transport prior to additional metabolic steps. More broadly, the interfacing of membrane editing with organelle membrane proteomics using proximity labeling represents a strategy for revealing mechanisms governing lipid homeostasis.

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