UC San Diego

Clathrin-mediated endocytosis (CME) constitutes the major route for selective cargo internalization in higher eukaryotes, and as such is also central to cellular homeostasis. This process involves the concerted action of dozens of different proteins over the span of a minute, yet little is known about how it is regulated both temporally and spatially. In this dissertation we develop and use novel analysis methods to study both of these aspects of the regulation of CME. We first adapt the fluorescence fluctuation based Number & Brightness (N&B) analysis to study the GTPase-dependent regulation of the endocytic checkpoint by dynamin2. We find that this analysis is capable of measuring the relative brightness of particles in vivo and with single molecule sensitivity. We find that background fluorescence, unless corrected for, impedes this analysis from measuring absolute brightness values. Finally, we apply this analysis to study the recruitment of dynamin2 wild-type, as well as two dynamin2 mutants deficient in basal GTPase activity. In order to study the spatial regulation of CME, we spatially correlate sites of CME nucleation observed in live cells by florescence microscopy. We find that CME is spatially organized into hotspots that can effectively retain nucleation resources. Based on the effects that knock down of various CME accessory factors have on the organization of CME, we distinguish two classes of accessory proteins: nucleation factors and nucleation organizers. Finally, we observe that clustering of transferrin receptors spatially randomizes pit nucleation and thus reduces the role of hotspots. On the basis of these data, we propose that hotspots are specialized cortical actin patches that organize CCP nucleations from within the cell by more efficient recruitment and/or retention of the resources required for CCP nucleation partially due to the action of nucleation organizers