Fig. 6

PA-dependent Rap1-GTP localization at the plasma membrane induces the development of the front membrane. a (Left) The FRET-based Rap1 activity sensor-expressing BAF cells were stimulated with CXCL12 in the presence or absence of CAY10594. An image of mTurquoise/Venus ratio represents FRET efficiency. (Center) The FRET efficiency at 6 μm edge region of plasma membrane is shown after CXCL12 stimulation (n = 20). Values are normalized to the level at zero point. The blue arrow marks the addition of CXCL12. (Right) The percentages of cells showing more than 1.3-fold increase in the FRET efficiency at the plasma membrane at 120 s after CXCL12 stimulation are shown (n = 20). *P < 0.001 versus CXCL12-stimulated cells without CAY10594. b (Left upper) Co-localization of PASS-GFP (PA) and Ral-GDS-RBD-mCherry (Rap1-GTP) in BAF cells at 10 min after CXCL12 stimulation with or without CAY10594 is shown. (Lower) We measured the ratios of Ral-GDS localized in the cytoplasm, plasma membrane, and PASS-concentrated region of plasma membrane of the cells. The graph shows percentages of cells showing that more than 50% of Ral-GDS was localized in each region (n = 30). (Right upper) Localization of Spa1-GFP and Ral-GDS-RBD-mCherry in BAF cells treated with CXCL12 is shown. (Lower) We measured the ratios of Spa1 localized in the cytoplasm, plasma membrane, and Ral-GDS-concentrated region of plasma membrane of the cells. The graph shows percentages of cells showing that more than 50% of Spa1 was localized in each region (n = 30). c Distribution of the Ral-GDS-RBD-mCherry and FITC-conjugated anti-CD44 in BAF cells on CXCL12 and ICAM-1-coated surface with (lower) or without (upper) CAY10594 is shown. Time 0 represents the first time-lapse image; subsequent images were obtained in the same focal plane. The asterisk symbol (*) shows the certain position. d Localization of PASS-GFP and Ral-GDS-RBD-mCherry in BAF cells on CXCL12 and ICAM-1-coated surface is shown. Each bar graph represents the means ± SEM. Scale bar, 5 μm