Abstract
The endosomal sorting complex required for transport (ESCRT) machinery mediates cargo sorting, membrane deformation and membrane scission on the surface of endosomes, generating intraluminal vesicles (ILVs) to degrade signaling receptors. By live-cell imaging of individual endosomes in human cells, we find that ESCRT proteins are recruited in a repetitive pattern: ESCRT-0 and -I show a gradual and linear recruitment and dissociation, whereas ESCRT-III and its regulatory ATPase VPS4 display fast and transient dynamics. Electron microscopy shows that ILVs are formed consecutively, starting immediately after endocytic uptake of cargo proteins and correlating with the repeated ESCRT recruitment waves, unraveling the timing of ILV formation. Clathrin, recruited by ESCRT-0, is required for timely ESCRT-0 dissociation, efficient ILV formation, correct ILV size and cargo degradation. Thus, cargo sorting and ILV formation occur by concerted, coordinated and repetitive recruitment waves of individual ESCRT subcomplexes and are controlled by clathrin.
Highlights
The endosomal sorting complex required for transport (ESCRT) machinery mediates cargo sorting, membrane deformation and membrane scission on the surface of endosomes, generating intraluminal vesicles (ILVs) to degrade signaling receptors
The HRS770 mutant showed a moderate increase in small ILVs (Supplementary Fig. 7D). These results indicate a role for clathrin in the formation of ESCRT-dependent ILVs, which is important for epidermal growth factor receptors (EGFRs) degradation
By following endosomes containing EGFR as a cargo, we have been able to combine live and electron microscopy to reveal that the formation of an multivesicular endosomes (MVEs) is mediated by characteristic and repetitive concerted recruitment waves of the whole ESCRT machinery at endosomes, starting immediately after cargo internalization
Summary
The timing of endosomal ESCRT recruitment and ILV biogenesis has remained unknown, as has the mechanistic function of the endosomal clathrin coat. Neck width of Ω-shaped buds n.s. Phase 1: The slow and linear accumulation of HRS on the endosomal membrane (duration approximately 120 s) reflects cargo sorting: ESCRT-0, together with clathrin, sequesters cargo on the limiting membrane of an endosome, forming a sorting microdomain which is visible as an electron-dense coat[5,36] Since ESCRT-I/II were shown to deform membranes in vitro[48], and ESCRT-0 and ESCRT-I are synchronously recruited to endosomes, we suggest that cargo sorting and membrane deformation may occur in parallel, starting in phase 1 This is strengthened by our finding that clathrin, which is recruited in phase 1, is required for the normal size of pits and U-shaped buds.
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