Figure 5: Vps35 and Serp colocalize at Rab9-enriched domains in the late-endosome membrane.

(a,b) S2 cells expressing GFP-Rab9 and Vps35-mRFP. Yellow and white arrowheads show the colocalization of Vps35-mRFP with GFP-Rab9-enriched membrane domains of small endosomes and vacuolar endosome, respectively. (c) Fluorescent intensity of GFP-Rab9 (green) and Vps35-mRFP (magenta) in an endosomal membrane. (d) Budding of GFP-Rab9 endosomes in S2 cells. White arrowheads indicate small vesicle formation, budding and release from the endosome (Supplementary Movie 1). (e) Tracheal cells co-expressing GFP-Rab9 and myc-Vps35. White arrowheads in the insets indicate myc-Vps35 puncta localized on the Rab9 endosomal membrane. (f) Colocalization of Serp-GFP with RFP-Rab9 in S2 cells. White arrowheads indicate that the Serp-GFP colocalizes with RFP-Rab9 in the endosomal membrane. Blue arrowheads indicate the colocalization of Serp-GFP puncta with small RFP-Rab9 vesicles in the cytoplasm. (g,h) S2 cells were co-transfected with GFP-Rab7 and Vps35-mRFP, then treated with control (g) and Rab9 (h) dsRNA. (i,j) S2 cells were co-transfected with GFP-Rab7 and RFP-Rab9, then treated with control (i) and Vps35 (j) dsRNA. Yellow arrowheads in (i) showed the tubule structures extending from endosomes. (k) Reverse transcription PCR of Rab9 and Vps35 fragment from the control and dsRNA-treated cells. rp49 was amplified for the internal control. Scale bar, 10 μm for a, b and e–j and 2 μm for d.