The protein architecture and mechanical properties of the clathrin-mediated endocytic machinery

• Applicant: Dr. Michal Skruzny, Ph.D.
• Subject Area: Biophysics ; Cell Biology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323495273

Clathrin-mediated endocytosis is the main vesicle trafficking route from the plasma membrane to the cytoplasm with an essential role in many cellular activities. It is required for nutrient uptake, cellular homeostasis and signaling during growth and development. Accordingly, it represents a primary pathway to study processes of membrane reshaping and trafficking, fundamental traits of all eukaryotic cells.During endocytosis a small area of the plasma membrane folds inward to form an endocytic vesicle. Reshaping of the plasma membrane requires mechanical force, which is provided by concerted action of many endocytic proteins sequentially assembled beneath the membrane. Although the timeline of their assembly is known, their functional arrangement at the endocytic site is poorly understood. Such information is however critical to understand a key step of the endocytic process: the force-dependent remodeling of the plasma membrane during vesicle budding.An analysis of the protein architecture of the endocytic site is not an easy task given that multiple copies of ~50 proteins assemble at this diffraction-limited spot for a short time. Electron and fluorescence microscopy provided recently some insights into its organization, being however only partially successful due to its limited spatiotemporal resolution. Notably, protein densities at the endocytic site seem to be well suited for mapping its organization by Förster/fluorescence resonance energy transfer (FRET). FRET, a powerful tool to detect proximities between fluorescently labeled proteins separated by less then 10 nm, has been successfully applied to many molecular cell biology problems. Importantly, our preliminary results show that it can be used to map the protein organization of the endocytic site.To analyze the functional architecture of the endocytic machinery we will perform a systematic FRET-based proximity screen using the yeast endocytic model. We will tag key endocytic proteins by GFP/RFP and assess their proximities in the native endocytic site by FRET. Next, we will follow potential rearrangements of the machinery during vesicle budding by tracking specific proximal protein pairs. Finally, we will introduce advanced FRET biosensors to study how endocytic proteins apply and transmit mechanical force for membrane invagination and scission. By these sensors we will analyze role of individual membrane-reshaping factors (clathrin, actin, BAR proteins etc.), plasma membrane or environmental cues to successful vesicle formation.The implementation of high-resolution live-cell imaging and the state-of-the-art force biosensors should allow us to draw a comprehensive picture of dynamic endocytic machinery and shed light on its key mechanical properties. Such information will be of great interest to many fields of current biomedical research and its applied pharmaceutical and bioengineering areas.

• DFG Programme: Research Grants