Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal novel actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among those proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins were highly recruited first at the midbody then at the abscission site, and their depletion delayed abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 were essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, revealing novel common requirements in cytokinesis, exosome formation and HIV budding.

The Flemmingsome reveals an ESCRT-to-membrane coupling required for completion of cytokinesis