1. Volume 24, Issue 11, Pages 3017-3032 (September 2018)
Granule-Dependent NK Cell Killing of Cryptococcus Requires Kinesin to Reposition the Cytolytic Machinery for Directed Cytotoxicity 
Henry Ogbomo, Martina Timm-McCann, Tavish Barnes, Richard F. Xiang, Khusraw Jamil, Anutosh Ganguly, Danuta Stack, Shaunna M. Huston, Shu Shun Li, Pina Colarusso, Christopher H. Mody 
Cell Reports 
Volume 24, Issue 11, Pages (September 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 3017-3032DOI: (10.1016/j.celrep.2018.08.027)
Copyright © 2018 The Authors Terms and Conditions

3. Figure 1 NK Cells Kill C. neoformans More Slowly Than Raji Cells
(A) Individual images obtained from time-lapse images of co-cultures of live YT-MAP4-GFP cells with C. neoformans (top panel, see also Video S1) or Raji cells (bottom, see also Video S2) with an E/T ratio of 2:1. C. neoformans are easily discriminated based on their smaller size, whereas live Raji cells were stained with DiD (magenta). Red fluorescence indicates uptake of PI and decreased viability or death. Representative images of three independent experiments are shown. Scale bar, 30 μm.
(B) Quantification of PI-positive C. neoformans or Raji cells over time. 20–30 cells from three biological replicates were assessed. Data are represented as mean ± SEM; one-way ANOVA; ∗p < 0.01.
(C) Individual images taken from the videos of co-cultures of live YT-MAP4-GFP cells with live C. neoformans (see also Video S3). Black arrows indicate contact of the “touch-and-go” type (under 10 min); white arrows indicate a firm contact that lasted 110 min. A representative image of three biological replicates is shown. Scale bar, 30 μm.
(D) Quantification of images in (C) showing the percentage of YT-MAP-4-GFP in contact with C. neoformans versus duration of contact.
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4. Figure 2 Real-Time Granule Movement in Response C. neoformans
(A) Individual images from time-lapse imaging of co-cultures of YT-MAP4-GFP cells with Raji cells (top panel; see also Video S4) or C. neoformans (bottom; see also Video S5). The YT-MAP4-GFP cell granules were pre-labeled with Lysotracker red before co-culture with target cells. A representative of four separate experiments is shown. Scale bar, 30 μm.
(B–E) Distances of granules to the MTOC (convergence) (B), granule-to-granule (congregation) (C), granules to the NKIS (granule polarization) (D), and MTOC to the NKIS (MTOC polarization) (E) were quantified from conjugate formed with either Raji cells or C. neoformans. One conjugate was maintained in focus for the duration of each experiment. Statistical analysis was performed using the distances at times 0 and 15 min and times 15 (time at counter-convergence and counter-polarization, representing the furthest distance away from the NKIS) and 120 min for C. neoformans and times 0 and 120 min for Raji cells. Data are represented as mean ± SEM of four biological replicates; paired t test ∗p < 0.05; ∗∗p < 0.009; unpaired t test; #p < ns, not significant.
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5. Figure 3
Perforin and Granulysin Polarize to the NKIS following Activation with C. neoformans
(A and C) YT or primary NK cells that had formed conjugates with C. neoformans were imaged for expression of granulysin (YT: A, middle column; pNK: C, middle column) and perforin (YT: A, left column; pNK: C, left column). The merge of granulysin and perforin antibody staining is shown in the right columns. Analyzed images are maximum intensity projections. pNK indicates primary NK cell.
(B and D) Distance and density histograms for YT (B) and pNK (D) cells were determined for granulysin and perforin at each time point. The pixels from each image were placed into groups or bins depending on how far they were from the NKIS. Each bin is 5% of the diameter of the YT or NK cell. The horizontal axis of each histogram is the bin number (the higher the number the further the pixels in that bin were from the NKIS). The vertical axis is the mean frequency of the pixel (number of pixel in each bin)/(total number of pixels in all bins). A leftward shift of the pixels (toward “histogram bin 1”) in the histogram indicates polarization of perforin or granulysin toward the NKIS. Images shown are a representative of three biological replicates. Scale bar, 5 μm. Data are represented as mean ± SEM; n = 22 conjugates; three biological replicates.
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6. Figure 4
Perforin, but Not Granulysin, Degranulated in Response to C. neoformans
(A–E) YT cells were cultured with (B, D, and E) or without (A and C) C. neoformans at an E/T ratio of 1:10 for 0, 4, 24, or 48 hr. Cells were labeled using immunofluorescence with antibody to perforin (A and B) or granulysin (C and D). Histograms are from one of five representative experiments. The amount of perforin or granulysin released into the co-culture supernatant (E). Data are represented as mean of duplicates from one experiment. The experiment was repeated twice with similar results.
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7. Figure 5
Inhibition of Eg5-Kinesin and Dynein Reduced the NK Cell Killing Capacity of C. neoformans, but Not that of Tumor Cells
(A and B) YT cells that were treated with 2 or 3 different Eg5-siRNA sequences (see also Figure S1) were tested for their ability to kill Raji cells (A) or C. neoformans (B). As controls, mock (nuclease-free water) or negative control (NC) siRNA were used. Two biological replicates produced similar results. Data are represented as mean ± SEM of four technical replicates; one-way ANOVA; ∗∗∗p <
(C–H) Expanded primary NK cells treated with either monastrol (C) or ciliobrevin D or YT cells (G) treated with either monastrol (D) or ciliobrevin D (H) were tested for their anticryptococcal activity. Cytotoxicity of monastrol-treated primary NK cells against Raji cells (E) and monastrol- or ciliobrevin D-treated primary NK cells against K562 cells (F). One representative of three biological replicates is shown. Data are represented as mean ± SEM of four technical replicates; one-way ANOVA; ∗p < 0.01; ∗∗p < 0.002; ∗∗∗p < ns, not significant.
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8. Figure 6
Eg5-Kinesin Was Required for Perforin Association with the Microtubules and Was Responsible for the Initial Granule Counter-convergence and MTOC Counter-polarization
(A) Super-resolution SIM of YT cells pretreated with either DMSO or 40 μM monastrol for 30 min prior to co-culture with C. neoformans. YT cells were labeled with perforin (red), Eg5-kinesin (blue), and α-tubulin (green); images are shown as maximum intensity projection. Phase contrast was obtained by confocal microscopy, and images are shown as a single stack. Magnification of the inset is represented as a single stack, whereas 3D rendering of the inset is shown as maximum intensity projection. Dashed circles represent the fungal cell border. Solid scale bar, 5 μm; dashed scale bar, 1 μm.
(B–D) Pearson correlation coefficient from confocal micrographs of YT cells pretreated with DMSO or 40 μM monastrol for 30 min before co-culture with C. neoformans for 5, 10, or 15 min between Eg5-kinesin and α-tubulin (B), perforin and Eg5-kinesin (C), and perforin and α-tubulin (D). Scatterplots are an average of 20–30 conjugates from two biological replicates. Data are represented as mean ± SEM; one-way ANOVA; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p <
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9. Figure 7
Eg5-Kinesin and Dynein Were Required for Granule Congregation, Convergence, and Polarization during Killing of C. neoformans
(A) Individual images from videos of LysoTracker red-labeled YT-MAP4-GFP cells were pretreated with DMSO control (top panel; see also Video S6), monastrol (middle; see also Video S7), or ciliobrevin D (bottom; see also Video S8) before co-cultured with live C. neoformans. Scale bar, 30 μm.
(B–E) Mean distances of granule to MTOC (B), granules-to-granules (C), granules to NKIS (D), and MTOC to NKIS (E) were quantified as described in the Experimental Procedures. Data are represented as mean ± SEM from three biological replicates. Statistical analysis (paired t test) was performed using the distances at times 15 (time at counter-convergence and counter-polarization, representing the furthest distance away from the NKIS) and 120 min. One conjugate was maintained in focus and measured during each experiment.
(F) The amount of perforin released into the co-culture supernatant. Data are represented as mean ± SD from two biological replicates; two-way ANOVA; ∗p < 0.01; ∗∗∗p < ns, not significant.
Cell Reports  , DOI: ( /j.celrep )
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