Figure 7: WASH is required for tubular abscission and Serp recycling.

(a) Tracheal cells expressing lifeact-mEGFP stained with WASH. White arrowheads in the inset indicate WASH colocalized with actin patches. (b) Tracheal cells expressing GFP-Rab9 stained with WASH. WASH localized at the Rab9 endosomal membrane. White arrowheads in the inset indicate one or two WASH puncta localized at one endosome. (c) Tracheal cells expressing GFP-Rab9 stained with WASH and Serp. Each intracellular Serp granule was associated with one or two WASH patches (white arrowheads in the inset in c). (d,e) S2 cells expressing GFP-Rab9 stained with WASH (d), and both phalloidin and WASH (e). WASH localized on Rab9-enriched subdomain of endosomal membrane (white arrowheads in d) and colocalized with actin puncta on the Rab9 endosomal membrane (white arrowheads in e). (f,g) S2 cells were co-transfected with RFP-Rab9, GFP-WASH (f) and RFP-Rab9, GFP-WASH-ΔVCA (g), respectively. Arrowheads in f show GFP-WASH puncta localized at the Rab9 endosomal membrane. White arrowheads in g show the tubular and membrane cluster structures in Rab9 endosomes. (h,i) rab9- and vps35-mutant embryos stained with Serp and WASH, respectively. White arrowheads show that WASH puncta were associated with Serp granules in rab9- (h) and vps35- (i) mutant DT. (j,k) Control and wash^Δ185-mutant embryos stained with CBP. (l) Quantification of DT length in control and wash mutation. Double (**) asterisks represent a significant difference between wash mutant and the control by Student’s t-test (0.001<P<0.01). (m,n) Control and wash-mutant embryos stained by CBP and Serp. White arrowheads in n show decrease of Serp but not chitin expression in the lumen. (o) Composition and organization of retrieval machinery for vesicle budding. WASH, actin, Rab9 and Vps35 sequentially localize at the endosomal tubular structure. They constitute the main components of retrieval machinery for vesicle budding from endosomes. (p) Two major pathways of vesicle trafficking regulate luminal materials. At the late stage of Drosophila tracheal development, the endocytosed luminal protein Serp is sorted into endosomal-budding membranes enriched with WASH, actin, Rab9 and Vps35, and recycled to the TGN for secretion by a Dia-independent pathway, whereas the secretion of other luminal markers, such as 2A12 and Pio, uses a Dia-dependent pathway. Thus, two distinct luminal protein trafficking pathways separately regulate tube morphogenesis. See text for details. Scale bar, 10 μm.