Quantitative analysis of the dynamics of protein-complex formation during clathrin-mediated endocytosis

• Applicant: Professor Dr. Marko Kaksonen
• Subject Area: Cell Biology
• Term: from 2008 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 85747477

Clathrin-mediated endocytosis (CME) is a major cellular trafficking pathway that is conserved throug-hout eukaryotes. CME is involved in a wide variety of biological processes including nutrient uptake, polarization of cells and neural signaling. In budding yeast, the molecular machinery driving CME is composed of up to 50 different proteins. During the course of an endocytotic event, components of this machinery dynamically assemble at the plasma membrane, with different proteins arriving and leaving the endocytic site in a precisely orchestrated sequence. The complex network of regulated protein-protein and protein-lipid interactions that underlies these processes, however, is not well understood. To obtain a better mechanistic understanding of endocytosis, quantitative data of the abundance of individual proteins in the cytoplasm and at the vesicles, as well as the localization and regulation of protein-complex formation is needed. This data has to be correlated quantitatively with the different steps of vesicle formation. Our recent developments in fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in combination with FCS calibrated quantitative image analysis of dynamic processes has opened venues that allow the acquisition of such quantitative data that report on the dynamics of protein-protein interactions in living cells. We will apply these methods in combination with genetic approaches in budding yeast to study quantitatively the dynamics of protein complex formation in order to increase our understanding of the mechanisms of clathrin-mediated endocytosis.

• DFG Programme: Research Grants
• Participating Person: Professor Dr. Michael Knop