![Figure 6. Rab27-mediated intracellular LFA-1 redistribution mediates differential behavior of first-division cells. (A) Representative images showing both surface and intracellular staining (permeabilized with 0.05% saponin) of LFA-1 (green: anti-CD11a, clone M17/4) and α-tubulin (red: clone 11H10). Bars, 2 µm. Pearson’s correlation coefficients from naive CD11a-mYFP/OT-I/Rab27 KO CD8+ T cells (right). Each dot represents mean PCC from one mouse (30–45 cells/mouse). (B) Representative mYFP fluorescence intensity from the CD11a-mYFP/OT-I/Rab27 KO CD8+ T cell surface (top). YFP fluorescence intensity is shown in a pseudocolor scale (from low [black] to high [red]). +/− 180°, rear of cell; 0°, leading edge; white lines depict the T cell–APC interface; arrowheads indicate the beginning of the T cell–APC contact. Quantification of relative fluorescence intensity of mYFP from CD11a-mYFP/OTI (WT) versus CD11a-mYFP/Rab27KO/OT-I (Rab27 KO) cells at the contact site (bottom). Data are expressed as mean of total 35–55 cells. *, P < 0.001. (C) Flow cytometry measuring LFA-1 surface levels (anti–LFA-1 antibody) on T cells from OT-I (WT) or OT-I/Rab27 KO after indicated times of T and N4-, D7-, or PBS-loaded APC contacts. Data are expressed as mean ± SEM of three separate experiments (8–10 mice/group). *, P < 0.0001. (D, top) Representative flow cytometry of CD11a-mYFP/OT-I/Rab27 KO CD8+ T cell division in x31-OVA infected mice 56 hpi (n = 9). (D, bottom) The homing index of Div1 Rab27 KO was calculated as the ratio between CD62L-treated mice and (CD62L + FTY720)–treated mice. Circles represent individual mice from three independent experiments (one mouse per experiment) with mean shown as a line. (E) Frequency distribution of migration velocity and displacement measured from WT-OTI (WT Div1) and OT-I/Rab27 KO (KO Div1) first-division or undivided OT-I/Rab27 KO (KO Undiv) CD8+ T cells migrating on ICAM-1+CCL21–coated plates. Data collected from three independent experiments (one mouse per experiment; 40–65 cells/mouse) were fit to nonlinear regression, and multimodality was assessed with the Kolmogorov–Smirnov test. Asterisk indicates significance between WT Div1 and KO Div1 (*, P < 0.01).](https://cdn.rupress.org/rup/content_public/journal/jcb/216/11/10.1083_jcb.201609072/6/jcb_201609072_fig6.jpeg?Expires=1771683407&Signature=F1xiukJLtlUeVHbzLwYrVuXqXsbzmAkQwT0s7RWYQxBszsJ4SL8qxR1GbaxBupum8HUblthkyrHyuYdY6ESWFl8JmyMDJFGu-yL-Tf3-fEYn4Yrm02hEUl-5ViyT2uxsk7BxF8CR2wIbrzUKCdDvPq9Ju1QRoCSN0uqmiCsCq1ZJTYC0BSfLhZxypKDAWv1ExGG7cq54-OyRcHEsEuyZgwpEoinhwfNQVyFosH1WN0C4sspMqgL7QZYK6k7jFOZoI2oCWjofmNDKMKunEAsEix1nwcb35dhpuOyRs-qrepTVdlzVRJH95APpGHHW7WrZNgTItN7iOE-ht8XTU9m9Mw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Rab27-mediated intracellular LFA-1 redistribution mediates differential behavior of first-division cells. (A) Representative images showing both surface and intracellular staining (permeabilized with 0.05% saponin) of LFA-1 (green: anti-CD11a, clone M17/4) and α-tubulin (red: clone 11H10). Bars, 2 µm. Pearson’s correlation coefficients from naive CD11a-mYFP/OT-I/Rab27 KO CD8+ T cells (right). Each dot represents mean PCC from one mouse (30–45 cells/mouse). (B) Representative mYFP fluorescence intensity from the CD11a-mYFP/OT-I/Rab27 KO CD8+ T cell surface (top). YFP fluorescence intensity is shown in a pseudocolor scale (from low [black] to high [red]). +/− 180°, rear of cell; 0°, leading edge; white lines depict the T cell–APC interface; arrowheads indicate the beginning of the T cell–APC contact. Quantification of relative fluorescence intensity of mYFP from CD11a-mYFP/OTI (WT) versus CD11a-mYFP/Rab27KO/OT-I (Rab27 KO) cells at the contact site (bottom). Data are expressed as mean of total 35–55 cells. *, P < 0.001. (C) Flow cytometry measuring LFA-1 surface levels (anti–LFA-1 antibody) on T cells from OT-I (WT) or OT-I/Rab27 KO after indicated times of T and N4-, D7-, or PBS-loaded APC contacts. Data are expressed as mean ± SEM of three separate experiments (8–10 mice/group). *, P < 0.0001. (D, top) Representative flow cytometry of CD11a-mYFP/OT-I/Rab27 KO CD8+ T cell division in x31-OVA infected mice 56 hpi (n = 9). (D, bottom) The homing index of Div1 Rab27 KO was calculated as the ratio between CD62L-treated mice and (CD62L + FTY720)–treated mice. Circles represent individual mice from three independent experiments (one mouse per experiment) with mean shown as a line. (E) Frequency distribution of migration velocity and displacement measured from WT-OTI (WT Div1) and OT-I/Rab27 KO (KO Div1) first-division or undivided OT-I/Rab27 KO (KO Undiv) CD8+ T cells migrating on ICAM-1+CCL21–coated plates. Data collected from three independent experiments (one mouse per experiment; 40–65 cells/mouse) were fit to nonlinear regression, and multimodality was assessed with the Kolmogorov–Smirnov test. Asterisk indicates significance between WT Div1 and KO Div1 (*, P < 0.01).
