Rab32 and Rab38 are primarily present in immature DGs. (A-C) DGs were labeled with mepacrine in live MEG-01 cells expressing Cherry-Rab38 and visualized by spinning-disk confocal fluorescence microscopy; 95% ± 2% of structures containing Cherry-Rab38 (40 cells) also contain mepacrine. (A) A structure containing the highest amount of mepacrine and low Cherry-Rab38 levels is indicated with a green arrowhead, a structure with high concentration of Cherry-Rab38 and low mepacrine with a red arrowhead, and a structure with intermediate amounts of both markers with a yellow arrowhead (bar represents 5 μm). (B) Both organelles and vesicles are labeled with Cherry-Rab38, which is shown as an inset in panel C (bar represents 5 μm). (C) Although mepacrine is only present in the organelles, Cherry-Rab38 is also present in the vesicle (bar represents 500 nm). (D) Live MEG-01 cell coexpressing LAMP2-Cherry, as a DG marker, and GFP-Rab38 imaged by spinning-disk confocal fluorescence microscopy; 88 ± 4% of structures containing GFP-Rab38 (37 cells) also contain LAMP2-Cherry. Bar represents 5 μm. (E) A close-up view of structures from panel D shows a reverse correlation between the amount of Rab38 and the DG marker. (F) Fluorescence intensity line scan of the structures shown in panel E (merge panel). A.U., arbitrary units. (G-L) Immunogold electron microscopy images of immature DGs from MEG-01 cells subjected to HPF using antibodies against LAMP2 (18 nm) and Rab32 (12 nm). LAMP2 is present in 73% of the organelles label with Rab32 (n = 84 organelles). Original magnifications were ×15,000, ×20,000, ×15,000, ×20,000, ×25,000, and ×20,000, respectively. Bars represent 200 nm.