REGULATION OF CORTICAL ACTIN CYTOSKELETON ASSEMBLY DURING POLARIZED CELL-GROWTH IN BUDDING YEAST

We have established an in vitro assay for assembly of the cortical act in cytoskeleton of budding yeast cells. After permeabilization of yeas t by a novel procedure designed to maintain the spatial organization o f cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is simil ar to the cortical actin distribution in vivo. Actin assembly in the b ud of small-budded cells requires a nucleation activity provided by pr otein factors that appear to be distinct from the barbed ends of endog enous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical a ctin cytoskeleton function, suggesting a possible role for these prote ins in the nucleation reaction. The rate and the extent of actin assem bly in the bud are increased in permeabilized Delta cap2 cells, provid ing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GT P-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding prote in required for bud formation. Furthermore, the lack of actin nucleati on activity in the cdc42 mutant can be complemented in vitro by a cons titutively active Cdc42 protein. These results suggest that Cdc42 is c losely involved in regulating actin assembly during polarized cell gro wth.