Mingli Zhu

Cilia-derived extracellular vesicles (EVs) mediate cell-cell communication and remove signaling molecules from cilia. The Hedgehog receptor Smoothened (SMO) is selected for packaging into ciliary EVs by ubiquitin chains, but how cargo selection is coupled to EV budding remains unknown. In this work, working in mouse kidney cells, we found that the ubiquitin reader endosomal sorting complex required for transport–0 (ESCRT-0) orchestrates both cargo selection and EV biogenesis. Unlike the conventional ESCRT pathway, SMO secretion did not require the ESCRT-III membrane scission machinery. Instead, ESCRT-0 condensed at the ciliary tip, where it clustered ubiquitinated SMO to drive EV biogenesis. Disrupting ESCRT-0 condensation blocked SMO secretion, and combining forced recruitment of ESCRT-0 with optogenetic clustering of SMO was sufficient to reconstitute EV budding. Thus, ESCRT-0 condensation and cargo clustering constitute a minimal module for ESCRT-III–independent reverse-topology budding.