Abstract
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 actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these 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 are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are 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, uncovering common requirements in cytokinesis, exosome formation and HIV budding.
Highlights
Cytokinesis requires the constriction of endosomal sorting complexes required for transport (ESCRT)-III filaments on the side of the midbody, where abscission occurs
ALIX directly interacts with ESCRT-III40 and we previously showed that syntenin can bind directly and simultaneously to the cytoplasmic tail of syndecans in vitro[41,42]
Differential centrifugations helped to enrich intact MidBody Remnant (MBR) from EDTA-treated HeLa cells (“Midbody Remnant Enriched fraction” or “MBRE”, Fig. 1a) expressing the midbody-localized kinesin green fluorescent protein (GFP)-MKLP245
Summary
Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. The endosomal sorting complexes required for transport (ESCRT) machinery plays a critical and evolutionarily conserved role in cytokinetic abscission, both in Eukaryotes and in Archea[20,21,22,23,24,25,26,27,28,29,30,31] This machinery is composed of several protein complexes (ESCRT-0 to III) and culminates with the polymerization of filaments made of ESCRT-III components that contract in the presence of the ATPase VPS4 and ATP31–35. TSG101 and ESCRT-associated protein ALIX, the entire ESCRT machinery[29,37] After this initial recruitment to the midbody itself and prior to abscission, the ESCRT-III machinery is progressively enriched to the future abscission site on the midbody side[12,13,20,21,25,30,31,36,38,39]
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