Figure 1. Rab11 compartments of Drosophila secondary cells contain intraluminal vesicles with specific cargos.

• A
  Schematics illustrate the fly accessory glands and associated secondary cells, highlighting their acidic and non‐acidic exosome‐forming compartments. First panel shows male fruit fly and its accessory glands. Boxed region is enlarged in second panel, revealing secondary cells (SCs; black dots), with red line indicating the plane of section through the AG lumen, which is used to generate the transverse view through a SC and surrounding main cells within the epithelial layer in the third panel. The unusually large, acidic late endosomes and lysosomes (Rab7‐positive; magenta) and non‐acidic compartments, characteristic of SCs, which we demonstrate here contain intraluminal vesicles (ILVs, some of which are Rab11‐positive, yellow), are labelled. The red line in the third panel shows the basal SC plane of section in the fourth panel and images in this figure.
• B–F
  Basal views through living SCs, with dashed white circles approximating the outline of a single SC, and acidic compartments marked by the vital dye LysoTracker® Red (magenta). In merge images, a single non‐acidic (C, D and F) and acidic (E) compartment containing intraluminal vesicles (ILVs) is boxed and magnified in the right panel (Zoom). ILVs appear as membrane‐delineated vesicles, using super‐resolution 3D‐structured illumination (3D‐SIM) microscopy for the brighter overexpressed GFP‐tagged constructs (yellow; C and F). However, ILVs appear only as puncta, using lower resolution wide‐field microscopy for the fainter endogenously expressed YFP‐tagged Rab GTPases (yellow; D and E). (B) Wide‐field fluorescence image, including differential interference contrast (DIC), of SC expressing a GFP‐tagged version of human CD63 (CD63‐GFP). CD63‐GFP expression is apparent on the limiting membranes of non‐acidic compartments and their ILVs and also on the limiting membranes of the enlarged acidic compartments. Most large non‐acidic compartments are Rab11‐positive (D) and contain dense‐core granules, which have a “fried egg” appearance with DIC (arrowheads) (Corrigan et al, 2014; Redhai et al, 2016). (C) 3D‐SIM image of CD63‐GFP-expressing SC. Arrow highlights CD63‐GFP-marked ILVs (Zoom). Many more ILVs are apparent in non‐acidic compartments in a complete Z‐stack of a non‐acidic compartment (Movie EV1). (D) Wide‐field fluorescence image of an SC expressing a YFP‐Rab11 gene trap. YFP‐Rab11 marks the limiting membranes of most non‐acidic compartments and internal puncta (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1B). (E) Wide‐field fluorescence image of SC expressing a YFP‐Rab7 gene trap. YFP‐Rab7 marks the limiting membranes of acidic compartments and internal puncta (arrow in Zoom). Enlarged acidic compartments are also present in adjacent main cells. (F) 3D‐SIM image of SC expressing a GFP‐tagged version of Breathless (Btl‐GFP). Btl‐GFP marks the limiting membranes of non‐acidic compartments and their ILVs (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1C). Images from 6‐day-old male flies shifted to 29°C at eclosion. This induces GAL4/UAS-dependent SC transgene expression in (B, C and F). The genotypes of flies carrying multiple transgenes are as follows: w; P[w ⁺ , UAS‐CD63-GFP] P[w ⁺ , tub‐GAL80 ^ts ]/+; dsx‐GAL4/+ (B and C); w; P[w ⁺ , tub‐GAL80 ^ts ]/+; dsx‐GAL4/P[w ⁺ , UAS‐btl-GFP] (F).

Data information: Scale bar in B–F (5 μm) and in C–F, Zoom (2 μm).