Abstract Neutrophils, which are essential for host defense against bacterial and fungal infections, induce inflammation following tissue necrosis or ischemic injury. If not properly resolved, neutrophilic inflammation is the driving force behind a plethora of human inflammatory diseases. Neutrophils, delivered via the bloodstream, arrive at the site of injury by first rolling along the vessel wall and then arresting. Arrest is triggered by chemokines that induce the high-affinity conformation of β2 integrins. Two FERM domain proteins, kindlin-3 and talin-1, are required for neutrophil arrest. It is well known that talin-1, an adaptor protein that binds the actin cytoskeleton, activates integrins by binding and altering the topology of the β transmembrane domain. We recently reported that kindlin-3 is recruited to the plasma membrane through its pleckstrin homology domain prior to neutrophil arrest. However, the mechanism underlying neutrophil spreading and the role of kindlin-3 in this process are poorly understood. Moreover, how kindlin-3 cooperates with talin-1 to activate integrins and whether kindlin-3 also functions as an adaptor protein by directly binding to actin are completely unknown. Our overarching hypothesis is that, during neutrophil spreading, both kindlin-3 and talin-1 are simultaneously recruited to the plasma membrane, where kindlin-3 organizes high-affinity integrin activation. To test this hypothesis, we generated reporter mouse lines for simultaneous detection of β2 integrin activation and imaging of kindlin-3 and talin-1 in mouse neutrophils. In Aim 1, we will test the hypothesis that kindlin-3 organizes a ring of clustered high-affinity β2 integrins during neutrophil spreading under flow conditions; in Aim 2, we will test the hypothesis that kindlin-3 regulates integrin activation by directly binding to the actin cytoskeleton; and in Aim 3, we will determine the role of β2 integrin activation in ischemia-reperfusion injury (IRI). The proposed research is conceptually innovative and highly significant because it will resolve the enigma of how kindlin-3 organizes high-affinity integrin activation and define the role it plays in IRI-induced inflammation. Successful completion of this proposal will establish molecular mechanisms of integrin activation and provide mechanistic insight into neutrophil spreading and vascular inflammation. 1