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. Author manuscript; available in PMC: 2019 Sep 20.
Published in final edited form as: Cell. 2018 Sep 13;175(1):254–265.e14. doi: 10.1016/j.cell.2018.08.030

Figure 3. Dynamic TMCC1 domains accumulate at the position and timing of endosome bud fission.

Figure 3.

(A-D) Merged images of Cos7 cells expressing BFP-Sec61β (ER in red), mCh-Rab7 (LE in blue) and low levels of (A) GFP-TMCC1, (B) GFP-TMCC1 (Δ1-570), (C) GFP-Protrudin, or (D) GFP-VAPA (green). Magnified images of the boxed regions show examples of endosome pre (t=0s) and post (t=2s) fission. White arrowheads highlight vacuolar ER-LE MCS and white arrows highlight the bud ER-LE MCS where fission occurs. (A) Note example of a GFP-TMCC1 dynamic domain (green) that accumulates at a MCS marking the site of fission (arrow). (B) In contrast, GFP-TMCC1(Δ1-570) signal (green) localizes throughout the ER and does not accumulate at the MCS with the bud. (C) Conversely, GFP-Protrudin domains (green) accumulate at vacuolar ER-LE MCS but not at the bud MCS. (D) GFP-VAPA distribution (green) resembles that of a general ER protein. (E) Cartoon diagrams how vacuolar ER-LE MCS and bud ER-LE MCS regions of interest (ROIs) were drawn on images like those shown in (A-D) in order to quantitatively analyze and graph relative protein distribution as shown in (F). (F) TMCC1, TMCC1(Δ1-570), Protrudin and VAPA enrichments quantified at each separate ROI by comparing each fluorescent signal to the general ER marker fluorescent signal in the pre fission frame of multiple fission events (n=41, n=32, n=34, and n=32 fission events respectively). (G) Quantification of TMCC1, TMCC1(Δ 1-570), Protrudin and VAPA protein signal enrichment at LE fission events described in (F) by comparing bud fission ROI to the vacuole ROI (Bud/Vacuole). TMCC1 protein gives a higher Bud/Vacuole enrichment ratio (2.07). Protrudin has a significantly lower Bud/Vacuole ratio (0.83) emphasizing more contact with the vacuole than with the bud. Mutant TMCC1 and VAPA have an equal ratio of Bud/Vacuole suggesting no MCS enrichment. Scale bars in A-D = 5 μm and 1μm in zooms. In the graphs in F-G, median=black line, mean=red line. *p<0.05, *** p<0.0005, N.S. not significant, two-tailed test.