Vesicle-mediated transport of ALIX and ESCRT-III to the intercellular bridge during cytokinesis

Abstract Cellular abscission is the final step of cytokinesis that leads to the physical separation of the two daughter cells. The scaffold protein ALIX and the ESCRT-I protein TSG101 contribute to recruiting ESCRT-III to the midbody, which orchestrates the final membrane scission of the intercellular bridge. Here, we addressed by which mechanisms ALIX and the ESCRT-III subunit CHMP4B are transported to the midbody. Structured illumination microscopy revealed gradual accumulation of ALIX at the midbody, resulting in the formation of spiral-like structures extending from the midbody to the abscission site, which strongly co-localized with CHMP4B. Live-cell microscopy uncovered that ALIX appeared together with CHMP4B in vesicular structures, whose motility was microtubule-dependent. Depletion of ALIX led to structural alterations of the midbody and delayed recruitment of CHMP4B, resulting in delayed abscission. Likewise, depletion of the kinesin-1 motor KIF5B reduced the motility of ALIX-positive vesicles and caused delayed recruitment of ALIX, TSG101 and CHMP4B to the midbody, accompanied by impeded abscission. We propose that ALIX, TSG101 and CHMP4B are associated with endosomal vesicles transported along microtubules by kinesin-1, leading to their directional transport to the cytokinetic bridge and midbody, thereby contributing to their function in abscission.