Fig. 5: Persistent recruitment of ESCRT-III to the abscission site depends on the syndecan4-syntenin-ALIX module. | Nature Communications

Fig. 5: Persistent recruitment of ESCRT-III to the abscission site depends on the syndecan4-syntenin-ALIX module.

From: The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

Fig. 5

a Cells treated with the indicated siRNAs were stained for endogenous CHMP4B and acetylated-tubulin. CHMP4B localization in late cytokinetic bridges was classified into three categories: (1) no staining, (2) CHMP4B localized only at the midbody, or (3) CHMP4B localized both at the midbody and at the abscission site (see representative images). The proportion of each category was quantified in control and depleted cells. n = 49–83 cells, N = 3 independent experiments. b–d Cells were depleted for either ALIX (b), syntenin (c), or syndecan-4 (d) and transfected with control plasmid (−) or with plasmids encoding either wild type or mutant versions of ALIX, syntenin, or syndecan-4. Upper panels: western blots were revealed with the indicated antibodies. Loading controls: GAPDH or β-tubulin. Lower panels: percentage (mean ± SD) of bridges with CHMP4B at the abscission site in each condition. b n = 28–31 cells; c n = 32–103 cells, d n = 33–56 cells. N = 3 independent experiments. e Cells were treated with control, ALIX, syntenin, syndecan-4, TSG101 siRNAs, alone or in combination. CHMP4B localization was quantified as in (a). n = 26–66 cells, N = 3 independent experiments. f–i HeLa cells stably expressing CHMP4B–GFP were treated with either control (f), ALIX (g), syntenin (h), or syndecan-4 (i) siRNAs and recorded by spinning-disk confocal time-lapse microscopy every 10 min. Zooms of the intercellular bridges are displayed. Time 0 corresponds to the frame preceding the arrival of CHMP4B at the midbody. Brackets mark the midbody. Arrows point toward pools of CHMP4B on the midbody side. Red arrows correspond to the CHMP4B leading to abscission (last time frame). Yellow and cyan arrows point to transient and unstable CHMP4B pools observed in depleted cells. See corresponding Supplementary Movies 5–8. j Quantification of cytokinesis with abnormal CHMP4B-GFP behavior (disappearance of signal on the side of the midbody and fragmented cones) for each condition reported in (f–i). n = 24–38 cells from 5 independent experiments. One-sided Fisher’s exact tests. a, f–i Scale bars: 2 μm. Brackets mark midbodies in (a, f–j). Panels a–e: means ± SD and one-sided Student’s t tests. NS nonsignificant.

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