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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CellStructure (Structural cell biology in situ using superresolution microscopy)

Teaser

Summary

The overall aim of CellStructure is to establish optical superresolution microscopy as a technique to determine the structural organization of multi-protein assemblies in their cellular and functional contexts. To this end, we are developing innovative optical, biological and computational imaging tools. We are applying them to investigate key questions on endocytosis in yeast, which is paradigmatic for many supramolecular machines regarding molecular organization, assembly and functional conformational dynamics. These tools will narrow the methodological gap that currently exists between cell biology and structural biology, and pave the way for answering biological questions not accessible by any other technique.

Work performed

To extend the application of superresolution microscopy to questions in structural biology, we need the highest 3D resolution. Thus, we optimized a method developed in the lab, called â€œSupercritical Angle Localization Microscopyâ€ to substantially improve its 3D resolution. Additionally, we developed software to greatly increase the resolution in standard 3D single-molecule localization microscopy (SMLM) (Li et al, Nature Methods, 2018). Another prerequisite is the ability to count the number of proteins in a complex. To this end we developed an accurate counting technique based on nuclear pores as robust reference standards. Using our technologies, we could address long-standing questions in the field of endocytosis. We automated our superresolution microscopes and imaged >100 000 endocytic structures, from which we could determine the average structural organization of 23 endocytic proteins (Mund et al, Cell, 2018). Additionally, we developed a timing approach based on correlative superresolution and diffraction-limited microscopy to obtain dynamic information. In collaboration, we developed a computational model of endocytosis. Based on these results, we could propose a model of how the actin machinery provides the force to invaginate the membrane.

Final results

By developing SALM and improved fitting algorithms, we could improve the z resolution of SMLM compared to the state of the art, opening the possibility of addressing structural biology questions with superresolution microscopy. We expect a fully optimized version of SALM to have an even twofold better resolution. Compared to currently used methods in SMLM, our approach to count protein copy numbers is very robust and with a relative error of approx. 10% very accurate. Our microscope was one of the first SMLM microscopes to be completely automated to acquire data around the clock without supervision, which enabled us to obtain data on the endocytic machinery in yeast with unprecedented size and scope. This substantially increased our understanding of the endocytic process. Currently we are working on reconstructing the dynamic architecture of the yeast endocytic machinery by including temporal information. In the future we will extend this approach to include 3D positions and hope that by the end of the project we will have a superresolution localization map of >20 endocytic proteins with sub-second temporal and ~20 nm 3D spatial resolution.