ABSTRACT Cell migration is central to many physiological and pathological processes, including morphogenetic movements during embryogenesis, wound healing, tissue regeneration and tumor metastasis. Migration is a cyclical, multistep process in which cells establish integrin-mediated adhesions at the leading edge, use those adhesions to pull themselves forward, and disassemble them at the cell rear, allowing translocation in the direction of movement. Many studies have shown that adhesion disassembly at the cell rear requires calcium influx, but our understanding of how this process is controlled is still largely incomplete. In most migrating cells, the STIM1/Orai complex is an essential mediator of Ca2+ influx. This bipartite sensor/channel assembles at endoplasmic reticulum-plasma membrane (ER-PM) contacts when it senses decreased Ca2+ in the ER and triggers focal influx of extracellular Ca2+ to the cytosol. STIM1 and Orai are coordinately upregulated in a wide variety of metastatic cancers, and pharmacological inhibition blocks migration and metastasis in model systems. In preliminary studies that form the basis of this proposal, we have uncovered a signaling cascade between STIM1/Orai activation at ER-PM contacts and focal adhesion disassembly that is mediated by the small GTPase Arf5, its guanine nucleotide exchange factor (GEF) IQSec1 and ORP3, a member of the Oxysterol binding protein Related family of proteins that mediate direct lipid exchange between the ER and other organelles. We find that ORP3 is recruited to ER/PM contact sites in response to STIM1/Orai activation, binds robustly to IQSec1 in a Ca2+-dependent manner and is essential for the Ca2+-induced activation of Arf5. Strikingly, individual knockdown of ORP3, IQSec1, or Arf5 yields the same phenotype; flattened cells with enlarged focal adhesions, slowed adhesion disassembly and dramatically reduced cell motility. Based on these observations, we hypothesize that calcium signaling at the trailing edge triggers the local ORP3-dependent activation of Arf5 by IQSec1, and that Arf5 activity is essential for focal adhesion disassembly and cell motility. This model will be tested in three Specific Aims: 1) to determine how Ca2+ influx triggers recruitment of ORP3 to ER/PM contact sites, 2) to determine how Ca2+ influx controls the interaction of ORP3 with IQSec1 and modulates its guanine nucleotide exchange activity, and 3) to determine how Arf5 promotes focal adhesion turnover and cell motility. In this context, Arfs are known to be allosteric activators of the type I PI5-kinase PIPKIβ, which has been implicated in focal adhesion disassembly through its recruitment of the clathrin-based endocytic machinery. The Arf GEF IQSec1 has been reported to promote metastasis in both breast cancers and melanomas, but its mechanism of action is poorly understood. Completion of these studies will provide a mechanistic understanding of how IQSec1 promotes tumor metastasis (and migration in other ...