Cytotoxic lymphocytes, which include NK cells, kill target cells by releasing the content of secretory lysosomes at the site of contact with target cells, which has been called the cytotoxic immune synapse. To understand the dynamics and control of cytotoxic immune synapses we imaged human primary NK cells on lipid bilayers carrying ligands of activation receptors. These were the first live images of cytotoxic immune synapses during degranulation, and of NK cell immune synapses. Formation of an organized synapse was dependent on the presence of the beta2 integrin ligand ICAM-1. Ligands of coactivation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Lysosomal protein LAMP-1 that was exocytosed during degranulation accumulated in a large and spatially stable cluster. A striking feature of NK cell cytotoxic immune synapses formed in the presence of ICAM-1 is that LAMP-1 delivered to the cell surface did not escape and diffuse over the plasma membrane. Instead, LAMP-1 accumulated in the central cluster, which overlapped with a site of membrane internalization. Imaging of NK cells at the earliest stages of degranulation suggested that ICAM-1 promotes LAMP-1 exocytosis at the center of the synapse, rather than LAMP-1 retrieval from the periphery. Furthermore, live imaging of lysosomal compartments showed that they reached the plasma membrane at focal points directly adjacent to centrally accumulated LAMP-1. This stable region of membrane dynamics is the defining feature of cytotoxic immune synapses, as it could occur even in the absence of an organized distribution of receptors at the synapse. Therefore, our work has shown that cytotoxic immune synapses include a central region of bidirectional vesicular traffic, which is controlled by the integrin LFA-1. Integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion, but also polarization of cytolytic granules in NK cells. We tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adapter molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, while NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes. Cellular signaling in response to mechanical forces exerted through cell surface receptors and the cytoskeleton, referred to as mechanotransduction, has been gaining recognition. Cells sense force at points of attachment, such as beta1 integrin binding to fibronectin. Force contributes to conformational and affinity changes in integrins. As force sensing by LFA-1 would depend on tethering of its ligands, it is possible that a loss of mechanotransduction due to release of ICAM from the cytoskeleton in target cells is at the base of adhesion and granule polarization defects in NK cells.
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