Inflammation is the critical and fundamental process responsible for responding to infection and repairing tissue damage. In response to an inflammatory stimulus the local blood vessels dilate, their endothelium is activated by cytokines and chemokines and circulating leukocytes are recruited to move out of blood vessels into sites of tissue damage. This involves a series of carefully regulated molecular interactions between the endothelial cells of the vasculature and leukocytes to allow them to adhere and pass through into the tissue. The first irreversible (and hence arguably the most important) step in this process is transendothelial migration (TEM) when the leukocyte squeezes across endothelial junctions to reach the inflamed tissues. An interconnected reticular network of membrane vesicles just underneath the surface of the endothelial plasma membrane, known as the lateral border recycling compartment (LBRC) is essential for providing proteins that facilitate the transmigration process. During TEM, membrane from the LBRC is trafficked to the sites of transmigrating leukocytes in a process known as targeted recycling (TR). We identified IQ-domain GTPase-activating protein (IQGAP1) as a molecule specifically enriched in fractions of the LBRC. Our preliminary data show that it is critically involved in facilitating TR and TEM. IQGAP1 is a multidomain, scaffolding protein that has been reported to interact with over 135 cellular proteins. Knocking down its expression prevents TR and TEM; however, it is not known which protein(s) it interacts with. As a first step toward identifying its mechanism of action, in Aim 1A we will determine which of its domains are essential for TR and TEM. Since its interacting partners bind to specific domains, defining which domains are important will help us quickly narrow down potential candidate molecules and eventually mechanism. Preliminary data show that the IQ domain is critical for TEM. In Aim 1B, we will test the hypothesis that calmodulin, the best demonstrated ligand for the IQ domain, is critically involved in IQGAP1 function in TEM. In order to fully understand the role of IQGAP1 in inflammation, we will study precisely how it functions spatio-temporally to regulate leukocyte TEM. In Aim 2A we will study the role of IQGAP1 in inflammation using mice selectively deficient in IQGAP1 in non-hematopoietic cells. With our novel 4-dimensional imaging system, we possess a unique and powerful tool to study TEM events in real time, in vivo at an unprecedented resolution. We will quantify TEM of neutrophils in the cremaster muscle circulation in response to cytokines and in response to ischemia/reperfusion injury. In Aim 2B we will extend our studies to a clinically-relevant model of inflammation: Myocardial infarction after ischemia and reperfusion. Our studies will provide a more thorough understanding of IQGAP1 and its role in inflammation. Identifying the binding partners of IQGAP1 that are critical for ...