Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules.

1,2-Dimyristoyl-sn-glycero-3-phospho-rac-glycerol has been suggested to form at intermediate temperatures and at high concentrations in low-salt solutions as a continuous sponge phase (Heimburg, T.; Biltonen, R. L. Biochemistry 1994, 33, 9477-9488). In the present study, the changes in signals seen for a range of fluorescent probes during phase transformations of this phospholipid indicate continuous melting and a change in lipid packing, in accordance with previous reports. However, in accordance with Lamy-Freund and Riske (Lamy-Freund, M. T.; Riske, K. A. Chem. Phys. Lipids 2003, 122, 19-32), no enhancement of lipid mixing within the putative sponge phase region was seen, suggesting a lack of a connected lipid surface. Accordingly, a typical sponge phase cannot account for the properties of the intermediate phase. The low scattering intensities of the latter have also been taken as evidence for disaggregation. While dynamic light scattering and data for membranes containing poly(ethylene glycol)-ylated lipids could lend credence to disaggregation, the most likely explanation for the scattering data would appear to be a shape transition without significant changes in neither vesicle aggregation nor bilayer connectivity. An abrupt change in light scattering and signals from some of the fluorescent probes used reveals a new transition at Tt approximately 43 degrees C, with the formation of a more ordered interface.

Natural killer (NK) cells secrete lytic granules to directly kill virus-infected or transformed cells and secrete cytokines to communicate with other cells. Threedimensional super-resolved images of F-actin, lytic granules, and IFN-␥ in primary human NK cells stimulated through different activating receptors reveal that both IFN-␥ and lytic granules accumulated in domains where the periodicity of the cortical actin mesh at the synapse opened up to be penetrable. Ligation of some activating receptors alone (eg, CD16 or NKG2D) was sufficient to increase the periodicity of the actin mesh, but surprisingly, ligation of others (eg, NKp46 or CD2) was not sufficient to induce cortical actin remodeling unless LFA-1 was coligated. Importantly, influenza virus particles that can be recognized by NK cells similarly did not open the actin mesh but could if LFA-1 was coligated. This leads us to propose that immune cells using germline-encoded receptors to directly recognize foreign proteins can use integrin recognition to differentiate between free pathogens and pathogen-infected cells that will both be present in blood. This distinction would not be required for NK cell receptors, such as NKG2D, which recognize host cell-encoded proteins that can only be found on diseased cells and not pathogens. (Blood. 2012;120(18):3729-3740) IntroductionNatural killer (NK) cells are innate lymphocytes whose responses are controlled through the balance of signals from germlineencoded activating and inhibitory receptors. 1 Integration of signals occurs across a structured interface, termed the immune synapse, between NK cells and target cells. 2,3 The spatial and temporal organization of the synapse is important for coordinating interactions between immune cell receptors, kinases, phosphatases, and adaptors as well as for directing the interaction between cells. [4][5][6] When signals downstream of activating receptors dominate, a cytolytic NK cell synapse can be assembled across which lytic molecules are secreted toward the target cell. [7][8][9] One of the best characterized NK cell activating receptors is NKG2D, which recognizes stress-induced ligands, such as MHC class I chain-related protein A (MICA), and triggers NK cells to stop migrating, spread symmetrically, and activate cytoskeletal reorganization. 8,10,11 Super-resolution microscopy revealed that remodeling of cortical actin occurs in domains within the central region of the synapse establishing secretory domains where lytic granules dock. 12,13 It is also established that cytokines can be secreted directionally across immune synapses. 14 In particular, IFN-␥ has been demonstrated to be directionally secreted in vitro from T cells to antigen-presenting cells and in vivo between T cells and brain cells. [15][16][17][18] NK cell-mediated IFN-␥ secretion is important for shaping the Th1 immune response, modulating dendritic cell and macrophage activation and stimulating antiproliferative effects in virus-infected or transformed cells. [19][20][21][22] NK cell expression of IFN-␥ c...

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