Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery.

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.

Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation.

Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

The VPS-20 subunit of the endosomal sorting complex ESCRT-III exhibits an open conformation in the absence of upstream activation.

Members of the endosomal sorting complex required for transport (ESCRT) machinery function in membrane remodelling processes during multivesicular endosome (MVE) biogenesis, cytokinesis, retroviral budding and plasma membrane repair. During luminal vesicle formation at endosomes, the ESCRT-II complex and the ESCRT-III subunit vacuolar protein sorting (VPS)-20 play a specific role in regulating assembly of ESCRT-III filaments, which promote vesicle scission. Previous work suggests that Vps20 isoforms, like other ESCRT-III subunits, exhibits an auto-inhibited closed conformation in solution and its activation depends on an association with ESCRT-II specifically at membranes [1]. However, we show in the present study that Caenorhabditis elegans ESCRT-II and VPS-20 interact directly in solution, both in cytosolic cell extracts and in using recombinant proteins in vitro. Moreover, we demonstrate that purified VPS-20 exhibits an open extended conformation, irrespective of ESCRT-II binding, in contrast with the closed auto-inhibited architecture of another ESCRT-III subunit, VPS-24. Our data argue that individual ESCRT-III subunits adopt distinct conformations, which are tailored for their specific functions during ESCRT-mediated membrane reorganization events.

Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly.

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts.