Figure 5.

Myo9b is required for dermal T cell entry into epidermis. (A) 2PM image of dsRed⁺ OT-I and GFP⁺ Myo9b^−/− OT-I T cells in skin on day 30 after HSV-1_TOM-OVA infection. The right panel identifies individual OT-I (white circles) and Myo9b^−/− (red circles) OT-I T cells. (B) Percentage of WT and Myo9b^−/− OT-I T cells per field of view. (C) Ratio of WT and Myo9b^−/− OT-I T cells in epidermal layer. Each dot represents one field of view collected from six mice in two independent experiments. Dotted line represents equal number of OT-I versus Myo9b-deficient OT-I T cells in a field of view. (D) 2PM image of OT-I and Myo9b^−/− OT-I T cell. (E–H) Dynamic motility parameters in epidermis. Sphericity (E), mean protrusion speed (F), single cell speeds (G), and turning angles (H) of epidermal OT-I and Myo9b^−/− OT-I T cells. (I) Exemplary synthetic WT (gray) and Myo9b^−/− (red) T cell tracks derived from 2PM datasets and projected over a 24-h period. (J) Motility coefficient of epidermal OT-I and Myo9b^−/− OT-I T cells. (K–N) Dynamic motility parameters in dermis. (K) 2PM image of dermal T cells (>25 µm below the epidermal T_RM layer). Arrowheads depict Myo9b^−/− OT-I T cells. (L) Single cell speeds of dermal OT-I and Myo9b^−/− OT-I T cells. (M) 2PM image of failed translocation event of a dermal Myo9b^−/− OT-I T cell (arrowhead). The red-circled cell represents a spontaneously rounding epidermal Myo9b^−/− OT-I T cell. Time in min and s. (N) Quantification of attempted epidermal translocation events of dermal WT and Myo9b^−/− OT-I T cells compiled from eight image sequences in three independent experiments. Mean protrusion rates at the cell periphery were collected from three image sequence movies of two different experiments. Statistical analysis: (B) paired Student’s t test; (F, G, and L) unpaired Student’s t test; (E and H) Mann-Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Horizontal bars depict mean. Bars: 30 µm (A); 10 µm (D); 40 µm (K); 20 µm (M).