%0 Generic
%A Höß, Philipp
%C Heidelberg
%D 2022
%F heidok:29646
%R 10.11588/heidok.00029646
%T Towards a dynamic reconstruction of the endocytic machinery by dual-color localization microscopy
%U https://archiv.ub.uni-heidelberg.de/volltextserver/29646/
%X During the process of clathrin-mediated endocytosis (CME), an invagination of the plasma membrane is formed which finally gets pinched off to form an intracellular vesicle. CME is essential for cellular function as it is important for the uptake of nutrients, regulation of signaling, and membrane homeostasis. The machinery mediating CME consists of more than 50 different proteins which are conserved from yeast to mammals. Dynamic high-resolution studies of CME remain challenging due to the small scale (≈ 250 nm) and the fast sequence (≈ 20 s) of the process. This leaves the underlying mechanisms by which spatial rearrangements of endocytic components drive membrane invagination poorly understood.    In this work, we established a new approach to dynamically reconstruct the mobile phase of CME in the budding yeast Saccharomyces cerevisiae at the nanoscale from static super-resolution snapshots. The approach is based on dual-color single-molecule localization microscopy (SMLM) of hundreds of endocytic sites that have been fixed at random points along the regular timeline of endocytosis. We introduced a reference structure composed of two endocytic proteins to sort the sites temporally and align them spatially by fitting a geometric model. Imaging of this reference structure in the first channel alongside an endocytic (query) protein in the second channel allowed us to reconstruct the temporal rearrangements of the query protein.    To obtain large datasets with high quality, we increased the throughput of dual-color SMLM by testing different imaging buffers. Sealing of the sample holder turned out to be crucial to maintain good imaging conditions. Furthermore, we developed a computational pipeline to automatically segment and coarsely align endocytic sites from high-throughput datasets. The segmented sites were then fitted with a geometric model based on previous knowledge from super-resolution and electron microscopy studies. This fitting process helped us to retrieve parameters from the SMLM data which were used for spatial and temporal alignment. Importantly, the fitting is only performed on the reference channel, imposing no structural assumptions on the query protein.    We first validated that the reference structure can be used to temporally sort endocytic sites and then successfully demonstrated that our approach can faithfully recover the structural rearrangements of four proteins exemplary for different endocytic modules. Future progress will enable us to integrate multiple datasets into a single dynamic reconstruction of yeast CME with multiple query proteins.