J Immunol 161:836C842. via NDR1 for recruitment of kindlin-3 and IS organization. imaging study revealed a requirement for LFA-1 and ICAM-1 for T cell arrest on APCs and memory responses (3), suggesting that TCR signals control LFA-1 adhesiveness. However, the mechanisms that regulate LFA-1 and ICAM-1 binding in phased T-APC interactions remain unclear. Antigen-specific T-APC interactions have been extensively studied in the immunological synapse (IS), which is composed of a central supramolecular activation cluster (cSMAC) of TCR-peptide major histocompatibility complex (pMHC) surrounded by a peripheral ring of LFA-1/ICAM-1 and associated talin (pSMAC) and a distal supramolecular activation cluster (dSMAC) of F-actin (4,C6). The dynamic formation of the cSMAC and pSMAC was revealed using a supported planar lipid bilayer (SPLB) incorporating pMHC complexes and ICAM-1 (5). Total PMCH internal reflection fluorescence (TIRF) microscopy demonstrated that the agonist pMHC induced a constant generation of peripheral TCR microclusters with sustained active TCR signaling that were transported into the center of the structure (7, 8). The cSMAC has been increasingly recognized as the site of signal termination (7, 8), endocytosis of engaged TCR, and targeted secretion (8, 9). TCR/CD3 complexes were recycled to the IS using intraflagellar and vesicle transport components (10, 11) and released to the extracellular space of the cSMAC as TCR-enriched microvesicles in an ESCRT (endosomal sorting complex required for transport)-dependent manner (12). Compared to TCR-pMHC interactions, our understanding of the regulatory mechanisms for LFA-1/ICAM-1 binding within the IS is still limited. TCR ligation triggers rapid activation of LFA-1 via inside-out signaling (13) and shifts the equilibrium of LFA-1 conformations from low/intermediate to high affinity for ICAM-1, and it initiates cell surface clustering (14, 15). Inside-out signaling activates the key integrin activators talin and kindlin-3 (16,C19), which interact with integrin cytoplasmic regions, leading to enhanced LFA-1 ligand-binding affinity (16). Ligand binding induces/stabilizes high-affinity conformations of LFA-1 as well as triggers outside-in signaling to activate integrin-dependent functions (20). TCR-stimulated T cells that were deficient for talin1 failed to adhere through LFA-1/ICAM-1 (21). In T cells, kindlin-3 is required for stabilization of LFA-1/ICAM-1 following TCR triggering (22) and during extravasation (23). It is generally thought that inside-out signals cause direct binding of talin-1 and kindlin-3 to the tail region of the subunits, leading to a D-(+)-Xylose separation of / integrin cytoplasmic tails, which induces conformational changes to the stalk and headpiece D-(+)-Xylose regions, resulting in a shift from bent low-affinity to extended intermediate- and high-affinity conformations (16, 20, 24, 25). It is still unclear how heterogeneous binding events of LFA-1 and ICAM-1 are regulated by inside-out signals and IS formation through talin and kindlin-3. The small GTPase Rap1 is a potent activator of integrins, including LFA-1 (26). We previously demonstrated that mammalian Hippo kinase Mst1 was associated with and activated by the Rap1-GTP binding protein RAPL, which in turn formed a complex with and activated LFA-1 (27, 28). Furthermore, ADAP/SKAP1 formed a complex with Mst1 and RAPL (29) or with RIAM and talin (30). Interestingly, lymphocytes and thymocytes from Mst1-deficient mice had impaired LFA-1-dependent adhesion and migration and exhibited defective self-tolerance (28, 31,C33). Mst1/Mst2-deficient mice also exhibited aggravated trafficking phenotypes (34). The emerging roles of Mst1/Mst2 in lymphocyte trafficking, adhesion, and cell polarity are distinct from the canonical Hippo-LATS-YAP pathway to restrain cell proliferation and are consistent with phenotypes of mutations identified in human immunodeficiencies with recurrent infection and autoantibody production (35). Recently, several regulators downstream of Mst1/Mst2 that mediate lymphocyte trafficking were reported, including DOCK8 (34), Rab13 (36), and D-(+)-Xylose LATS homolog NDR kinases (37). However, the role of TCR-triggered Rap1 signaling to Mst1 for LFA-1 activation and formation of the IS remains unknown. To date, there is limited information regarding the dynamics and regulation of LFA-1/ICAM-1 binding events for intracellular signaling during IS development in primary T cells recognizing physiological ligands. We analyzed LFA-1/ICAM-1 binding dynamics on supported lipid bilayers presenting pMHC and clarified the role of Rap1 signaling during IS formation at the single-molecule level. D-(+)-Xylose High-affinity binding preferentially occurred at.