Figure 6. Dynamin is required for maturation of engulfed apoptotic cells into Rab5-coated endosomes.

Arrows and arrowheads indicate apoptotic cells or protein recruited around engulfed apoptotic cells in phagosomes. Neither Dyn2^K44A nor Dyn2^RNAi had any obvious effect on staining of endogenous endosomal/lysosomal structures with Lysotracker Red or Rab5. Error bars represent s.d. Scale bar, 10 Îm.

(aâl) NIH/3T3 fibroblasts transfected with GFP (aâd), HA-Dyn2^K44A (eâh), or GFP-Rab5^S34N (iâl) were incubated with apoptotic thymocytes in the presence of Lysotracker Red to determine the efficiency of phagosome maturation. In the majority of GFP-transfected cells, internalized apoptotic thymocytes (arrows) co-stained with Lysotracker red (d, inset); cells transfected with Dyn2^K44A (h, inset) or Rab5^S34N (l, inset) showed decreased numbers of engulfed thymocytes co-staining with Lysotracker. Apoptotic cells incubated at 4 ÂC with phagocytes did not stain strongly with Lysotracker Red (u, Supplementary Figure S9). HA-Dyn2^K44A expressing cells were stained with anti-HA and an Alexa 488-conjugated secondary antibody for visualization of transfected cells.

(mât) Apoptotic cells were incubated with NIH/3T3 fibroblasts, transfected with either GFP alone (as control) or HA-Dyn2^K44A and the localization of endogenous Rab5 was monitored. The majority of engulfed apoptotic thymocytes inside GFP-transfected cells were in endosomes coated with Rab5 (o, arrowheads, p, inset). In Dyn2^K44A transfected cells, phagosome maturation was disrupted and decreased numbers of engulfed thymocytes were in Rab5 coated endosomes (s, t, arrowhead, inset). Apoptotic cells incubated at 4 ÂC with phagocytes did not stain strongly for Rab5 (w, Supplementary Figure S9)

(u) Quantitation of experiment shown in (aâl). 4 ÂC n=20 (2 experiments), 37 ÂC, n=60 (3 experiments).

(v) NIH/3T3 fibroblasts were electroporated with a control siRNA or dynamin-2 (dyn2) siRNA and assayed as in (aâl). Dyn2^siRNA cells showed decreased co-localization with Lysotracker as compared to control^siRNA cells. n=50 (2 experiments).

(w) Quantitation of experiment shown in (mât). 4 ÂC n=20 (2 experiments), 37 ÂC, n=60 (4 experiments).