FIGURE 2. Time sequence of actin and Rab5 accumulation, Rab5 activation, and breakdown of apoptotic thymocytes.

a, b, Swiss3T3/integrin _v3 cells expressing EGFP–actin (a) or mEGFP–Rab5 and mRFP (b) were cultured together with apoptotic thymocytes. c, Swiss3T3/integrin _v3 cells were cultured together with apoptotic thymocytes derived from green mice. In a and c, the top panels show phase-contrast (PC) and fluorescent images at the indicated time points (min). In the fourth column of b, ratio images of mEGFP–Rab5/mRFP are shown in pseudocolour mode. In a–c, the bottom panels show the images of engulfment sites marked by white squares in the top panels. Scale bars, 20 m. d–g, Black lines indicate PC intensity; magenta lines indicate the fluorescence intensity of EGFP–actin (d), the FRET/CFP ratio of Raichu-Rab5/PM (e), the mEGFP–Rab5/mRFP ratio (f), and the fluorescence intensity of EGFP-expressing thymocytes (g) in the circular regions (diameter 7.3 m) containing engulfment sites (n = 8 in each panel). PC intensity (d–g), EGFP–actin intensity (d), the FRET/CFP ratio (e) and the mEGFP–Rab5/mRFP ratio (f) were normalized by dividing by their average values obtained before zero time (for EGFP–actin intensity (d) the average value between -10 and -5 min was used). EGFP-expressing thymocyte intensity (g) was normalized by dividing by the reference value, which was averaged over the period during which the EGFP signal had been stable before the quenching started. h, Control or Rab5-S34N-expressing Swiss3T3/integrin _v3 cells were cultured together with apoptotic thymocytes derived from green mice. The half-life of EGFP-expressing thymocyte fluorescence was divided into seven equal 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 < 0.01; n > 25).