David G. Drubin
The membrane actin cytoskeleton undergoes a carefully coordinated series of changes in organization that direct the morphological development of a yeast cell. To understand how the organization of the cytoskeleton changes in response to a variety of signals, we have biochemically identified several yeast proteins, including cofilin, fimbrin and Abp1p, that bind to actin filaments and regulate the assembly and/or organization of actin filaments. We have genetically tested the roles of these proteins in the living cell, and have used selections and screens to identify the genes that regulate these proteins. As a result of this work, and the work in several other laboratories, a large number of genes that control cellular morphogenesis in budding yeast has been identified. The challenge now is determine how the products of a large number of genes cooperate to control the development of cell polarity and cell shape.
To this end, we have established a permeabilized cell model for polarized actin assembly. By gently permeabilizing yeast under conditions that preserve the arrangement of intracellular constituents, we have established an in vitro assay that allows the assembly of exogenously added fluorescent actin into the precise regions of the cell that incorporate actin in vivo. This assembly is dependent on the functions of Sla1p and Sla2p, two gene products that function in the in vivo incorporation of actin at the cell cortex. Moreover, we have obtained evidence for the requirement of GTP-binding proteins for the actin nucleation activity. Finally, the assembly of actin in permeabilized yeast cells can be modulated by the cell cycle control cyclin-dependent kinase/cyclin complex in a manner that mimics the in vivo control. We are using the permeabilized cell assay to elucidate the full regulatory pathways that control actin assembly in living cells. Since actin and the proteins that bind to actin are highly conserved, the results that we obtain in yeast are likely to be applicable to more complex eukaryotes as well.
Elucidation of the molecular mechanisms used to regulate actin assembly will require a detailed knowledge of how actin subunits assemble into long polymers. Recently, atomic models for the actin monomer and actin filament have been developed. These advances provide a unique opportunity to determine the mechanisms of actin mediated processes at the level of atomic interactions. We are performing a structure-function analysis of actin by mutating specific residues and assaying the effects of these mutations on actin assembly in vitro and in vivo. We are developing novel genetic and biochemical approaches to mapping the binding sites of actin-binding proteins on the actin filament, and to relating specific aspects of actin structure to actin function in the living cell. Finally, genetic, biochemical and structural studies of the low molecular weight (16 kD) actin filament severing protein cofilin are being performed to determine the molecular mechanism of filament severing and to elucidate the regulatory pathways that control the activity of this protein.