A Cdc42-directed/formin-driven actin remodeling machine

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DESCRIPTION (provided by applicant): The Diaphanous-related formins (Drfs) and Rho family GTPases associate during the regulation of cell shape, motility, and cell division in response to growth factors and extracellular stimuli; Specific Drf/GTPase pairs co-localize to specific subcellular actin-based structures. This proposal focuses on the mammalian Drf india2 and its interaction with the GTPase Cdc42 in promoting filopodia or microspike formation. Our preliminary data indicate that mDia2 acts as a Cdc42 effector in the generation of filopodia and is localized to the tips of microspikes. We propose that Cdc42 directs the formation of an mDia2-associated protein complex leading to mDia2 targeting to filopodia. Experiments proposed here aim to define molecular determinants driving Cdc42-dependent mDia2 filopodial targeting, to ascertain which proteins associate with mDia2 and to understand how Cdc42-directed proteins contribute to mDia localization to the leading edge of migrating cells. Fluorescent resonance energy transfer (FRET) technology will be used to measure spatial and temporal dynamics of mDia2/Cdc42 interactions within cells and to determine mDia2 domain requirements for targeting. Cdc42-directed mDia2 collaborators affecting targeting will be identified by mass spectroscopy (MS) and co-localization and direct interactions demonstrated by FRET. As dynamic remodeling of the actin cytoskeleton facilitates changes in cell shape, migration and invasion within specific tissues, the results should provide insight as to how the interaction between Cdc42 and mDia2 can function in both normal and migrating tumor cells. Ultimately, we plan on exploiting the DRF family as targets for cancer therapy. Understanding the nature of the mDia2/Cdc42 interaction is a fundamental step towards that goal.