from Rab27a-KD-A1 (red) and control shRNA (black) were compared using histograms (C) and cumulative probability plots (D). The relative occurrence of each type of movement for each indicated condition is summarized in a bar plot (E). The numbers of tracking events (n) are listed on the right. The data are representative of at least two independent experiments. Found at: doi:10.1371/journal.pone.0012870.s007 (0.82 MB TIF) Table S1 Rab27a decreases the velocity of the directed movement of LG. aVelocity in mm/s. bNKL cells expressing GFP-FasL. cMouse NK cells labeled with LysoTracker Red DND- 99. Found at: doi:10.1371/journal.pone.0012870.s008 (0.04 MB DOC) Video S1 Rab27a Defect Reduces the Number of LG at the Plasma Membrane. Time-lapsed TIRF imaging of LG in NK cells. Left panel: representative TIRF image of paraformaldehyde- fixed NKL-GFP-FasL cells. Middle panel: representative TIRF image of untreated NKL-GFP-FasL cells. Right panel: represen- tative TIRF image of Rab27a-KD in NKL-GFP-FasL cells. Images were acquired at 100 ms/frame. Scale bar represents 3.0 mm. Found at: doi:10.1371/journal.pone.0012870.s009 (8.23 MB MOV) Video S2 3-D Tracking of LG in a Live Untreated NK Cell by High-speed Spinning Disc Confocal Microscopy. LG in an untreated NKL-GFP-FasL cell are shown in Green. Images were acquired for 20 time points of about 1 second each. Each time point contains 40 axial steps of 0.2 micron in the z - direction. Each grid represents 1.0 mm. Found at: doi:10.1371/journal.pone.0012870.s010 (0.79 MB MOV) Video S3 3-D Tracking of LG in Live NK Cell Treated with LatB by High-speed Spinning Disc Confocal Microscopy. Time- lapsed 3D spinning disc confocal microscopy of live NKL-GFP- FasL cells. LG in NKL-GFP-FasL cells treated with 10 mM LatB are shown in Green. Images were acquired for 20 time points of about 1 second each. Each time point contains 40 axial steps of 0.2 micron in the z - direction. Each grid represents 1.0 mm. Found at: doi:10.1371/journal.pone.0012870.s011 (1.63 MB MOV) Video S4 3-D Tracking of LG in a Live NK Cell Treated with Nocod by High-speed Spinning Disc Confocal Microscopy. Time- lapsed 3D spinning disc confocal microscopy of live NKL-GFP- References 1. Lodoen MB, Lanier LL (2006) Natural killer cells as an initial defense against pathogens. Curr Opin Immunol 18: 391–398. 2. Bossi G, Griffiths GM (2005) CTL secretory lysosomes: biogenesis and secretion of a harmful organelle. Semin Immunol 17: 87–94. 3. Trapani JA, Smyth MJ (2002) Functional significance of the perforin/granzyme cell death pathway. Nat Rev Immunol 2: 735–747. 4. Bossi G, Griffiths GM (1999) Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells. Nat Med 5: 90–96. 5. Stinchcombe JC, Griffiths GM (2007) Secretory mechanisms in cell-mediated cytotoxicity. Annu Rev Cell Dev Biol 23: 495–517. 6. Carpen O, Virtanen I, Lehto VP, Saksela E (1983) Polarization of NK cell cytoskeleton upon conjugation with sensitive target cells. J Immunol 131: 2695–2698. 7. Kupfer A, Dennert G, Singer SJ (1983) Polarization of the Golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets. Proc Natl Acad Sci U S A 80: 7224–7228. 8. Katz P, Zaytoun AM, Lee JH, Jr. (1982) Mechanisms of human cell-mediated cytotoxicity. III. Dependence of natural killing on microtubule and microfila- ment integrity. J Immunol 129: 2816–2825. 9. Lyubchenko TA, Wurth GA, Zweifach A (2003) The actin cytoskeleton and cytotoxic T lymphocytes: evidence for multiple roles that could affect granule exocytosis-dependent target cell killing. J Physiol 547: 835–847. Granules Movement in NK Cells FasL cells. LG in NKL-GFP-FasL cells treated with 10 mM Nocod are shown in Green. Images were acquired for 20 time points of about 1 second each. Each time point contains 40 axial steps of 0.2 micron in the z - direction. Each grid represents 1.0 mm. Found at: doi:10.1371/journal.pone.0012870.s012 (0.92 MB MOV) Video S5 The Distribution of Actin and Perforin Containing LG by 3D Confocal Microscopy. Serial confocal images taken through an NKL cell bound to a poly-L-lysine coated coverslip with DMSO vehicle control. Actin and perforin are shown in Green and Red, respectively. Perforin-containing lytic granules are not directly surrounded by accumulated F-actin. The signal from F- actin is weak. Found at: doi:10.1371/journal.pone.0012870.s013 (8.24 MB MOV) Video S6 A Dense Actin Meshwork Restricts LG in NKL cells with Jasp treatment. Serial confocal images taken through one NKL cell bound to a poly-L-lysine coated coverslip with 1.0 mM Jasp treatment. Actin and perforin are shown in Green and Red, respectively. Perforin-containing granules are directly caged by accumulated F-actin. The signal from F-actin is strong. Found at: doi:10.1371/journal.pone.0012870.s014 (4.28 MB MOV) Video S7 3-D Tracking of LG in Live Rab27a-KD NKL-GFP- FasL Cell by High-speed Spinning Disc Confocal Microscopy. Time-lapsed 3D spinning disc confocal microscopy of live NKL- GFP-FasL cells. LG in Rab27a-KD in NKL-GFP-FasL cells are shown in Green. Images were acquired for 20 time points of about 1 second each. Each time point contains 40 axial steps of 0.2 micron in the z - direction. Each grid represents 1.0 mm. Found at: doi:10.1371/journal.pone.0012870.s015 (1.28 MB MOV) Acknowledgments We thank M. Peterson, M. March, J. Brzostowski, D. Li, S. Rajagopalan, and P. Tolar for advice and help, J. Hammer for providing Ashen mice, G. Griffiths for a GFP-FasL plasmid, and B. Wehrle-Haller for GFP-actin plasmid. Author Contributions Conceived and designed the experiments: DL TM EOL. Performed the experiments: DL. Analyzed the data: DL. Contributed reagents/materials/ analysis tools: DL TM. Wrote the paper: DL TM EOL. 10. 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Griscelli C, Durandy A, Guy-Grand D, Daguillard F, Herzog C, et al. (1978) A syndrome associating partial albinism and immunodeficiency. Am J Med 65: 691–702. 16. Sanal O, Ersoy F, Tezcan I, Metin A, Yel L, et al. (2002) Griscelli disease: genotype-phenotype correlation in an array of clinical heterogeneity. J Clin Immunol 22: 237–243. PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12870