Actin-Regulatory Complexes in Yeast

Suny, Upstate Medical University, Syracuse NY

Investigators

Abstract

In this study, several basic cell biological questions are being addressed: (i) how does a cell establish polarity; (ii) what signals convey this decision-making process to components of the cell that are responsible for carrying out the necessary morphological changes; and (iii) once this message is conveyed, how are morphological changes controlled in an orderly fashion? In yeast cells, morphological changes in response to internal or external signals generally involve rearrangements of the cell's actin cytoskeleton. The focus of this research project is on the link between the signaling transduction pathways and the downstream cytoskeletal reorganization. In particular, the emphasis of this project is on the role played by the small actin-binding protein, profilin. Through genetic and physical interaction screens, multiple links between the actin-regulatory protein profilin and small G-protein signaling pathways have been established. These results, coupled with data from other laboratories, suggest that profilin binds to multiple protein complexes in response to activation (by binding to GTP) of Ras, Cdc42, or Rho proteins. This study focuses on one of these protein complexes to determine its effects on actin polymerization and to better understand the general role of profilin in promoting cytoskeletal changes. One particular protein complex in yeast is likely responding to the Ras-adenylate cyclase signaling pathway to induce cytoskeletal changes. The interactions between yeast profilin, adenylate cyclase, and adenylate cyclase-associated protein (CAP/Srv2p), and the relevance of these interactions to Ras-induced cytoskeletal changes, will be examined through biochemical and two-hybrid analyses. It will be determined how these proteins participate in a larger protein complex, and the effects of such a complex on actin polymerization will be examined in vitro and in vivo. Results from this study will advance our understanding of how actin cytoskeletal changes are brought about in response to specific cellular signals. Since profilin has been linked to multiple protein complexes, characterization of its biochemical activity in one of these actin-regulatory complexes, and determination of its role in nucleating or extending actin filaments, will be widely applicable to the analysis of related complexes. Upon understanding the core activity (i.e. the actin nucleating/elongating function) of these complexes, one can extend the studies to investigate the role of additional proteins in modulating the three-dimensional structure and function of actin networks promoted from a given regulatory complex. The studies described in this project will largely be carried out by the Principal Investigator and undergraduate students under his direction. The nature of the experiments is such that undergraduate students can learn a substantial number of techniques and can be of abundant assistance to the Principal Investigator. If past success is repeated, it is likely that the more senior and proficient undergraduate students will perform aspects of the described studies in a more independent fashion.