We further analyzed how disruption of the cytoskeleton changed the fraction of LG within each type of movement. LatB alone, Nocod alone, and LatB + Nocod treatment increased the fraction of immobile LG and decreased the fraction of LG with directed movement (Figure S2). LatB alone increased the fraction of immobile LG from 42% (Control, n = 3633) to 86.9% (LatB treatment, n = 2013), as did Nocod alone (87.2%, Nocod treatment, n = 1611) and LatB + Nocod (93.4%, LatB + Nocod treatment, n = 1529). LatB and Nocod each decreased the fraction of LG with directed movement from 26% (Control) to 8% (LatB alone or Nocod alone treatment), as did the combination of LatB and Nocod (4%, n = 1529). Therefore, the actin cytoskeleton and MT are required for the movement of LG at the PM. We conclude that F-actin, at physiological concentrations, promotes LG movement at the PM. Rab27a does not promote arrest (or stable docking) at the PM, but enhances actin- and MT-dependent directed granule movement at the PM. The Cytoskeleton Regulates LG Dynamics in the Cytosol To visualize 3D movement of LG deeper inside the cells, we set up a spinning disc confocal microscopy imaging system capable of recording a 3D data set every second (which is the time for acquiring each 3D stack). At each time point 40 axial steps separated by 0.2 mm in the z-direction were collected into a 3D- stack. Movement of LG was tracked in live, unstimulated NK cells Granules Movement in NK Cells by combining this data set with 3D particle tracking (Fig. 3 and Video S2). A striking observation was that LG in unstimulated NK cells (i.e. in the absence of cytokines or sensitive target cells) are mobile. The proportion of mobile LG, including those with random, directed, caged, and complex movement, was about 70%. After treatment with LatB, LG movement became more peripheral (Fig. 3 and Video S3). However, LatB was not sufficient to promote association of LG with the PM, given that the average number of LG visible by TIRF did not increase (Fig. 1). MT disruption in NKL cells treated with Nocod caused a significant decrease in the length of tracks (Fig. 3 and Figure S3), consistent with the role of MT in LG movement [32,33]. At a higher concentration (10 mM), Nocod almost completely abolished long- range movement (Figure S3 and Video S4). We further analyzed whether disruption of F-actin and MT affected straightness and track lengths in each condition. LatB did not change the straightness of the tracks (Figure S4A). The track lengths were slightly increased at higher concentrations of LatB treatment (control group: 1.160.012 mm, n = 925; 10 mM LatB treatment: 1.360.01 mm, n = 547) (Figure S5). To further evaluate the role of F-actin in the movement of LG in the cytosol, NK cells were also treated with different concentrations of Jasplakinolide (Jasp), a membrane-permeable compound that binds to and stabilizes actin filaments [34,35]. Exposure of NKL cells for 30 min to 1.0 mM Jasp resulted in an Figure 3. 3D movement of single LG tracked in live NK cells. Single vesicle tracks are superimposed on maximum intensity projections of image stacks as used for the tracking analysis (see Material and Methods). The x-y plane (which is shown) is projected in the z direction. NKL cells were either untreated (control), or treated with 10 mM LatB, 10 mM Nocod, or after Rab27a-KD, as indicated. LG were labeled by expression of FasL- GFP. The colors of tracks indicate the time, according to the color bar (start = blue, end = yellow). Scale bar is 1.0 mm. doi:10.1371/journal.pone.0012870.g003 PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12870