| Actin dynamics are fundamentally important for a wide-range of biological processes in living cells. Actin filament disassembly, a key step in actin dynamics, is important for endocytic trafficking to the vacuole in budding yeast. However, the precise role of actin filament disassembly during endocytosis is unclear. In addition, the contribution of actin filament disassembly factors to actin filament dynamics in vivo has remained unclear due to the lack of a tractable model for studies of their functions in live cells. In this work, I defined the role of the major actin filament disassembly factor, cofilin, during endocytic trafficking to the vacuole. My work showed that cofilin-mediated filament turnover is important for several steps of endocytosis, which include internalization from the cell cortex and trafficking to the vacuole. In addition, perturbing cofilin function blocks the disassembly of actin filaments, which coat membranes of endocytic vesicles. The dynamic behavior of cofilin and the cofilin cofactor, Aip1p, as well as their contribution to actin filament dynamics, were uncovered in live cells by measuring actin dynamics in sla2Delta cells. Cofilin and Aip1p were found to be recruited to actin filaments with a delay after initiation of actin filament assembly. Mutations in actin, which mimic the ADP-Pi intermediate of actin's nucleotide cycle, block the efficient recruitment of cofilin to actin in vivo. These data suggest that cofilin is recruited after Pi is released from actin subunits within filaments. Furthermore, cofilin mutations reduce the actin flux rates through filament networks, which suggests that the actin monomer pool is limiting in yeast and that cofilin-mediated actin disassembly is rate-limiting for this process. The actin monomer pool is replenished not only by cofilin, but also by the action of Aip1p, which when deleted does not change actin flux dynamics. Instead, actin assembly becomes resistant to the monomer sequestration activity of latrunculin A. In vitro studies showed that Aip1p stimulates the conversion of actin oligomers into actin monomers. These data taken together suggest that an actin assembly pathway using actin oligomers exists in vivo and pave the way for future studies investigating this novel actin assembly pathway. |
