Figure 1.

Early endosome fission occurs at ER contact sites. (A) Merged images of a live Cos-7 cell expressing mCh-Rab5 (EE in cyan), GFP-Rab4 (EE in red), and BFP-Sec61β (ER in green) that is pulse-labeled with EGF conjugated to Alexa Fluor^® 647 (cargo in blue). (B) Magnified time-lapse images of the region boxed in A shows an example of early endosome fission. Merged images show the relative location of Rab4, Rab5, ER and EGF, as indicated over time. The exiting Rab4⁺ bud is marked by a yellow arrowhead. See also Movie S1. (C) Trace outline of the endosome shown in B and the corresponding line-scan analysis of the relative fluorescence intensity (FI) of Rab4, Rab5, ER and EGF for time points: t=0s (Pre-ER), t=10s (Pre-Fission), and t=15s (Post-fission). Images in B and line-scan in C reveal that a dynamic ER tubule (marked by a blue arrow) is recruited to the divide between Rab4 compartments just before fission (at t=10s). (D) Merged images taken of a live Cos-7 cell transfected as in A that is instead pulse-labeled with Tf conjugated to Alexa Fluor^® 647 (cargo in blue). (E) Zoom from D shows the relative localization of Rab4, Rab5, ER and Tf as indicated over time. An ER tubule (marked by blue arrow) is again recruited to the divide between Rab4 compartments just before fission (t=35s). An exiting Rab4⁺/Tf⁺ bud is marked by a yellow arrowhead. See Movie S2. (F) Line-scan analysis of relative FI depicts the position of a dynamic ER tubule to the position and timing of endosome fission for time points: t=0s (Pre-ER), t=35s (Pre-Fission), and t=45s (Post-Fission). Scale bars = 5 μm in A and D; 1 μm in B and E. s = seconds.