FIGURE 3. Involvement of microtubules and Gapex-5 in Rab5 activation at engulfment sites.

a, b, Swiss3T3/integrin _v3 cells expressing Raichu-Rab5/PM were pretreated with 0.1 M nocodazole (a) or 10 nM paclitaxel (b) for 5 min and cultured together with apoptotic thymocytes. Time sequences in the top panels show the phase-contrast (PC), FRET/CFP ratio and CFP images at engulfment sites. The bottom panels show the time course of the normalized PC intensity (black) and FRET/CFP ratio (magenta). c, In dimethylsulphoxide (DMSO)-treated and paclitaxel-treated cells, at least 14 events of phagocytosis were observed in each experiment (n = 4). The percentage of cells showing reactivation (mean and s.e.m.) is shown (*P < 0.01). d, Swiss3T3/integrin _v3 cells treated with siRNA for the indicated Rab5 GEFs were transfected with pRaichu-Rab5/PM and cultured together with apoptotic thymocytes. The maximum relative increase in Rab5 activity during phagocytosis was divided into seven segments. A histogram plotting the percentage of cell number in each segment is presented in the left panel. In some experiments we also used Swiss3T3/integrin _v3 cells stably expressing Gapex-5 resistant to Gapex-5 siRNA (+ rescue). The Rab5 peak activation (mean and s.e.m.) is shown in the right panel (*P < 0.001; n > 40). e, Swiss3T3/integrin _v3 cells treated with siRNA for the indicated Rab5 GEFs were cultured together with apoptotic thymocytes derived from green mice. The half-life of EGFP-expressing thymocyte fluorescence was divided into eight segments. Left: histogram plotting the percentage of cell numbers in each segment. Right: half-life of EGFP-expressing thymocyte fluorescence (mean and s.e.m.) (*P < 0.001; n > 40).