ABSTRACT The actin cytoskeleton is regulated by members of the p21 Rho family of small GTPases by propagating signals to downstream effectors including the mammalian diaphanous-related formin 1, mDia1. The formin mDia1 directly modulates the actin cytoskeleton by nucleating actin and assembling linear actin filaments. In addition to the enzymatic activity, mDia1 can recruit proteins containing SH3 domains such as non-receptor tyrosine kinase, Src. There is a limited understanding of the mDia1-specific, RhoGTPase-mediated functions that contribute to cellular protrusive structures, specifically at the leading edge. To protrude, cells require polymerization of actin filaments to mechanically extend the leading edge. The newly extended protrusion can become stabilized by formation of adhesion sites or retracted by disassembly of adhesions. In this proposal we seek to delineate RhoGTPase paralogs that regulate mDia1 and its subsequent downstream functions in leading edge cell protrusions. We hypothesize that the RhoGTPases RhoC and/or RhoA are activating mDia1 at the leading edge to recruit Src to sites of adhesion and to polymerize actin filaments, respectively. To gain understanding into the RhoGTPase regulation of mDia1, in Aim 1 we will develop Förster Resonance Energy Transfer (FRET) based biosensor tools capable of reporting the activity status of mDia1 in living cells. We will focus on the development of Near-Infra red (NIR) fluorescent protein biosensor of mDia1 to allow simultaneous detection of cyan-yellow FRET RhoGTPase biosensors or to use the blue-green optogenetic tools targeting RhoGTPases and their upstream regulators, in single living cells. We will use this approach to determine the RhoC and RhoA regulation of mDia1 at the leading edge. For Aim 2, we will uncover the downstream function of mDia1 by recruitment of Src to sites of adhesion and how this impacts adhesion turnover during protrusive events. Additionally, we will elucidate the spatiotemporal dynamics of mDia1 in mediating linear actin assembly at the leading edge protrusions. We will discern the mDia1-specific, localized activities and functions using our newly developed mDia1 biosensor in WT and RhoGTPase perturbed cells. Our approach will allow us to delineate the spatiotemporal dynamics of mDia1 and the respective RhoGTPase paralogs regulating them in cells during leading edge protrusions.