The endosomal sorting complex required for transport (ESCRT) protein machinery comprises four complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, that facilitate receptor sorting into the lumen of multivesicular endosomes (MVEs) in order to terminate signalling receptors for final degradation within the lysosomes. Even though ESCRT proteins appear to be essential for the biogenesis of MVEs in Saccharomyces cerevisae, it is not clear whether ESCRT-independent pathways for MVE biogenesis exist in higher organisms. In this study we maximized inhibition of ESCRT-dependent pathway by depleting cells of key subunits of all four ESCRTs and followed MVE formation and epidermal growth factor (EGF) receptor (EGFR) traffic using electron and confocal microscopy. There was a dramatic alteration in the morphology of components of the endocytic pathway in ESCRT-depleted cells, but early and late endosomes stayed clearly differentiated. Importantly, although EGFinduced formation of MVEs was highly sensitive to ESCRT depletion, EGF-independent formation of MVEs could still occur. The MVEs remaining in ESCRT-depleted cells contained enlarged intralumenal vesicles into which EGFRs were not sorted. Our observations suggest that both ESCRT-dependent and ESCRT-independent mechanisms of MVE biogenesis exist in mammalian cells.

At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus. During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei. How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair, is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA (ATPase associated with diverse cellular activities) ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III functions in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope-microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission.

The discovery of the endosomal sorting complex required for transport (ESCRT) machinery through studies of a subgroup of Saccharomyces cerevisiae vacuolar protein sorting (vps) mutants by Emr and colleagues (1-3) has opened new avenues for understanding how receptor ubiquitination is coordinated with lysosomal (vacuolar) degradation. As reviewed elsewhere (4,5), the ESCRT machinery consists of four subcomplexes, ESCRT-0, -I, -II and -III, plus the ATPase VPS4 and accessory factors that promote disassembly and recycling of ESCRT-III oligomers. ESCRT-0, -I and -II contain ubiquitin-binding subunits, and recent studies with giant unilamellar vesicles and recombinant ESCRTs have suggested that ESCRT-0 sequesters ubiquitinated cargo, ESCRT-I and -II mediate invaginations of the endosomal membrane into which cargo is sorted, and ESCRT-III functions to pinch off the neck of the invagination so that an intraluminal vesicle (ILV) is formed (6) (Figure 1). Deubiquitinating enzymes that are recruited by ESCRT-III serve to deubiquitinate cargo at the stage of ESCRT-III engagement so that the cargo molecules that enter the ILVs are without ubiquitin (7). This ensures that the ESCRT pathway does not cause a depletion of the cytosolic ubiquitin pool.Sorting of proteins into ILVs of multivesicular endosomes (MVEs) can have several alternative purposes, although the ultimate consequence of such sorting is the exposure of the ILV and its content to hydrolases after fusion of the MVEs with lysosomes. The first ESCRT-dependent cargo to be studied was S. cerevisiae carboxypeptidase S, which uses the ESCRT pathway for its transport to the vacuole (lysosome) lumen where the ILV membrane becomes degraded and carboxypeptidase S proteolytically activated (1). Thus, in this example, the ESCRT pathway mediates correct delivery of a hydrolase to the vacuole lumen. Another function of the ESCRT pathway is as part of a quality control system for plasma membrane proteins. As first shown with diseaseassociated mutants of the cystic fibrosis transmembrane conductance regulator, misfolded plasma membrane proteins undergo ubiquitination and ESCRT-mediated lysosomal degradation (11). A third function of ILV sorting is downregulation of surface-exposed solute transporters in response to physiological cues. The epithelial Na + channel (ENaC) is an example of an ion channel that undergoes ESCRT-dependent degradation in lysosomes, a degradation that can be counteracted by hormones that increase apical Na + conductance through increasing the number of ENaC molecules in the apical plasma membrane (12). A further physiological role of ILV sorting of membrane proteins is the downregulation of adhesion molecules and gap junction molecules in response to physiological stimuli, as exemplified by E-cadherin, α5β1-integrin and connexin43 (13-15). Likewise, ubiquitination and ILV sorting of MHC-II molecules are important for controlling maturation and functions of antigenpresenting cells (16)(17)(18)(19). Finally, ILV sorting plays an important role in the regul...