Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity - PubMed (original) (raw)

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity

Amit Tuli et al. Mol Biol Cell. 2013 Dec.

Abstract

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

PubMed Disclaimer

Figures

FIGURE 1:

Arl8b localizes to lytic granules in NK cells. (A–D) Arl8b colocalizes with perforin, granzyme B, and LAMP-1, markers of lytic granules. YT-Indy cells were fixed and stained with anti-Arl8 (green), anti-perforin (red, A), anti-granzyme B (red, B), anti-LAMP-1 (red, C), and anti-EEA1 (red, D) antibodies, and analyzed by confocal microscopy. As shown in the inset, Arl8b strongly colocalizes with perforin, granzyme B, and LAMP-1 but not with EEA1 (marker for early endosomes). Scale bar, 10 μm. (E) Quantification of Arl8b-positive structures, showing colocalization with markers of lytic granules in YT-Indy cells. The graph displays the percentage of Arl8b colocalizing with perforin, granzyme B, LAMP-1, and EEA1. The values were obtained from the analysis of the following numbers of cells: Pfn, n = 33; GzmB, n = 33; LAMP-1, n = 19; and EEA1, n = 31. Error bars, SD. (F) Arl8b associates with lytic granules. Lytic granules were isolated from YT-Indy cells by density gradient separation and collected in seven equal-volume fractions (from least to most dense). Arl8b was identified in the lytic granule preparation by Western blot (second from top) and comigrated with granzyme B (top) and LAMP-1 (third from top). PNL and CLF, postnuclear lysate and crude lysosomal fraction, respectively, generated in preparing the starting material for the density gradient. The fractions were also probed for actin to demonstrate the general lack of cellular contamination during lytic granules isolation (bottom).

FIGURE 2:

Arl8b regulates NK cell cytotoxicity. (A) Stable silencing of Arl8b expression in NK cells using two distinct shRNA sequences (407 and 921). Left, Western blot analyses of Arl8b (top), perforin (middle), and actin (bottom) levels in YT-Indy (untreated) and YT-Indy stably expressing lentiviral vector–driven control shRNA, Arl8b 407 shRNA, or Arl8b 921 shRNA, as indicated. HeLa and 721.221 cell lysates were used as a negative control for perforin, and actin was used as a protein loading control. (B) qRT-PCR analyses of Arl8b and Arl8a levels in control shRNA–, Arl8b 407shRNA–, and Arl8b 921shRNAxexpressing YT-Indy cells. (C–E) Down-regulation of Arl8b impairs NK cytotoxicity. Cytotoxicity of YT-Indy cells stably expressing control shRNA– or Arl8b-specific shRNA-407 and -921 (C), control- or Arl8b siRNA–treated (72 h) primary human NK cells (D), and YT-Indy cells stable expressing control shRNA, Arl8b-specific shRNA, or Rab27a-specific shRNA (E) was tested against 721.221 target cells by 51Cr-release assay at various E:T ratios. Data show mean ± SEM from triplicates of one representative experiment of three performed; *p < 0.05. (F) Silencing of Arl8b does not affect conjugate formation. YT-Indy cells (control or Arl8b shRNA transduced) and 721.221 target cells were stained with PKH26 (Red Fluorescent Cell Linker) and PKH67 (Green Fluorescent Cell Linker), respectively. Labeled cells were coincubated at a 2:1 E:T ratio for 20 min, fixed in 4% PFA, and analyzed by flow cytometry. Events positive for red and green fluorescence were considered conjugates, and the percentage of conjugation was calculated as (red + green fluorescence/red fluorescence only) × 100. Data show mean ± SD of three independent experiments. Differences between groups were not significant. (G) Cell surface levels of CD11a and CD18 receptors remain unchanged in NK cells lacking Arl8b. YT-Indy cells (control or Arl8b shRNA transduced) were stained with isotype control, anti-CD11a-PE (left), or anti-CD18- fluorescein isothiocyanate (right) antibodies for 30 min on ice and analyzed by flow cytometry.

FIGURE 3:

Ar8b is required for lytic granule polarization to the immune synapse in NK cells. (A–C) Lytic granules fail to polarize at the immune synapse in the absence of Arl8b. Confocal analysis was performed on YT-Indy-CD2-GFP cells (effector) stably transduced with control shRNA or Ar8b-specific shRNA (407 and 921) and mixed with 721.221 cells (target) for 20 min at the E:T ratio of 2:1. Conjugates were fixed, and lytic granules were stained using anti-perforin antibody (red). Immune synapse was identified in true conjugates by accumulation of CD2-GFP signal (white arrowhead). Scale bar, 10 μm. (D) The percentage of lytic granule polarization in control vs. Arl8b-silenced-YT-Indy cells. Bar graph, mean ± SD of three independent experiments; at least 100 conjugates were evaluated in each experiment. Asterisks indicate statistical significance (*p < 0.05 by Student's t test) as compared with control. (E, F) Transmission electron microscopy of control shRNA– or Arl8b shRNA–transduced YT-Indy cells (effector) incubated with 721.221 target cells (target). Black arrowhead, immune synapse; magnified boxed area highlights the presence of lytic granules (LGs) in close proximity to the immune synapse in conjugate between control shRNA–transduced YT-Indy cell and 721.221 target cell. Scale bar, 2 μm (E, left), 500 nm (E, right), and 2 μm (F, left and right). The analysis is based on evaluation of at least five conjugates in three separate experiments.

FIGURE 4:

MTOC polarization to the immune synapse is inhibited in Arl8b-silenced NK cells. (A–C) Loss of MTOC polarization upon Arl8b silencing. Confocal analysis was performed on YT-Indy cells (effector) stably transduced with control shRNA or Ar8b-specific shRNA (407 and 921) and mixed with 721.221 cells (target) for 20 min at the E:T ratio of 2:1. Conjugates were fixed, and lytic granules were stained using anti-perforin antibody (green); the MTOC was determined based on pericentrin staining (red). Immune synapse is marked by white arrowhead. (D) Percentage of MTOC (marked by pericentrin staining) polarization in control vs. Arl8b-silenced-YT-Indy cells. Bar graph, mean ± SD of three independent experiments; at least 50 conjugates were evaluated in each experiment. Asterisks indicate statistical significance (*p < 0.05 by Student's t test) as compared with control. (E, F) Transmission electron microscopy of control shRNA– or Arl8b shRNA–transduced YT-Indy cells (E) incubated with 721.221 target cells (T). To better highlight the position of lytic granules (LGs) and MTOC (red arrow) in control vs. Arl8b-silenced NK cell–721.221 conjugates, the boxed area was magnified and is shown at the bottom. Black arrowheads indicate an immune synapse. Scale bar, 2 μm (top) and 500 nm (bottom). The analysis is based on evaluation of at least five conjugates in three separate experiments. (G, H) Control siRNA– and Arl8b siRNA–treated primary human NK cells (effector) were mixed with 721.221 cells (target) for 20 min at the E:T ratio of 2:1. Conjugates were fixed, costained for perforin (green) and pericentrin (red), and analyzed by confocal microscopy.

FIGURE 5:

Dynamics of MTOC movement relative to the immune synapse and lytic granule movement in control vs. NK cells lacking Arl8b. (A, B) Quantitative analyses of lytic granule movement relative to the MTOC as measured by mean MTOC to granule distance ± SD as a function of time (left) or averaged over time (right) in unconjugated control or Arl8b-silenced cells and in conjugates between control or Arl8b-silenced NK cells and susceptible 721.221 target cells (n = 5 for all conditions). All mean distances of lytic granules from the MTOC were significantly less in conjugates from unconjugated NK cells (p < 0.05), and distance of lytic granules from the MTOC was significantly greater in control unconjugated NK cells than in Arl8b-silenced NK cells (p < 0.05). (C) Quantitative analyses of the mean distance ± SD between the MTOC and the immune synapse normalized to the largest distance of the MTOC from the immune synapse in that cell, as a function of conjugation time in conjugates between control NK cells and susceptible 721.221 target cells (red line) and Arl8b-silenced NK cells and susceptible 721.221 target cells (blue line). n = 5 for all conditions. (D) Time-lapse images of MTOC movement in (top) a YT-Indy GFP-tubulin cell transduced with control shRNA conjugated with a susceptible 721.221 target cell and (bottom) a YT-Indy GFP-tubulin cell transduced with Arl8b shRNA conjugated with a susceptible 721.221 target cell. In each pair of images, transmitted light is shown on the top and confocal immunofluorescence in the plane of the MTOC is shown on the bottom. Green fluorescence represents GFP–α-tubulin and red LysoTracker-loaded acidified organelles.

FIGURE 6:

KIF5B, a kinesin motor protein recruited by Arl8b, regulates polarization of lytic granule–MTOC complex to the immune synapse in NK cells. (A, B) Arl8b is required for Kif5B localization to lysosomes. Control (A) or Arl8b siRNA–treated (B) HeLa cells were transfected with Kif5B-YFP. Post 24-h transfection, cells were fixed, costained with anti-Arl8b (blue) and anti–LAMP-1 (red) antibodies, and analyzed by confocal microscopy. The Kif5B-YFP–transfected cell is marked with dashed white boundary, and the inset shows colocalization between Kif5B-YFP, endogenous Arl8b, and lysosome marker, LAMP-1. Scale bar, 10 μm. (C, D) Arl8b associates with Kif5B in YT-Indy cells. Bacterially expressed, purified recombinant GST-Arl8b (WT), GST-Arl8b (Q75L), GST-Arl8b (T34N), GST-Arl3 (WT), or GST only was incubated with lysates from YT-Indy cells. The bound proteins were resolved by SDS–PAGE, transferred to PVDF, and immunoblotted with anti-Kif5B antibody (top). A weaker exposure (middle) was taken to better visualize the Kif5B signal in the input lane. For the assay, all the purified proteins were taken in equal amounts as shown at the bottom. The graph in D represents densitometric analyses performed on three independent experiments. Briefly, the ratios of band intensity of Kif5B pulled down by GST-Arl8b (Q75L) and GST-Arl8b (T34N) to that of GST-only were calculated in each experiment. Note that the actual numerical values for ratios of Kif5B band intensity varied with each experiment; for GST-Arl8b (Q75L) to GST they produced values of 37, 8.4, and 22, and for GST-Arl8b (T34N) to GST they yielded values of 36, 11, and 31. However, as these values are arbitrary in nature (depending on film exposure times, etc.), by comparing Arl8b to GST in each individual experiment, it was possible to normalize GST value at 1 (see dotted line in bar graph) and represent a mean fold increase in Kif5B pull down by Arl8b (Q75L) and Arl8b (T34N) for all three experiments (including SE bars). No significant difference was observed between Kif5B pulled down by GST-Arl8b (Q75L) or GST-Arl8b (T34N). (E–G) Kif5B is required for lytic granule–MTOC complex polarization to immune synapse in NK cells. Confocal analysis was performed on YT-Indy cells stably transduced with control shRNA or Kif5B-specific shRNA (#1 and #2) and mixed with 721.221 target cells for 20 min at the E:T ratio of 2:1. Conjugates were fixed, lytic granules were stained using anti-perforin antibody (green), and the MTOC was marked by anti-pericentrin antibody staining (red). (G) The percentage of lytic granule polarization in control vs. Kif5B-silenced YT-Indy cells. Bar graph, mean ± SD of three independent experiments; at least 100 conjugates were evaluated in each experiment. Asterisks indicate statistical significance (*p < 0.05 by Student's t test) as compared with control. (H) Silencing of Kif5B reduces NK cell cytotoxicity. YT-Indy cells were stably transduced with control shRNA or Kif5B-specific shRNAs (shRNAs #1 and #2), and their ability to kill 721.221 target cells was measured by 51Cr-release assay at various E:T ratios. Data show mean ± SEM from triplicates of one representative experiment of three performed; *p < 0.05.

FIGURE 7:

Lytic granule–MTOC complex polarization to the immune synapse is inhibited in SKIP-silenced NK cells. (A–C) SKIP is required for lytic granule–MTOC complex polarization to immune synapse in NK cells. Confocal analyses was performed on YT-Indy cells stably transduced with control shRNA or SKIP-specific shRNA and mixed with 721.221 target cells for 20 min at the E:T ratio of 2:1. Conjugates were fixed, lytic granules were stained using anti-perforin antibody (green), and the MTOC was marked by anti-pericentrin antibody staining (red). (D) The percentage of lytic granule (marked by perforin staining) polarization in control vs. SKIP-silenced YT-Indy cells. Bar graph, mean ± SD of three independent experiments; at least 50 conjugates were evaluated in each experiment. Asterisks indicate statistical significance (*p < 0.05 by Student's t test) as compared with control. (E) Silencing of SKIP in YT-Indy cells. Stable silencing of SKIP was achieved in YT-Indy cells by introducing lentiviral vector–driven shRNA against SKIP (shRNAs #1 and #2), and qRT-PCR was performed to check the efficiency of SKIP silencing.

References

1. Bagshaw RD, Callahan JW, Mahuran DJ. The Arf-family protein, Arl8b, is involved in the spatial distribution of lysosomes. Biochem Biophys Res Commun. 2006;344:1186–1191. - PubMed
1. Benado A, Nasagi-Atiya Y, Sagi-Eisenberg R. Protein trafficking in immune cells. Immunobiology. 2009;214:507–525. - PubMed
1. Bonifacino JS. Insights into the biogenesis of lysosome-related organelles from the study of the Hermansky-Pudlak syndrome. Ann NY Acad Sci. 2004;1038:103–114. - PubMed
1. Burkhardt JK, Hester S, Lapham CK, Argon Y. The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments. J Cell Biol. 1990;111:2327–2340. - PMC - PubMed
1. Casey TM, Meade JL, Hewitt EW. Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome. Mol Cell Proteomics. 2007;6:767–780. - PubMed

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity - PubMed (original) (raw)