Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation

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.

An ESCRT-LEM domain inner nuclear membrane protein surveillance system is poised to directly monitor the integrity of the nuclear envelope barrier and nuclear transport system

“…As the latter impact the recruitment of Vps4, it is possible that the sequence of events of nuclear envelope sealing are similar to those found at ILVs during MVB formation with a fundamental difference being that a nuclear envelope hole instead of a single continuous lipid bilayer, is the likely initial "substrate" for ESCRT-III action. Indeed, in the more artificial scenarios that we present here, be it at sites of chm7OPEN accumulation or Chm7 in vps4Δ cells (Figures 6 and 7), there are obvious parallels between the morphologies observed at the INM and those at the plasma membrane (von Schwedler et al, 2003;Hanson et al, 2008;Morita et al, 2011;Cashikar et al, 2014;Jackson et al, 2017), and at endosomes (Adell et al, 2014;Wenzel et al, 2018) in the context of mutants that stall or inhibit membrane scission. They also resemble more physiological circumstances like in the ILVs of A. thaliana (Buono et al, 2017) and C. elegans (Frankel et al, 2017), which resemble beads on a string.…”

A toolbox for the immunomagnetic purification of signaling organelles

“…Occurrence of clathrin at later time points seems odd at first glance, but besides being involved in clathrin‐mediated endocytosis clathrin is also involved in trafficking from endosomes and the trans‐golgi network and formation of MVB. These processes are regulated by other adaptor proteins . Hence, we cannot distinguish between clathrin derived from PM derived vesicles or vesicles derived from intracellular organelles.…”

“…These processes are regulated by other adaptor proteins. [55][56][57] Hence, we cannot distinguish between clathrin derived from PM derived vesicles or vesicles derived from intracellular organelles. Such analyses should be made more detailed on the basis of the respective adaptor proteins.…”

ESCRT subunit CHMP4B localizes to primary cilia and is required for the structural integrity of the ciliary membrane

“…CHMP4B polymers induce the curvature of the endosomal membrane during the formation of multivesicular bodies 50 and also maintain a narrow tubular shape of the plasma membrane before viral budding. 51 CHMP4B localization at primary cilia suggests a direct role of CHMP4B in the stabilization of the highly curved ciliary membrane.…”