Fig. 3: ALIX directly recruits syntenin and syntenin directly recruits syndecan-4 to the cytokinetic bridge. | Nature Communications

Fig. 3: ALIX directly recruits syntenin and syntenin directly recruits syndecan-4 to the cytokinetic bridge.

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

Fig. 3

a Western blots of protein extracts from cells treated with control, ALIX, syntenin (Synt), or syndecan-4 (SDC4) siRNAs revealed with the indicated antibodies. Loading control: β-tubulin. b Left panels: representative intercellular bridges from control, ALIX or syndecan-4 siRNA-treated cells stained for acetylated-tubulin and endogenous syntenin, as indicated. Right panel: percentage (mean ± SD) of bridges positive for syntenin after control, ALIX and syndecan-4 depletion. n = 31–53 cells, N = 3 independent experiments. (c) Left panels: representative intercellular bridges from control- or ALIX-depleted cells expressing either control (Empty), wild-type ALIX or ALIX F676D mutant (unable to interact with syntenin), and stained for acetylated-tubulin and endogenous syntenin, as indicated. Right panel: percentage (mean ± SD) of bridges with syntenin recruitment in the corresponding conditions. n = 25–31 cells, N = 3 independent experiments. d Left panels: representative intercellular bridges from syntenin-depleted cells expressing either GFP-syntenin wild-type, GFP-syntenin ΔALIX (unable to interact with ALIX) or GFP-syntenin ΔSDC (unable to interact with syndecan-4). Acetylated-tubulin and GFP signals are shown. Right panel: percentage (mean ± SD) of bridges with GFP-tagged syntenin recruitment in the corresponding conditions. n = 14–33 cells, N = 4 independent experiments. e Left panels: representative intercellular bridges from control, ALIX or syntenin siRNA-treated cells and stained for acetylated-tubulin and endogenous syndecan-4, as indicated. Right panel: percentage (mean ± SD) of bridges positive for syndecan-4 after control, ALIX and syntenin depletion. n = 30–40 cells, N = 3 independent experiments. f Left panels: representative intercellular bridges from cells expressing either GFP-syndecan-4 wild-type, GFP-syndecan-4 ΔECD (deleted from its entire extracellular domain) or GFP-syndecan-4 ΔSynt (unable to interact with syntenin). Acetylated-tubulin and GFP signals are shown. Right panel: percentage (mean ± SD) of bridges with GFP-tagged syndecan-4 recruitment in the corresponding conditions. n = 23–41 cells, N = 3 independent experiments. b–f Scale bars: 2 μm. Brackets mark the midbody. Panels b–f: one-sided Student’s t tests. NS non significant.

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