Figure 2.

ER tubules define functional constrictions along dynasore-stalled tubular endosomes. (A) Images of live Cos-7 cells expressing mCh-Rab5 and GFP-Sec61β show that dynasore treatment elongates early endosomes while ER is unaffected. (B) Merged images of live Cos-7 cells expressing GFP-Rab5, BFP-Sec61β, and mCh-FAM21 following dynasore treatment. (C) Magnified image of endosome boxed in B. Images reveal that FAM21 concentrates at positions along the tubular endosome where Rab5-labeling is reduced and ER tubules intersect these FAM21 domains. (D) Line-scan analysis of relative FI of the endosome shown in C confirms that FAM21and ER co-localize with Rab5-labeled endosome constrictions (marked by corresponding blue and yellow arrows). (E) A model outlines the FRAP technique used to test for a cargo diffusion barrier (D.B.) along tubular endosomes. (F) A live Cos-7 cell expressing BFP-Rab5, mCh-Sec61β, and GFP-TfR (membrane-bound cargo) were treated with dynasore to elongate endosomes. Endosomes were photobleached in the region indicated (dotted yellow circle). Images were taken at times indicated during the recovery (see kymograph). See also Movie S3. (G) A graph of relative FI during time points shown reveals a D.B. limits the recoveryof TfR at the position of an ER-marked constriction (at yellow arrow). The D.B./constriction maintains contact with the ER over time (marked by yellow arrows). (H-I) Another example as in F-G. Scale bars = 5 μm in A and B; 1 μm in C, F, and H.