The proposed experiments are aimed at understanding how the actin cytoskeleton becomes polarized, thereby causing growth of yeast to be polarized (i.e., direct to the bud). Late in the G1 phase of the cell cycle, the actin cytoskeleton becomes oriented: actin cables align and point to the incipient bud site, and end in patches of actin at the cell cortex (cortical patches) where the bud will emerge. The process of bud site selection and actin cytoskeleton polarization requires several proteins. Central to the mechanism that polarizes actin cables and patches is the small G-protein Cdc42. It is thought that activation of Cdc42 by the Cdc28 CDK causes it to orient actin. How this is achieved is not known. Among the several proteins that have been implicated in this process is Aip3, which Amberg identified as an actin-interacting protein with the 2-hybrid technique. He believes that Aip3 is involved in actin polarization for two reasons. First, deletion of AIP3 disrupts actin polarization. Mutants can initiate bud formation, but cannot maintain actin polarity, resulting in depolarized growth, inefficient targeting of secretory vesicles to the bud site, enlarged, disorganized cells, poor septum formation, random bud-site selection in diploids, defective nuclear segregation in mitosis. Second, Aip3 localizes to sites that overlap cortical actin, suggesting that it is associated with polarized actin and could play a role in the process. The idea that Aip3 plays a role in directing actin assembly at the bud site is supported by the observation that Aip3 localizes to the bud neck well before actin cortical patches assemble there. However, Aip3 is not absolutely required for actin assembly at the bud site, nor for septin assembly at the bud site, since both of these processes occur (but with less efficiency and fidelity) in Aip3 mutants.
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