Figure 1.
Figure 1. In vitro characterization of primary Myo9b−/− T cells. (A) Outline of nonpolarized versus polarized T cell with small GTPase activity and Myo9b distribution, including a scheme of its RhoGAP “cargo.” The red arrow of the polarized cell indicates direction of movement, whereas the green arrow indicates uropod contractility. (B) Myo9b gene expression of naive and vesicular stomatitis virus (VSV)-OVA–activated effector and memory T cells isolated from spleen. Data are from ImmGen database. (C) Flow cytometry plot of RhoA-GTP levels in resting WT (gray line) and Myo9b−/− T cells (red line). The black line depicts background, numbers indicate percentage of positive cells. (D) MFI of RhoA-GTP in WT and Myo9b−/− T cells. Shown is mean ± SEM. Pooled from three mice per genotype in one experiment. (E) MFI of pMLC in WT and Myo9b−/− T cells. Pooled from five mice in two independent experiments. (F) Experimental layout of AFM-based surface elasticity measurement. The gray and red lines are representative measurements of cantilever deflection after bead contact to WT or Myo9b−/− CD8+ T cells. (G) Young’s modulus of elasticity of WT and Myo9b−/− CD8+ T cells. Pooled from two independent experiments. (H) Fluorescent images of WT and Myo9b−/− T cells before and after CCL21 stimulation. Arrows indicate F-actin–containing membrane protrusions. Bars, 5 µm. (I) Polarization index of WT (gray line) and Myo9b−/− (red line) T cells. Pooled from two independent experiments with a total of 202 WT and 142 Myo9b−/− T cells. (J) Transwell migration of WT and Myo9b−/− T cells toward CCL21, CCL19, and CXCL12. Shown is mean ± SEM. Pooled from two to five independent experiments. (K) Scheme of Rho signaling pathways and selected inhibitors. (L) Transwell migration of WT and Myo9b−/− T cells to CCL21 in presence of indicated inhibitors. Shown is mean ± SEM. Pooled from four independent experiments. (M) Migration speeds of WT and Myo9b−/− T cell blasts in a 3D collagen matrix. (N) Arrest coefficient of WT and Myo9b−/− T cell blasts in a 3D collagen matrix. Data in G and H are pooled from four mice per genotype in two independent experiments. (O) Under agarose speeds of WT and Myo9b−/− T cell blasts from one experiment. (P) Sustained shear-resistant adhesion of naive WT and Myo9b−/− T cells in flow chamber. Shown are percentages of T cells that resisted detachment from ICAM-1 + CCL21 after 10 min of high shear. Pooled from two independent experiments. (Q) Crawling speeds of WT and Myo9b−/− T cells on ICAM-1 + CCL21 under physiological shear. Pooled from four independent experiments. Statistical analysis: unpaired Student’s t test (D, E, J, M, N, and O); Mann-Whitney test (G, I, and Q); paired t test (P); ANOVA with Sidak’s multiple comparison test against “no inhibitor” (L). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Horizontal bars depict mean.

In vitro characterization of primary Myo9b−/− T cells. (A) Outline of nonpolarized versus polarized T cell with small GTPase activity and Myo9b distribution, including a scheme of its RhoGAP “cargo.” The red arrow of the polarized cell indicates direction of movement, whereas the green arrow indicates uropod contractility. (B) Myo9b gene expression of naive and vesicular stomatitis virus (VSV)-OVA–activated effector and memory T cells isolated from spleen. Data are from ImmGen database. (C) Flow cytometry plot of RhoA-GTP levels in resting WT (gray line) and Myo9b−/− T cells (red line). The black line depicts background, numbers indicate percentage of positive cells. (D) MFI of RhoA-GTP in WT and Myo9b−/− T cells. Shown is mean ± SEM. Pooled from three mice per genotype in one experiment. (E) MFI of pMLC in WT and Myo9b−/− T cells. Pooled from five mice in two independent experiments. (F) Experimental layout of AFM-based surface elasticity measurement. The gray and red lines are representative measurements of cantilever deflection after bead contact to WT or Myo9b−/− CD8+ T cells. (G) Young’s modulus of elasticity of WT and Myo9b−/− CD8+ T cells. Pooled from two independent experiments. (H) Fluorescent images of WT and Myo9b−/− T cells before and after CCL21 stimulation. Arrows indicate F-actin–containing membrane protrusions. Bars, 5 µm. (I) Polarization index of WT (gray line) and Myo9b−/− (red line) T cells. Pooled from two independent experiments with a total of 202 WT and 142 Myo9b−/− T cells. (J) Transwell migration of WT and Myo9b−/− T cells toward CCL21, CCL19, and CXCL12. Shown is mean ± SEM. Pooled from two to five independent experiments. (K) Scheme of Rho signaling pathways and selected inhibitors. (L) Transwell migration of WT and Myo9b−/− T cells to CCL21 in presence of indicated inhibitors. Shown is mean ± SEM. Pooled from four independent experiments. (M) Migration speeds of WT and Myo9b−/− T cell blasts in a 3D collagen matrix. (N) Arrest coefficient of WT and Myo9b−/− T cell blasts in a 3D collagen matrix. Data in G and H are pooled from four mice per genotype in two independent experiments. (O) Under agarose speeds of WT and Myo9b−/− T cell blasts from one experiment. (P) Sustained shear-resistant adhesion of naive WT and Myo9b−/− T cells in flow chamber. Shown are percentages of T cells that resisted detachment from ICAM-1 + CCL21 after 10 min of high shear. Pooled from two independent experiments. (Q) Crawling speeds of WT and Myo9b−/− T cells on ICAM-1 + CCL21 under physiological shear. Pooled from four independent experiments. Statistical analysis: unpaired Student’s t test (D, E, J, M, N, and O); Mann-Whitney test (G, I, and Q); paired t test (P); ANOVA with Sidak’s multiple comparison test against “no inhibitor” (L). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Horizontal bars depict mean.

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