in the cytosol. Therefore, actin is not required for LG movement along MT inside the cells. In contrast, treatment of NK cells with LatB increased the fraction of immobile LG at the PM and reduced the directed movement of mobile LG at the PM. The high level of F-actin induced by Jasp treatment reduced LG movement, consistent with a role of cortical F-actin as a barrier that controls the delivery of LG to the PM in degranulating cells [39,40,41]. Whether Rab27a interacts with cytoskeletal elements, thereby influencing LG movement needs to be further investigated. MT-dependent long-range movement of intracellular organelles is well-documented in neuronal and endocrine cells, in which long- range transport is mediated by MT and short-range transport is mediated by actin [42,43,44]. In polarized cytotoxic T cells, LG move along microtubules in a dynein-mediated minus-end movement toward the centrosome, which is pulled towards the cytotoxic immunological synapse [38]. MT integrity is required for NK cell cytotoxic activity [6,7,8,10]. In unstimulated NK cells, Nocod treatment, but not LatB treatment, decreased the velocity, diffusion coefficient, length, and displacement of long-range directed movement of LG in the cytosol. Disruption of MT by Nocod decreased both the number of LG at the PM and the fraction of LG with directed movement at the PM. A pre-docking role for microtubules in the process leading to degranulation in 3T3-L1 adipocytes has been proposed [45]. The loss-of-function of the retinal pigment epithelium Rab27a–Myrip–MyoVIIa complex in Ashen mice leads to a reduced constraint on MT-based melanosome motility [46]. Our data demonstrated that movement of LG at the PM of unstimulated NK cells is dependent on both F-actin and MT. Rab27a is a key component of the vesicle transport machinery in melanocytes [47] and is essential for exocytosis of LG by cytotoxic T lymphocytes [17,18,48], of multivesicular endosomes by HeLa cells [49], and of azurophilic granules by neutrophils [50]. Rab27a also plays a role in mediating the tight docking of insulin granules at the PM during glucose stimulation in mouse pancreatic b cells [51]. A mutation in Rab27a causes the defect in the transport of melanosomes in ashen mice [20]. Knockdown of Rab27a expression in unstimulated NKL cells and a Rab27a mutation in NK cells from Ashen mice resulted in a greatly decreased number of LG at the PM. Although clearly reduced in number as compared to wild-type cells, association of LG with the PM in Rab27a-deficient cells implies that some LG reach the PM stochastically, or that a specific subset of LG reaches the PM by a Rab27a-independent pathway. The defect in LG tethering (or docking) at the PM of Rab27a-deficient cells [18,22,52] suggests that Rab27a has a role in the final transport step to the PM and/or retention at the PM. We show here that Rab27a in NKL cells and primary NK cells enhances directed LG movement at the PM, which is consistent with a role of Rab27a in retention, but not stable docking, of LG at the PM. Rab27a increased the fraction of mobile LG at the PM. This could be interpreted either as a direct effect of Rab27a on LG movement at the PM, or as a distinct property of a LG subset (with greater mobility) that is delivered to the PM by a Rab27a-dependent mechanism. To test the latter hypothesis, we carefully analyzed the properties of LG at the PM by calculating the diffusion coefficient of LG in both Rab27a wild- type and Rab27a deficient NK cells. The diffusion coefficient of LG in Rab27a wild-type NK cells is not higher than that of LG in Rab27a deficient NK cells (data not shown), which does not support the existence of distinct subsets of LG. Therefore, this direct effect of Rab27a on LG movement at the PM could occur through its binding to synaptotagmin-like proteins (Slps), which are expressed predom- inantly at the PM and promote exocytosis of LG in cytotoxic T lymphocytes [53,54]. A role for Rab27a interaction with Slp1 in anterograde transport of vesicles in axons and exocytosis of neutrophil granules has been reported [55,56]. Granules Movement in NK Cells Activating signals induce fusion of Rab27a-associated late endosomal vesicles with cytolytic granules in T cells [57], and enhance Rab27a recruitment to LG in NK cells [58]. We report here that Rab27a controls LG movement in the cytosol even in unstimulated cells. The apparent abundance of Rab27a in the cytosol of NKL cells and human resting NK cells, and its association with perforin-containing LG (data not shown), suggests that signal- specific recruitment of Rab27a to LG may not be required for its role in constraining LG movement in the cytosol. NKL cells with reduced Rab27a expression and primary NK cells from Rab27a mutant mice showed an increase in the proportion of mobile LG in the cytosol, implying that Rab27a reduces MT-dependent LG movement. Materials and Methods Cells, Inhibitors, and Reagents The human cell line NKL was cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% fetal calf serum, 1% L- glutamine, 1% sodium pyruvate, and 100 U/ml of recombinant IL-2 (Hoffmann-La Roche). NKL cells were transfected with a GFP-FasL plasmid (gift of G. Griffiths, Cambridge, UK) by Amaxa nucleofection technology (Amaxa, Cologne, Germany). Briefly, 26106 NKL cells and 4.0 mg DNA were resuspended in solution, and transfected using nucleofector kit V and program O- 17. After transfection, cells were transferred into 6-well plates at 37uC. After 72 hours, 1.0 mg/ml G418 was added. Transfected NKL cells were subcloned to obtain a stable NKL-GFP-FasL cell line. NKL cells were pre-incubated with the following inhibitors for 30 min at 37uC, where indicated: 0.5 and 10 mM Latrunculin B (BIOMOL International L.P, Plymouth Meeting, PA); 0.1 and 10 mM Nocodazole (BIOMOL International L.P.); 0.5 and 1.0 mM Jasplakinolide (Invitrogen, Carlsbad, CA). The GFP-actin construct was provided by Bernhard Wehrle-Haller (Geneva, Switzerland) [59]. Fixation and Permeabilization of NKL Cells Perforin staining in NK cells was described [11]. Briefly, 16106 NKL cells were fixed with 4% freshly prepared paraformaldehyde for 15–30 minutes at room temperature (RT), washed with PBS for 3 times. Cells were permeabilized in 0.5% Triton-X100 and 10% normal donkey serum (NDS) in PBS for 30 minutes at RT. Cells were stained with anti perforin monoclonal antibody mouse IgG2b (Clone: dG9, Pierce Chemical Co. Rockford, IL, USA) for 60 minutes at RT. The primary antibody was diluted with 0.05% Triton-X100 and 3% NDS in PBS (1:333 dilution). After three washes in PBS, cells were incubated for 1 hour at RT with appropriate secondary antibodies in 0.05% Triton-X100 and 3% NDS in PBS. Secondary antibody used was Alexa Fluor 647 conjugated goat anti-mouse IgG2b (1:1000 dilution) (Molecular Probes, Eugene, OR, USA). Mouse NK Cells Wild-type C3H/HeSnJ (C3H) mice were purchased from Taconic (Rockville, MD). Ashen mice were a gift of John A. Hammer (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD). Mice were maintained in the Comparative Medicine Branch of the NIAID, NIH (Twinbrook II Facility, Rockville, MD, USA). All animal protocols were approved by the NIAID Animal Care and Use Committee. The animal protocols number for this study is LIG-32. Unless otherwise specified, anti-mouse surface molecule antibodies were purchased from BD PharMingen. The antibodies were as follows: CD3 (clone, 145-2C11, Cat. No. 553062); CD49b (clone, KMC8, Cat. No. 553758); CD16 (clone, 2.4G2, Cat. No. 553144); NK1.1 PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12870