Granules Movement in NK Cells Three dimensional track analysis To quantify the mobility of single LG, we calculated diffusion coefficients (D) based on the least-square fits of the functions described below to the mean square displacement (MSD) as a function of time (MSD(Dt)) [26,60]. We defined five classes of motion, to characterize the mobility of each LG: random movement (MSD(Dt) is a straight line), directed motion superimposed on diffusion (MSD(Dt) deviates from a straight line and shows an upward curvature), and restricted movement in a ‘cage’ (MSD(Dt) shows a downward curvature). In addition, we defined conditions for which a LG was assigned to be immobile. Finally, if a track did not meet any of the above conditions within a certain error, its movement was considered ‘‘complex’’. To quantify the motion of the LG, we used the following equations to fit the MSD data: random diffusion with a diffusion coefficient D was fitted with MSDðDtÞ~6DDtzk directed diffusion at velocity v and with a diffusion coefficient D was fitted with: 1 ð Þ 2 ð Þ 3 ð Þ (clone, PK136, Cat. No. 557391). NK cells were isolated from mouse spleen by negative selection with an NK isolation kit (Miltenyi Biotec, Auburn, CA), and were .90% CD3 2 , NK1.1+, CD49b+ (DX5+), and CD16+. Briefly, spleen cells were treated in ACK lysing buffer (Cat No. 304–000, Biofluids) to lyse erythro- cytes for 5 minutes at room temperature. Cells were washed three times in RPMI 1640 (Gibco, Grand Island, NY, USA), and resuspended in RPMI 1640. Cells were negatively selected by passing through a MACS column. Freshly isolated mouse NK cells were resuspended in RPMI 1640 containing 10% fetal calf serum, 1% L-glutamine, 1% sodium pyruvate, 50 mM 2-mercaptoethanol (Sigma-Aldrich) and 800 U/ml of recombinant IL-2 (Hoffmann- La Roche). Rab27a shRNA A plasmid containing Rab27a shRNA (Mission shRNA plasmid DNA, pLKO.1-Puro lentiviral vector) was obtained from Sigma (St. Louis, MO). The Rab27a shRNA targeting the sequence 59- GGCAGGAGAGGTTTCGTAGCTTA- 39 in the 59 CDS of Rab27a gene was inserted into the pLKO.1-Puro lentiviral vector. A stable Rab27a shRNA clone was obtained by transfecting NKL- GFP-FasL cells with the Rab27a shRNA plasmid DNA. 72 hours after transfection, 0.5 mg/ml puromycin was added, and increased to 1.0 mg/ml after another 48 hours. Transfected cells were subcloned to obtain a stable NKL-GFP-FasL-Rab27a-shRNA cell line. Rab27a expression was assessed by Western blot and quantified with the Odyssey Infrared imaging system. The anti- human Rab27a antibody (Cat. No. ab55667) was purchased from Abcam (Cambridge, MA). Image acquisition Spinning disc confocal and TIRF imaging were performed on an Olympus inverted IX-81 microscope, ASI MS-2000 controller (Applied Scientific Instrumentation, Inc., Eugene, OR, USA) motorized stage for xyz movements, electron- multiplier charge-coupled devices (Photometrics QuantEM, Roper Scientific), Olympus TIRF module, and lasers launched in a single mode fiber via an AOTF (NEOS Technologies, Melbourne, Florida, USA). The 10061.45 N.A. objective from Zeiss (Zeiss, h plan-Fluar 100x/1.45 oil) was used for all experiments. Illumination was provided by the 488 nm line of an Argon laser (150 W, Laser Physics, Salt Lake City, UT, USA). The hardware on the microscope was controlled by Metamorph software (Downingtown, PA). TIRF images were recorded at 100 ms/frame. 3D image stacks with 40 single frames (25 ms/frame) and a total axial dimension of 8 mm were recorded at 1 sec/stack. Three-dimensional tracking of LG For three-dimensional particle tracking, Imaris 6.1 software (Bitplane AG, Zurich, Switzerland) was used. The estimated diameter for structures to be tracked was set to 0.3 mm, in accordance with the lateral optical resolution of our setup. Autoregressive motion was selected as the tracking algorithm. The parameters ‘‘MaxDistance’’ and ‘‘MaxGapSize’’ of the software were set to 0.5 and 2 mm, respectively. LG within a distance of 0.2 mm to the PM have been removed by selecting a 3D region of interest using Imaris 6.1 software. First, the PM is identified by superimposing maximum intensity projections of image stacks to outline the cell surface. Second, the region of interest, i.e. the cell interior, is selected in all x, y, and z directions. Tracks with a least 6 recorded steps were kept for further analysis. PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12870 MSDðDtÞ~6DDtzv2ðDtÞ2zk and restricted diffusion was fitted with h MSDðDtÞ~R2 1 a1exp 6a2DDt=R2 iz6DDtzk where R is the radius of the cage, D is the diffusion coefficient of the cage, and a1<0.99, a2<0.85 are constants [26,36]. In all equations, k is the constant offset of the MSD at all time steps [61], which is caused by the limited positional accuracy with which the center of LG has been determined by means of calculating the center of mass in 3D. The positional accuracy is mainly caused by the noise found in the recorded images. Sorting different types of movement To sort trajectories into the five categories of movement types (i.e. immobile, random, directed, caged, and complex), we first defined the maximum MSD value of a track for which we still considered a vesicle to be immobile. This value was based on the tracking of fixed LG: 95% of all LG recorded in the NKL-GFP- FasL fixed by 4% freshly prepared paraformaldehyde for 15–30 minutes at room temperature, were found to have a maximum 2 MSD value of 0.2 mm . Hence, all tracked LG, which never exceed this MSD value within the total observation time of 50 s, were defined as ‘‘immobile’’. To assign the remaining types of movements to the trajectories we fitted equations 1–3 to the MSDs and calculated the respective values of R2 for each fit (R2, correlation coefficient, indicating the strength and direction of a linear relationship between two random variables). In general, a track was assigned to the type of movement, which achieved the highest R2, i.e. the best fit. If R2 was below 0.33 for all fits, we defined the movement as ‘‘complex’’, to indicate, that it was not possible to mathematically describe it with one of the three basic types of diffusion. This was, however, never the case for more than 5% of analyzed trajectories. If R2 of the MSD fit to equation 2 was larger than fits to equation 1 or 3, the LG movement type ‘‘directed’’ was assigned. If the fit of the MSD to equation 3 achieved the highest R2, the LG was classified as showing ‘‘caged’’ movement. If equation 1 resulted in the highest value for R2, LG were considered to have ‘‘random’’ movement. In addition, if the standard deviation of all