Fig. 5: SEMA3A affects T cell actin dynamics through actomyosin II. | Nature Communications

Fig. 5: SEMA3A affects T cell actin dynamics through actomyosin II.

From: Semaphorin 3A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells

Fig. 5

a Representative flow cytometric analysis of F-actin content with varying exposure to SEMA3AS-P in stimulated OT-I T cells. b, c Representative brightfield (b) or confocal (c) images of stimulated LifeAct OT-I T cells adhering to ICAM-1/CD3 coated plates before and after SEMA3AS-P added to medium. Color of contour (c—right) indicates time from 0 to 300 s. Dashed white line indicate area used for (d). Scale bar = 10 μm. Independently performed three times. d Kymograph before (top) and after (bottom) SEMA3AS-P added. Dotted line denote data used for calculating (e). e F-actin velocity at cell edge before and after treatment with either SEMA3AS-P, Jasplakinolide or mutant SEMA3A (n = 33 cells in SEMA3AS-P group, n = 27 in Jasplakinolide group, and n = 24 in mutant SEMA3A group). f Intensity plot of LifeAct signal before and after SEMA3AS-P treatment of a OT-I T cell (left) or multiple cells exposed to SEMAAS-P (right) (n = 28 cells). g Cell width dynamics like (f) before (white background) or after (gray background) SEMA3AS-P, Jasplakinolide or mutant SEMA3A addition. h IRM area of OT-I T cells (gray lines) and average (red line) over time, with no treatment (leftmost white background), under treatment with SEMA3A (gray background) and Blebbistatin (rightmost white background). Representative contour plots of single cell under different treatments above, with color denoting time (150 sec total). I IRM area of individual OT-I T cells and contour plots as in (h), but with treatment with Blebbistatin (gray background) before SEMA3AS-P (rightmost white background). Abbreviations: A.U. arbitrary units, Min minutes, ns, not significant. Sec seconds, t time. Error bars are means ± SD from 1 (f) or combined from 2 (a) and 3 (e, h, i) independent experiments. *P = 0.016 (i), **P = 0.0011 (f), ***P < 0.0002 (H), ****P < 0.0001 (e, h) by two-tailed paired t-test (f) or Students t-test at time-points 90, 270 and 450 sec (h, i). Source data are provided as a Source Data file.

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