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. Author manuscript; available in PMC: 2015 Nov 20.
Published in final edited form as: Cell. 2014 Nov 20;159(5):1027–1041. doi: 10.1016/j.cell.2014.10.023

Figure 4.

Figure 4

Late endosome division occurs at ER contact sites. (A) A Cos-7 cell expressing mCh-Rab7 (late endosome) and GFP-Sec61β was pulse-labeled with EGF conjugated to Alexa Fluor® 647 (cargo in blue). (B) Magnified image of the region boxed in A shows an example of lateendosome fission. Merged images show the relative location of Rab7, EGF and the ER, as indicated over time. See also Movie S5. (C) Traced outline and the corresponding line-scan analysis of relative FI through the equator of the dividing endosome shown in B. Relative FI of Rab7, ER, and EGF were performed for time points: t=0s (Pre-ER), t=35s (Pre-fission), and t=40s (Post-fission). Note that a dynamic ER tubule is recruited to the position and timing of endosome constriction and fission (compare position of the ER tubule marked by an arrow at t=0s to t=20s in B and C). (D) Percent of late endosome division events that co-localize with ER tubules (n=29 from 24 cells). (E) Image of a Dnm2-depleted cell expressing mCh-Rab7 (red) and GFP-Sec61β (green) and pulse-labeled with EGF conjugated to Alexa Fluor® 647 (blue). (F) Magnified image of box in E shows an elongated endosome that moves until it becomes associated with an ER tubule (white arrow) at the site of endosome division (blue arrow). The location of the Rab7 endosome bud (yellow arrowhead) is shown. See also Movie S6. (G) The percent of late endosome fission events that co-localize with ER tubules in the absence of dynamin-2 (out of 12 events from 8 cells). (H) Immunoblot analysis shows efficient depletion of Dnm2 in cells transfected with Dnm2 siRNA (right lane) relative to control (left lane). (I) The endosome image surface covered by ER tubule crossing was measured for all 12 fission events. In example shown, the top row shows indicated fluorescence markers and bottom row shows thresholded images. (J) Table summarizes predicted frequency of ER-marked LE fission (21.24% based on coverage) versus the actual frequency of ER-marked fission (100%). ***, P<0.001, Fisher’s exact test. Scale bars = 5 μm in A and E; 1 μm in B and F.

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