Until recently, the actin cytoskeleton and the endocytic machinery were thought to operate independently. However, there is growing evidence in diverse eukaryotes that F-actin and the Arp2/3 complex play a direct role in endocytic internalization. Real-time image analysis of GFP-tagged proteins defined a molecular pathway for receptor-mediated endocytosis in budding yeast, creating unprecedented opportunities to develop a comprehensive understanding of this complex process using tools uniquely available in yeast. Features of the pathway are conserved widely, indicating that principles derived from yeast studies will apply to humans where endocytic defects can contribute to cancer and heart disease. The proposed studies will increase understanding of the role of actin in endocytosis, and how the order and timing of events in the endocytic pathway are achieved. Genomics-scale surveys will fuel focused, hypothesis-driven inquiries. Real-time image analysis using conventional wide-field and TIR fluorescence microscopy will be used to study the dynamics and order of appearance at endocytic sites of at least 60 GFP-tagged endocytic proteins, resulting in a comprehensive description of when each protein arrives at an endocytic site, and what specific motile behaviors of the endocytic complex are associated with the appearance of each protein. Chemical genetics, mass spectrometry and a fluorescence-imaging screen will identify additional endocytic proteins and phosphoregulation mechanisms. The dynamics of GFP-tagged early and late endocytic markers Sla1p and Abp1p, respectively, will be analyzed in mutants of at least 60 endocytic proteins to reveal specific functions. Focused studies, combining molecular genetics and live cell imaging, will provide new insights into the specific in vivo roles of several Arp2/3 activators and the linker protein Sla2p. Finally, real-time fluorescence light microscopy and electron microscopy analyses of novel derivatives of an endocytic cargo molecule will fully reveal the dynamics and ultrastructure of the receptor-mediated endocytosis pathway. ? ?
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