The actin cytoskeleton is a force-generating machine with numerous interconnected parts. By modulating the activities of these parts, actin can be assembled into specialized structures applied to diverse cellular processes, such as cell crawling, endocytosis, vesicular transport, and cytokinesis. In our lab, we are dissecting actin machines using an approach similar to overhauling a car engine. We isolate and dismantle the engine, identify the parts and their physical contacts, and rebuild the engine from purified parts.
We are performing these studies in budding yeast, where we can introduce rapidly mutations into any component and test the effects in vivo and in vitro. A critical breakthrough in our work was the development of an actin assembly assay in yeast extracts. This has allowed us to isolate the yeast actin cytoskeleton and initiate a comprehensive mass spectrometry analysis of its components. Further, by fractionating the isolated actin mixtures by gel filtration, we have begun to define new interactions and activities among components, and we are testing the relevance of these interactions in vivo using genetic analyses. Our long-term goal is to determine how upstream signals orchestrate changes in actin-associated protein activities and interactions to generate force for actin-dependent processes in vivo.