A new approach to reconstruct dynamics from static super-resolution images to study clathrin-mediated endocytosis

Philipp Hoess1, Yu-Le Wu1, Markus Mund2, Joran Deschamps1, Marko Kaksonen2, Jonas Ries1

1 EMBL Heidelberg, Cell Biology & Biophysics, Meyerhofstr. 1, 69117 Heidelberg, Germany
2 University of Geneva, Department of Biochemistry

As one of the super-resolution techniques, single-molecule localization microscopy (SMLM) has enabled new biological discoveries in the last decade. Highest spatial resolution in SMLM requires long acquisitions that typically last minutes to hours. Thus, it is required to chemically fix the sample so that the structure of interest is static and does not change during the acquisition.

However, many biological processes are highly dynamic and it is difficult to study them based on static super-resolution images. We aim to fill this gap by developing an approach to reconstruct dynamic information from individual snapshots. We acquire dual-color SMLM images and first align them in space and time using one color as reference structure. We then infer the structural distribution of a protein of interest from the second color channel. The approach includes high-throughput SMLM by automated microscopy and subsequent segmentation, alignment and averaging.

We apply this concept to study the spatial distribution of different proteins involved in clathrin-mediated endocytosis (CME) in budding yeast. This process is particularly well-suited to demonstrate our approach because it is well-known from conventional microscopy of live cells that the individual endocytic events progress with high spatial and temporal regularity. The dynamic reconstruction of the rearrangement of key players in this machinery will help us to understand their mechanistic function in CME.

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