Supplementary Materials Supplemental Materials (PDF) JCB_201704048_sm. phenotype. Introduction Metastasis is a multistep process where cells escape the primary tumor and disseminate through the body to establish secondary tumors at distant sites. To achieve this, cancer cells form actin-rich protrusions called invadopodia that, in their mature form, degrade the ECM and facilitate local invasion of the cells into Colchicine the surrounding tissue (Schmitz et al., 2000; Fidler, 2003; Condeelis et al., 2005; Yamaguchi et al., 2005). Although much progress has been made in understanding the molecular mechanisms that regulate invadopodia dynamics in recent years (Chen and Wang, 1999; Ayala et al., 2006; Buccione et al., 2009; Destaing et al., 2011; Linder Mouse monoclonal to ERBB3 et al., 2011; Courtneidge, 2012; Hoshino et al., 2013; Beaty and Condeelis, 2014; Bergman et al., 2014; Paz et al., 2014; Hastie and Sherwood, 2016), the mechanisms of how invadopodia transition from initial precursors Colchicine to mature degradative structures are not fully understood. Rac3, a member of the p21 Rho family of small GTPases, is an understudied paralog of the canonical Rac1 GTPase and has been implicated in cancer cell invasion (Baugher et al., 2005; Gest et al., 2013; Rosenberg et al., 2017). Rho-family GTPases are molecular switches that cycle between the GTP-bound on state and the GDP-bound off state, Colchicine regulated by guanine nucleotide exchange factors (GEFs) that activate and GTPase-activating proteins (GAPs) that inactivate them as well as the inhibitory guanine nucleotide dissociation inhibitor (GDI; Hall, 2005). In nonpathological circumstances, Rac3 is primarily expressed in the brain and neuronal tissues (Corbetta et al., 2009; Vaghi et al., 2012). However, up-regulation of Rac3 has been reported in aggressive breast carcinoma as well as prostate and brain cancers (Hwang et al., 2005; Engers et al., 2007; Gest et al., 2013). Despite 93% primary sequence identity between Rac3 and the canonical Rac1, there is evidence to suggest that these paralogs play antagonistic roles. In neuronal differentiation, Rac1 and Rac3 play opposing roles in which Rac3 functions as a negative regulator (Hajdo-Milasinovic et al., 2007). A specific role for Rac3 in autophagy has also been found (Zhu et al., 2011). In breast cancer, expression of Rac3 is linked to increased tumor invasion in vitro, although its mechanism of action is unknown (Baugher et al., 2005; Chan et al., 2005; Rosenberg et al., 2017). Furthermore, little work has been done to elucidate differential signaling networks involving Rac1 and Rac3. This is intriguing because the Switch I/II regions that mediate regulator and effector binding are identical and thus, they could interact with the same GEFs, GAPs, and downstream effectors. This suggests that differential regulation of these paralogs involves coordinated spatial and temporal control of upstream regulators, downstream effectors, and the GTPases themselves. In this study, we show that at invadopodia in metastatic breast cancer cells, Rac3 is required to integrate adhesion signaling and ECM degradation. Rac3 is recruited by its specific binding partner, CIB1, and promotes integrin activation at invadopodia. We developed a sensitive monomeric F?rster resonance energy transfer (FRET)-based fluorescent biosensor for Rac3 that allowed us to specifically probe the spatiotemporal dynamics of Rac3 activity at invadopodia. We found that activation of Rac3 is coordinated by two GEFs, Vav2 and PIX, and subsequently active Rac3 modulates vesicular trafficking of MT1Cmatrix metalloproteinase (MMP) through its effector GIT1. Moreover, we show that Rac3 significantly impacts breast tumor metastasis in vivo. We propose that Rac3 regulates the balance of adhesion and matrix degradation to promote tumor invasion and metastasis. Results Rac3 is enriched at invadopodia and required for matrix degradation Rac3 is.