University of Virginia Health System

Our research is focused on understanding how cells maintain the dynamic actin filament cytoskeleton required for numerous cellular processes, including, cell migration, determination and maintenance of shape, and intracellular protein trafficking. To investigate molecular mechanisms of actin assembly that contribute to these different functions, we developed a system to visualize actin assembly in living cells using GFP-tagged probes for dynamic actin.  We also use biochemical approaches to probe the regulation of actin assembly by individual components, with the ultimate goal of understanding how they contribute to cell dynamics and membrane homeostasis.  Initial observations of actin assembly in living cells revealed some surprising new roles for actin dynamics during a variety of cellular processes.  A pool of dynamic actin was observed at the cell periphery, as expected for extension of lamellipodia during the initial steps of cell migration. Dynamic actin was also observed at other sites on the plasma membrane and in association with intracellular endosomal compartments. We are testing the hypothesis that these sites of actin assembly provide filaments that facilitate endocytosis and post-endocytic traffic.
Dynamin and actin assembly. A survey of proteins that biochemically link the endocytic and actin machineries reveals that the GTPase dynamin2 may play a key role in coordinately regulating actin and membranes. GTPase-defective mutant forms of dynamin2 inhibit actin assembly when expressed in cells.  Dynamin interacts directly with several proteins that regulate actin assembly, including profilin and cortactin. In addition, GTP hydrolysis by dynamin alters the organization of actin filaments formed in vitro by Arp2/3 complex and cortactin. We aim to determine the mechanisms by which dynamin2 regulates actin filament dynamics for endocytosis and for cell motility.
Actin dynamics and post-endocytic traffic. Observations in living cells of dynamic actin associated with endosomal compartments suggests that actin dynamics facilitates traffic through the endosomal system. Movies of living cells revealed foci of dynamic actin associated with vesicular compartments that can be loaded with endocytic tracers. Inhibition of actin assembly leads to the accumulation of the endocytic tracer. The function of actin filaments during trafficking, and the mechanism for regulation of the actin assembly on endocytic compartments is being investigated.
Regulation of actin assembly during cell motility. A long-standing interest is to understand the mechanisms of actin assembly that provides for cell motility. One focus has been on capping protein, a ubiquitous actin-binding protein that regulates polymerization by binding to and thereby "capping", the fast-growing end of actin filaments. We recently showed that proteins of the Ena/VASP family antagonize capping activity in vitro.  We are developing TIRF microscopy-based assays to determine the mechanism whereby Ena/VASP proteins regulate actin dynamics at actin filament barbed ends, including inhibiting barbed end capping.

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