Requirement and regulation of actin polymerization during endocytosis

Endocytosis, or cell 'eating,' is a process used by cells for functions such as ingesting foreign particles during an immune response, sensing environmental cues during development and fine-tuning communication between synapses during a neuronal transmission. Endocytosis also allows individual cells to internalize their own protein and membrane components from the plasma membrane to maintain polarized growth, recycle membrane-bound receptors and perform quality control within the cell by guiding damaged proteins for degradation. The unicellular model organism, fission yeast, is an excellent system to study conserved aspects of endocytosis. Clathrin-mediated endocytosis, the focus of this thesis, involves the formation of a clathrin coat and polymerization of a branched actin network around the endocytic site, which facilitates internalization of the plasma membrane. The assembly of over 50 proteins during this process occurs under a minute with very high precision. However not much is known about the precise temporal regulation of these steps.;In this thesis I report the discovery of a switch that regulates the timing of actin polymerization during endocytosis. I characterize a novel component of the endocytic machinery, dip1p, which is involved in regulating this switch. I highlight additional modes of activation of actin polymerization and endocytosis in dip1 mutants. In my assessment for the requirement for actin polymerization during endocytosis, I discover that one role of actin polymerization during the initial invagination step of endocytosis in yeast is to overcome the tremendous turgor pressure within the cell. I show that in certain mutants defective in actin polymerization, defects in endocytosis can be rescued by reducing turgor pressure. I also show that the cell wall does not contribute to forces required for endocytic internalization.;Finally, I report the fortuitous sighting of filamentous actin in the nuclei of a certain mutant fission yeast cell, namely dip1for3 double mutant cells. An excess of nuclear actin leads to defects in nuclear architecture and appears to cause defects in chromosome segregation. This finding establishes a model system to gain further insight into functions of nuclear actin.;In summary, this thesis provides insights into the requirement and novel mechanisms of regulation of Arp2/3-mediated actin polymerization and furthers the understanding of mechanisms of endocytosis. These discoveries can form the basis for further studies in other conserved processes, such as cell migration, microbial pathogenesis and cell division that require polymerization of actin filaments.