encoding shRNA against Rab27a, and clones with stable shRNA expression were isolated. Expression of Rab27a was monitored by Western blot analysis (Fig. 1A). In stably transfected NKL-GFP- FasL cells, most of the GFP-FasL-labeled granules contained perforin, as detected by intracellular staining of perforin (data not shown). The Pearson correlation coefficient was 0.7860.01 (n = 11) in control cells. Knock-down of Rab27a expression did not change the sorting of perforin and FasL, as their colocalization was still observed, with a Pearson correlation coefficient of 0.7760.02 (n = 10). The distribution of LG was investigated by TIRF microscopy (TIRFM). TIRFM has been widely used to track the movement of single vesicles within cells. TIRFM provides several advantages for imaging processes in living cells, such as low background, high temporal resolution, minimal photobleaching, and minimal phototoxicity, which have made it a powerful tool to study granule movement and fusion with the PM [23,24,25,26]. The evanescent field generated by TIRF illumination penetrates only ,200 nm from the coverslip in our microscope setup. Under these conditions, LG labeled by expression of GFP-FasL were easily observed in close proximity to the PM. As shown in Video S1, the majority of LG at the PM are mobile. Knockdown of Rab27a (Rab27a-KD) decreased the number of LG per cell contact detectable within the evanescent field from 42.663.1 (shRNA control, n = 26) to 13.160.95 (Rab27a-KD, n = 34) (Fig. 1B and Video S1). While Latrunculin B (LatB) treatment did not change the number of LG, depolymerization of MT by Nocodazole (Nocod) treatment significantly decreased the number of LG (Fig. 1C). No noticeable difference of the contact area to the poly- L-lysine-coated coverslip has been observed between untreated and inhibitor-treated cells, as well as between shRNA control and Rab27a-KD NKL cells (data not shown). Similar results were obtained from Rab27a mutant mice (Ashen) as compared to wild- Granules Movement in NK Cells type mice (C3H) (Fig. 1D). The LG of mouse NK cells are secretory lysosomes [27,28] which, as all acidic compartments, can be labeled with LysoTracker Red DND-99 [29,30] and imaged by TIRFM. The number of LG per cell contact decreased from 9.160.27 (C3H, n = 26) to 4.560.23 in the absence of Rab27a (Ashen, n = 25). Therefore, fewer LG reside near the PM in the absence of Rab27a, consistent with a role of Rab27a in transport to, or retention at the PM [20]. Rab27a Enhances Cytoskeleton Dependent Movement of LG at the PM Several parameters of LG movement were determined by automated image analysis of TIRF data to evaluate how Rab27a may affect granule movement in mouse NK cells. The length of tracking paths at the PM increased in Rab27a-mutant NK cells from Ashen mice (Fig. 2A and 2B), but displacement and straightness decreased (Fig. 2C–2F). The Rab27a mutation decreased the fraction of LG with directed movement from 42% (C3H wild-type, n = 594, in 27 cells) to 28% (Ashen, n = 584, in 24 cells). Similar results were obtained with Rab27a knockdown in NKL cells (Fig. 2G). Comparable results were observed with two independent Rab27a knockdown NKL clones (data not shown). Consistent with previous studies [18,22,31], NK cells lacking Rab27a contain acidic compartments of apparently normal size, number, and morphology (data not shown). These data imply that Rab27a enhances the movement of LG that are close to the PM. To test how the actin cytoskeleton and MT affect LG movement at the PM, NKL cells were treated with different concentrations of LatB and Nocod. LatB alone, Nocod alone, and the combination of LatB and Nocod (LatB + Nocod) decreased displacement and straightness of movement (Figure S1). There- fore, LG movement close to the PM is dependent on both actin and MT. Figure 1. Knockdown of Rab27a and disruption of microtubules decreased the number of granules docked at the PM. (A) Knockdown of Rab27a expression in NKL cells. The shRNA against Rab27a was stably transfected into NKL cells. Rab27a was detected by Western blot and visualized by Odyssey Infrared Imaging (Li-Cor Biosciences). (B) Number of granules within evanescent filed per NKL cell between control shRNA and Rab27a knockdown (Rab27a-KD) visualized by TIRF microscopy. (C) Number of granules within evanescent filed per NKL cell pretreated with 10 mM Latrunculin B (LatB) and Nocodazole (Nocod). (D) Number of acidic compartments within evanescent filed per NK cell from Ashen (Rab27a mutation) and C3H (wild-type) mice. The data are representative of at least 50 cells in three independent experiments. doi:10.1371/journal.pone.0012870.g001 PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12870