Figure 5.

The ER is recruited to FAM21-marked sorting domains prior to fission. (A) Merged image of a live Cos-7 cell expressing GFP-Rab7, BFP-Sec61β, and mCh-FAM21 that was also pulse-labeled with EGF conjugated to Alexa Fluor^® 647 (cargo in blue). (B) Magnified time-lapse images of an endosome in A shows the tip of an ER tubule tracking with a FAM21 punctum at the bud of a late endosome (yellow arrowhead). See also Movie S7. (C) The number of FAM21 puncta on late endosome buds that maintain contact with ER over a 2 min time course (165 puncta from 31 cells). (D) Merged image of a live Cos-7 treated as in A. (E) Magnified images of boxed region in D shows a late endosome undergoing fission. A bud labeled by FAM21 extends from the late endosome and undergoes constriction and division. An ER tubule (follow white arrow) is recruited to the FAM21 bud just before fission (at the blue arrow). The location of the exiting Rab7 endosome bud is marked by a yellow arrowhead. See also Movie S8. (F) Line-scan analysis of endosome shown in E relates the timing and position of ER tubule recruitment relative to Rab7, FAM21, and EGF. (G) The percent of FAM21-marked late endosome division events that co-localize with ER tubules during cargo segregation, (n=36 from 31 cells). Scale bars = 5 μm in A and D; 1 μm in B and E.