Myo9b deficiency causes a mild reduction of in vivo T cell homing and interstitial motility in LNs. (A) Homing ratio of Myo9b^−/− to WT T cells at 2 h after transfer. Data are pooled from four to six recipient mice of two to three independent experiments. Shown is median with 25 and 75% quartiles and minimum/maximum values. (B) 3DIF LN reconstruction of adoptively transferred WT (gray) and Myo9b^−/− (red) T cells at 40 min after transfer. HEVs are labeled blue. Square length, 35 µm. (C) Localization of adoptively transferred WT (gray) and Myo9b^−/− (red) T cells in LNs as percent of recovered cells. Pooled from two independent experiments. (D) Top, representative 2PM tracks of migrating WT and Myo9b^−/− T cells in LN interstitium. Square length, 15 µm. Bottom, representative time-resolved shapes of WT and Myo9b^−/− T cells. Bar, 20 µm. (E–J) Quantification of 2PM motility parameters. Pooled from seven image sequences in three independent experiments. Circularity analysis (E), mean track speeds (F), meandering index (G), arrest coefficient (H), turning angle distribution (I), and motility coefficient analysis (J) of WT and Myo9b^−/− T cells in LN interstitium. Horizontal bars in C, F, and G depict mean, whereas horizontal bar in H shows median. Statistical analysis: ANOVA with Tukey’s multiple comparison test (A); paired Wilcoxon test (C); Mann-Whitney test (E, G, H, and I); unpaired Student’s t test (F). **, P < 0.01; ***, P < 0.001.