CI-MPR segregates in SNX1/2–SNX5/6 tubular profiles which are not decorated with retromer. (A) Endogenous CI-MPR localizes to a highly packed vesicular cluster that colocalized with the TGN and partially colocalized with retromer-positive endosomes. HeLa cells were fixed and immunostained for endogenous TGN46 (TGN marker), VPS35 (retromer), SNX1, and SNX2 (both retromer-linked SNX-BARs). Bars: (main images) 20 µm; (zooms) 5 µm. (B, top) CI-MPR colocalizes with SNX2-positive subdomains on enlarged endosomes. HeLa cells were transfected with BFP-Rab5Q79L and immunostained for endogenous CI-MPR, SNX2, and VPS35 after 48 h. The dashed line in the top left image indicates the contour of the nucleus, and the dashed line on the top right refers to the enlarged endosome from which the intensity line scan was measured. Bars: (main images) 10 µm; (zooms) 5 µm. (B, bottom) Line scan of signal intensity across the circumference of enlarged endosome. (B, right) Distribution of CI-MPR in retromer subdomains; n = 3 independent experiments. CI-MPR signal was quantified in 36 enlarged endosomes (means ± SEM). (C) Overexpression of a WT GFP–CI-MPR chimera leads to the formation of extended CI-MPR tubules, some of which are positive for endogenous SNX1. HeLa cells were transfected with GFP–CI-MPR chimera WT and immunostained for SNX1 after 48 h. Bars: (main images) 20 µm; (zooms) 10 µm. (D) A subpopulation of GFP–CI-MPR chimera tubules is decorated with endogenous SNX1 but not endogenous VPS35. HeLa cells were transfected with GFP–CI-MPR chimera WT and immunostained for SNX1 and VPS35 after 48 h. Bars, 20 µm. (E) Some SNX1/2–SNX5/6-decorated CI-MPR–containing tubules were observed to emanate from VPS35-positive endosomes. HeLa cells were transfected with GFP–CI-MPR chimera WT and immunostained for SNX6 and VPS35 after 48 h. The box in the leftmost panel indicates the area depicted in the other four panels. Bars: (main images) 20 µm; (zooms) 2 µm.