== A, NK clones infected with WR or Dyn2 recombinant vaccinia virus were stimulated for the indicated times with anti-FcR and then lysed

== A, NK clones infected with WR or Dyn2 recombinant vaccinia virus were stimulated for the indicated times with anti-FcR and then lysed. findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity. Keywords:Natural Killer Cells, Signal Transduction, Cytotoxicity, Human == Introduction == Natural killer (NK) cells are critical components of the innate immune system that mediate direct killing of virally-infected or tumorigenic target cells through the release of pre-formed granules. These granules are a form of secretory lysosome and contain enzymes (including granzyme A, granzyme B, and perforin) that are Ceramide delivered to the target cell and initiate apoptosis (1,2). During the development of cell-mediated cytotoxicity the microtubule organizing center (MTOC) and granules polarize toward the target cell, and lytic granule release is controlled by specialized subcellular machinery, which facilitates vesicle plasma membrane fusion (1,3). The machinery of lysosome secretion in hematopoietic cells is known to utilize the regulator of vesicle docking, Rab27a (4), and the regulator of SNARE-mediated fusion, Munc 13-4 (5). However, the SNAREs and other proteins involved in the regulated secretion of this organelle are not well comprehended. Dynamin 2 (Dyn2)3is the ubiquitously-expressed isoform of the conventional dynamin family of large GTPases (also including Dyn1, found in the brain, and Dyn3, found in the lung, testis, and brain), which has been extensively characterized for its role in receptor-mediated endocytosis and membrane remodeling (6). Structurally, Dyn2 is made up of a GTPase domain name, a middle domain name involved in oliogmerization, a pleckstrin homology (PH) domain name, a GTPase effector domain name, and a proline-rich domain name (PRD) important for interacting with Src homology 3 (SH3) domains. In addition to its role in endocytosis, Dyn2 has been identified as a regulator of actin assembly, interacting with several components of the actin remodeling machinery including cortactin, Grb2, and Nck (7). We have previously demonstrated that an conversation between Dyn2 and Vav1 regulates actin polymerization in T cells and modulates T cell activation (8). More recently, the yeast homologue of Dyn2, Vps1p, was shown to interact with the t-SNARE, Vam3p, and participate in the fusion of yeast vacuoles (9). Here we describe a central role for Dyn2 in regulating Rabbit Polyclonal to BL-CAM (phospho-Tyr807) NK cell-mediated cytotoxicity by controlling the exocytosis of lytic granules. Dyn2 localizes with lytic granules upon NK cell activation and polarizes toward the NK target cytolytic synapse. Suppression of Dyn2 protein levels using RNA interference (RNAi) or pharmacological inhibition of Dyn2 impairs cell-mediated cytotoxicity. Moreover, the effects of Dyn2 are independent of the proximal signaling events that initiate the generation of a cytotoxic response. Instead, after granule polarization, Dyn2 regulates the terminal phases of granule release. Our results linking Dyn2 to the regulation of secretion in NK cells suggest a novel function for this protein in granule exocytosis as well as a broader role for Dyn2 in cell regulation. == Materials and Methods == == Reagents, cells and antibodies == All reagents were obtained from Sigma unless stated otherwise. The P815 murine mastocytoma line was obtained from American Type Culture Collection. The MHC class I-deficient B lymphoblastoid cell Ceramide line 721.221 was kindly provided by Peter Parham (Stanford University, Palo Alto, CA). Human NK cells were cloned and passaged as described (10). Rabbit polyclonal antibodies to PLC-2, SLP-76 and Vav1 were generated and characterized as previously described (1114). Rabbit polyclonal antibodies to Dyn2 were generated as described (8). Monoclonal anti-NKG2D (R&D Systems), antiphosphorylated tyrosine (4G10; Upstate), anti-LFA-1 (MHM23), anti-perforin, anti-CD107a-PE (BD Biosciences) and IgG1 (50327; MP Biomedicals) were purchased as indicated. Goat anti-mouse IgG F(ab)2was obtained from MP Biomedicals. == Plasmids & Recombinant vaccinia == The sequence encoding Dyn2 used to generate vaccinia Ceramide constructs were amplified, subcloned into the previously described pSP11.FLAG plasmid and recombinant vaccinia were generated by homologous recombination as previously described (8,14). The cDNA sequence of Dyn2 was verified by PCR. Infections with vaccinia were conducted for 5 hours in serum free media at an MOI of 20:1. == Cytotoxicity assay == The51Cr-release assays were performed as described previously (10). In some assays, NK cells were pre-treated with the Dyn2 inhibitor dynasore for 30 minutes at the indicated concentrations. == Conjugate formation == NK cells were labeled for 1 h at 37C with 100 M sulfofluorescein (Molecular Probes, Eugene, OR), and the K562 target cells were labeled for 1 h at 37C with 40 g/ml hydroethidene (Polysciences Inc., Warrington, PA). NK cells were pretreated with a final concentration of 10 g/l IgG1 control or anti-LFA-1 antibody for 10 minutes on ice. The cells were then washed and resuspended at a concentration of 5 106cells/ml. The effectors and targets (25 l of each) were mixed together, and allowed to incubate at 37C for 10 min before adding.