Skip to main content
. Author manuscript; available in PMC: 2020 Jul 8.
Published in final edited form as: Curr Biol. 2019 Jun 13;29(13):2174–2182.e7. doi: 10.1016/j.cub.2019.05.050

Figure 2. Assembly of the ESCRT machinery of abscission is spatiotemporally coupled to the septin double ring.

Figure 2.

(A) SEPT9 localization with respect to endogenous TSG101, VPS25, CHMP4B and GFP-CHMP6 in the outlined ICB areas (arrows) of MDCK cells. Arrowheads are fixed in position and point to SEPT9-ESCRT overlap.

(B-D) Western blot (B) of MDCK-GFP-CHMP4B cell lysate and time-lapse images of GFP-CHMP4B with SEPT9-mCherry (C) and mCherry-SEPT2 with GFP-CHMP2A (D). Plots show the mean pixel intensity of septins (magenta) and GFP-CHMP2A/CHMP4B (green) across the width of the midbody. Asterisks highlight fluorescence overlap and green arrows point to the formation of ESCRT-III cones.

(E) Mean fluorescence intensity of GFP-CHMP4B and SEPT9-mCherry rings after normalization to a scale of 0 (min) to 1 (max) prior to abscission (t=0; n = 3 cells).

(F-H) Quantification of the fluorescence intensity of SEPT9-mCherry (percent of maximum) in the frame preceding the emergence a GFP-CHMP4B cone (F), and timing of SEPT9-mCherry ring disassembly and GFP-CHMP4B cone formation with respect to the ICB cut (t = 0) in individual cells (G) and as an average ± SD (H; n = 12 cells). See also Figure S2 and Videos S3 and S4.