almost complete inhibition of granule movement (n = 2072 granules in 21 cells) (Figure S4). To further analyze the relationship between F-actin and 3D LG movement, we examined the distribution of actin and perforin containing LG by 3D confocal microscopy. Treatment of NKL with Jasp resulted in much stronger accumulation of actin, as detected by expression of actin-GFP (Fig. 4). In 3D renditions of the data (Video S5 and Video S6) perforin-containing LG appeared to be ‘‘caged’’ within actin-dense regions in Jasp-treated NKL cells. We conclude that high level of F-actin may hinder LG movement in the cytosol. MT disruption in NKL cells treated with Nocod caused a significant decrease in the straightness, length, displacement, and velocity of tracks (Figure S6). We further investigated how disruption of F-actin and MT affected each type of movement. Disruption of F-actin by LatB did not significantly change the fraction of LG showing directed movement. LatB treatment decreased the fraction of immobile LG from 30.8% (control group, n = 473, from 30 cells) to 22.6% (low concentration of LatB treatment group, n = 338, from 13 cells) or 23.8% (high concentration of LatB treatment group, n = 266, from 21 cells), respectively. However, disruption of MT by Nocod increased the immobilized fraction from 31% (Control) to 71% (Nocod treatment), and decreased the directed movement fraction from 42.2% (Control) to 18.5% (Nocod treatment), respectively (Fig. 5). Therefore, long-range 3D movement in the cytosol requires the integrity of MT but not actin. Rab27a Restricts LG Movement in the Cytosol The effect of Rab27a knockdown on the movement of LG in the cytosol was investigated. Control cells were either untreated NKL cells or NKL cells transfected with control shRNA. No significant difference in LG distribution or movement was observed between these two controls (data not shown). Knockdown of Rab27a did not change the overall distribution of LG in the cytosol (Video Granules Movement in NK Cells S7).We first analyzed whether knockdown of Rab27a affected straightness and track lengths. Knockdown of Rab27a clearly increased the straightness of tracking (Fig. 5A and 5B) and increased the track lengths (Fig. 5C and 5D), as measured in 10 second intervals (shRNA vector control: 1.060.01 mm, n = 925; Rab27a-KD: 1.560.01 mm, n = 814). To rule out the possibility of variability among separate clones of transfected cells, we generated and analyzed two additional independent clones of Rab27a-KD NKL cells (Rab27a-KD-A1 and Rab27a-KD-A2). Data from these two Rab27a-KD NKL cells were comparable (Figure S7). The effect of Rab27a knockdown on the track lengths in NKL clones was confirmed by analysis of Rab27a-mutant mice (Fig. 5E and 5F). The track lengths in 10 seconds time intervals were significantly increased (C3H wild-type: 1.360.09 mm, n = 260; Ashen: 1.760.04 mm, n = 224) (Fig. 5E). Straightness increased from 0.4260.01 (C3H wild-type, n = 261) to 0.4660.007 (Ashen, n = 659). Therefore, Rab27a restricts MT-dependent, directed LG movement in the cytosol. To further characterize and quantify the mobility of single vesicles, each of the 1532 3D trajectories, which were recorded in 32 control cells (Fig. 5G), was analyzed for its type of movement (i.e. immobilized, random, directed, caged or complex) [36]. The averaged MSD plots for non-treated NK cells are shown in Figure 6, where the characteristic shapes of the MSD plots for, random, directed and caged movement (straight line, upward curvature, downward curvature, respectively) are shown. Knock- down of Rab27a reduced the immobile fraction from 31% (shRNA control, n = 1532) to 15.2% (Rab27a-KD, n = 1706) and increased the directed movement fraction from 42.2% (shRNA vector control, n = 1532) to 55.1% (Rab27a-KD, n = 1706). Knockdown of Rab27a increased the velocity of directed movement (Supplementary Table S1). The velocities of directed movement derived from the MSD fitting are summarized in Supplementary Table S1. Similar results were obtained from Figure 4. High level of F-actin hinders LG movement in the cytosol. NKL cells were transfected with actin-GFP. Perforin containing LG were stained by anti-perforin monoclonal antibody followed by Alexa Fluor conjugated secondary antibody. The representative images from DMSO vehicle control (A) and 1.0 mM Jasp treatment (B) were shown individually. Scale bar is 2.0 mm. The data are representative of at least three independent experiments. doi:10.1371/journal.pone.0012870.g004 PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12870