Granules Movement in NK Cells Figure 5. Rab27a defect increased the mobility of cytosolic LG. The straightness of single vesicle trajectories observed in Rab27a knockdown (Rab27a-KD) NKL cells (red), NKL cells expressing control shRNA (control, black), as well as fixed cells (gray) are shown as histograms (A) and cumulative probability plots (B). For the same population of cells, the track lengths are shown as histograms (C) and cumulative probability plots (D). Note that there was no significant difference between control (black) and shRNA vector control (gray) in (C and D). Mean values of track lengths during 10 seconds were compared between Rab27a Knockdown and shRNA vector (C, inset). Finally, length (E) and straightness (F) of single trajectories in NK cells from Ashen (red) and C3H mice (black) were compared using cumulative probability plots. Insets show the mean values of track lengths and straightness during 10 seconds between Rab27a mutant mice (Ashen, red) and C3H-wt mice (Control, black). The relative occurrence of each type of movement for each indicated condition is summarized in a bar plot (G). The numbers of tracking events (n) are listed on the right. The data are representative of at least two independent experiments. doi:10.1371/journal.pone.0012870.g005 Rab27a mutant Ashen mice (Fig. 5 and Supplementary Table S1). Therefore, Rab27a plays a role in constraining LG movement along MT in the cytosol. Discussion LG in cytotoxic T lymphocytes from Rab27 mutant Ashen mice have a defect in docking at the PM and exocytosis [18,22]. In this study, we used live imaging of granule movement in unstimulated NK cells and tested the role of Rab27a in controlling movement by knock down in a human NK cell line and by using primary NK cells from Rab27a mutant Ashen mice. The movement of individual LG was investigated by high-speed 3D spinning disc confocal and 2D TIRF microscopy in combination with automated image analysis to track several thousands of LG. Applying both imaging techniques allowed us to obtain separate information on LG movement in the cytosol and at the PM. Our data show that while Rab27a enhanced the MT and actin-dependent movement of LG at the PM, it reduced the MT-dependent movement in the cytosol. Therefore, Rab27a has distinct functions in controlling LG movement in the cytosol and at the PM, and exerts its control in unstimulated NK cells in the absence of active granule polarization. F-actin dynamics is essential for cytotoxicity by T lymphocytes and NK cells [37,38]. In unstimulated NK cells depolymerization of the actin network by LatB had little effect on granule movement Figure 6. Three-dimensional mean square displacement (MSD) plots of each type of movement. Averages of all 3D-MSD plots from 32 control cells are shown for trajectories showing directed (A), random (B), caged (C), and complex (D) movement, as well as immobile vesicles (E) (see Materials and Methods). Note that the scales among the plots differ. doi:10.1371/journal.pone.0012870.g006 PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12870