Polymerization of monomeric actin into filaments provides the force for cell motility; aberrant cell motility contributes to disease, notably atherosclerosis and tumor metastasis. The key regulatory step required for the production of actin filaments in cells is the creation of a filament 'nucleus', which is achieved by proteins called actin nucleation factors. To date, only four classes of actin nucleation factors are known, including the Arp2/3 complex and Spire. I have discovered a new actin nucleation factor, called JMY. JMY is found only in vertebrate organisms, where it is most highly expressed in the heart, the brain and in motile cells (Shikama et al., 1999; preliminary results). I found that JMY promotes actin polymerization by two mechanisms: (1) direct nucleation of filaments and (2) activating the Arp2/3 complex. In most cells JMY localizes to the nucleus, but when cells differentiate into highly motile phenotypes, JMY leaves the nucleus and accumulates at the leading edge. My long term goal is to understand how JMY regulates actin nucleation in cells. Specifically, I will: 1.) Determine how full-length JMY is regulated. Proper regulation of actin assembly is essential for the survival of cells. Therefore, actin nucleation factors are tightly regulated in cells. Several mechanisms for blocking the interaction of actin with these proteins and their cofactors have been highly conserved throughout evolution. These include autoinhibition (like N-WASp and the formins) and trans-inhibition (like WAVE) by other proteins. Several known JMY-interacting proteins are known, and are thus good candidates for regulatory proteins. I hypothesize that JMY is regulated by one or both of these mechanisms. I will first determine whether JMY is autoinhibited by characterizing nucleation and Arp2/3 activation activities of the full-length protein. Then I will test whether JMY-dependent actin polymerization is regulated by known JMY-interacting proteins p300, Strap, and Mdm2. 2.) Characterize the role of JMY in cell motility. I found that JMY localizes to the actin-rich leading edge of motile cells, and silencing JMY expression causes reduced migration rates in scratch-wounding assays. To better understand the cellular role of JMY in cell motility, I will analyze motility defects in motile cells with reduced JMY, using RNA interference. Then I will determine the kinetics of JMY localization to the leading edge in living motile cells.
|Program type||Predoctoral Fellowship|
|Effective start/end date||07/01/2008 → 06/30/2009|