Project Summary Remodelling of the extracellular matrix (ECM) is a key process in cell migration during normal development as well as during cancer metastasis. This ECM remodelling is mediated via formation of structures, known as invadopodia and targeted secretion of enzymes, known as matrix metalloproteinases (MMPs). Invadopodia extension and degradation of ECM is dependent on coordinated localized actin polymerization as well as targeted secretion of MMPs at the tips of the invadopodia, which ultimately leads to cell migration. However, little is known about the mechanisms mediating targeted MMP secretion and how MMP secretion and actin dynamics are coordinated during cell migration. We recently identified Rab40b as a key regulator of targeted MMP secretion and invadopodia extension in breast cancer cells. We have shown Tks5 and SGEF are Rab40b binding proteins. Significantly, Tks5 and SGEF are both actin and in invadopodia regulating proteins. Additonally, we also demonstrated that Cullin-5 protein also binds to Rab40b and possibly mediate ubiquitination/degradation of invadopodial proteins. Based on all these data, we propose the following specific hypotheses: (1) Rab40b binding to SGEF coordinates actin polymerization and MMP secretion at the invadopodia during cell migration through the ECM; (2) Cullin-5 binding to Rab40b regulates dynamics of invadopodia extension/retraction during cell migration. The goal of this project is to directly test these hypotheses. First, we will define the roles of Rab40b and SGEF complex in regulating actin dynamics during invadopodia formation and cell migration in vitro. Second, we will elucidate the role of Cullin-5 binding to Rab40b in terminating invadopodia formation and cell migration. Third, we will test Rab40b role in regulating actin dynamics and cell migration in vivo. To that end, we will use neural crest cell migration during zebrafish development as experimental model that will allow us to analyse cell migration and ECM remodelling in live cells during embryogenesis. In summary, completion of this study will define new machinery governing and coordinating polarized membrane transport, cytoskeleton dynamics and ECM remodelling during cell migration in development and carcinogenesis.