"The plasma membrane represents the interface between cells and their neighborhood, mediating diverse processes such as cell–cell communication, cell–matrix interactions and uptake of nutrients. As a result, clathrin mediated endocytosis (CME) has a central role in maintaining cellular homeostasis and function and mis-regulation of endocytic traffic is linked to many human diseases. Yet, the mechanisms regulating CME remain poorly understood. We aim to define the role(s) of the adaptor protein-2 (AP2) in regulating CME. Using state-of-the-art molecular cell biology including siRNA knockdown and reconstitution through bicistronic retroviral mediated transfections, as well as genome editing we will generate stable cell lines expressing WT and functionally defined AP2 mutants. These will be analyzed by live cell total internal reflection fluorescence microscopy, in combination with sophisticated particle tracking algorithms and mathematical analyses to measure effects on discrete early stages of CME. We will determine how PI4,5P2, cargo, clathrin and endocytic accessory factor binding to AP2, as well as phosphorylation events trigger conformational changes in AP2 to regulate initial events in clathrin coated pit (CCP) assembly, stabilization of nascent CCPs and their maturation. Together these studies will test the hypothesis that allosteric conformational changes in AP2 play a central role in regulating early, critical events in CME. These interdisciplinary studies benefit from combining and transferring the most up-to-date experimental techniques and expertise in molecular cell biology, structural biology, microscopy, and mathematics. The collaboration between outgoing and European host ensures the expertise necessary to accomplish this complex goal. Most importantly it gives an opportunity to the applicant for training and career development in leading research institutions under the guidance of mentors who are world experts in their respective fields."
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