Biochemical Approaches to Studying Caenorhabditis elegans ESCRT Functions In Vitro.

Our fundamental understanding of the roles played by the endosomal sorting complex required for transport (ESCRT) machinery in cells comes from interdisciplinary approaches that combine numerous in vivo and in vitro techniques. Here, we focus on methods used to biochemically characterize Caenorhabditis elegans ESCRT components in vitro, including the production and characterization of recombinant ESCRT complexes and their use in membrane interaction studies. Key methodologies used include gel filtration chromatography, glycerol density gradient analysis, multi-angle light scattering, liposome co-flotation, and single-liposome fluorescence microscopy. Collectively, these studies have enabled us to define subunit stoichiometry of soluble C. elegans ESCRT complexes and to demonstrate that the late-acting ESCRT-III complex facilitates membrane bending and remodeling, at least in part by virtue of its ability to sense the curvature of lipid bilayers.

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.

Phosphoregulation of the C. elegans cadherin-catenin complex.

Adherens junctions play key roles in mediating cell-cell contacts during tissue development. In Caenorhabditis elegans embryos, the cadherin-catenin complex (CCC), composed of the classical cadherin HMR-1 and members of three catenin families, HMP-1, HMP-2 and JAC-1, is necessary for normal blastomere adhesion, gastrulation, ventral enclosure of the epidermis and embryo elongation. Disruption of CCC assembly or function results in embryonic lethality. Previous work suggests that components of the CCC are subject to phosphorylation. However, the identity of phosphorylated residues in CCC components and their contributions to CCC stability and function in a living organism remain speculative. Using mass spectrometry, we systematically identify phosphorylated residues in the essential CCC subunits HMR-1, HMP-1 and HMP-2 in vivo. We demonstrate that HMR-1/cadherin phosphorylation occurs on three sites within its ß-catenin binding domain that each contributes to CCC assembly on lipid bilayers. In contrast, phosphorylation of HMP-2/ß-catenin inhibits its association with HMR-1/cadherin in vitro, suggesting a role in CCC disassembly. Although HMP-1/α-catenin is also phosphorylated in vivo, phosphomimetic mutations do not affect its ability to associate with other CCC components or interact with actin in vitro. Collectively, our findings support a model in which distinct phosphorylation events contribute to rapid CCC assembly and disassembly, both of which are essential for morphogenetic rearrangements during development.

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.

TFG clusters COPII-coated transport carriers and promotes early secretory pathway organization.

In mammalian cells, cargo-laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER-Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII-coated transport carriers traverse a submicron, TFG (Trk-fused gene)-enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three-dimensional electron microscopy reveals the formation of flexible, octameric cup-like structures, which are able to self-associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII-coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII-coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII-coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.

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.

Spatial control of phospholipid flux restricts endoplasmic reticulum sheet formation to allow nuclear envelope breakdown.

The nuclear envelope is a subdomain of the endoplasmic reticulum (ER). Here we characterize CNEP-1 (CTD [C-terminal domain] nuclear envelope phosphatase-1), a nuclear envelope-enriched activator of the ER-associated phosphatidic acid phosphatase lipin that promotes synthesis of major membrane phospholipids over phosphatidylinositol (PI). CNEP-1 inhibition led to ectopic ER sheets in the vicinity of the nucleus that encased the nuclear envelope and interfered with nuclear envelope breakdown (NEBD) during cell division. Reducing PI synthesis suppressed these phenotypes, indicating that CNEP-1 spatially regulates phospholipid flux, biasing it away from PI production in the vicinity of the nuclear envelope to prevent excess ER sheet formation and NEBD defects.

The ESCRT machinery: from the plasma membrane to endosomes and back again.

The manipulation and reorganization of lipid bilayers are required for diverse cellular processes, ranging from organelle biogenesis to cytokinetic abscission, and often involves transient membrane disruption. A set of membrane-associated proteins collectively known as the endosomal sorting complex required for transport (ESCRT) machinery has been implicated in membrane scission steps, which transform a single, continuous bilayer into two distinct bilayers, while simultaneously segregating cargo throughout the process. Components of the ESCRT pathway, which include 5 distinct protein complexes and an array of accessory factors, each serve discrete functions. This review focuses on the molecular mechanisms by which the ESCRT proteins facilitate cargo sequestration and membrane remodeling and highlights their unique roles in cellular homeostasis.

Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure.

Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (TFG) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.

Roles of acidic phospholipids and nucleotides in regulating membrane binding and activity of a calcium-independent phospholipase A2 isoform.

Phospholipase A(2) activity plays key roles in generating lipid second messengers and regulates membrane topology through the generation of asymmetric lysophospholipids. In particular, the Group VIA phospholipase A(2) (GVIA-iPLA(2)) subfamily of enzymes functions independently of calcium within the cytoplasm of cells and has been implicated in numerous cellular processes, including proliferation, apoptosis, and membrane transport steps. However, mechanisms underlying the spatial and temporal regulation of these enzymes have remained mostly unexplored. Here, we examine the subset of Caenorhabditis elegans lipases that harbor a consensus motif common to members of the GVIA-iPLA(2) subfamily. Based on sequence homology, we identify IPLA-1 as the closest C. elegans homolog of human GVIA-iPLA(2) enzymes and use a combination of liposome interaction studies to demonstrate a role for acidic phospholipids in regulating GVIA-iPLA(2) function. Our studies indicate that IPLA-1 binds directly to multiple acidic phospholipids, including phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, and phosphorylated derivatives of phosphatidylinositol. Moreover, the presence of these acidic lipids dramatically elevates the specific activity of IPLA-1 in vitro. We also found that the addition of ATP and ADP promote oligomerization of IPLA-1, which probably underlies the stimulatory effect of nucleotides on its activity. We propose that membrane composition and the presence of nucleotides play key roles in recruiting and modulating GVIA-iPLA(2) activity in cells.

Phosphoinositide signaling during membrane transport in Saccharomyces cerevisiae.

Phosphatidylinositol (PI) is distinct from other phospholipids, possessing a head group that can be modified by phosphorylation at multiple positions to generate unique signaling molecules collectively known as phosphoinositides. The set of kinases and phosphatases that regulate PI metabolism are conserved throughout eukaryotic evolution, and numerous studies have demonstrated that phosphoinositides regulate a diverse spectrum of cellular processes, including vesicle transport, cell proliferation, and cytoskeleton organization. Over the past two decades, nearly all PI derivatives have been shown to interact directly with cellular proteins to affect their localization and/or activity. Additionally, there is growing evidence, which suggests that phosphoinositides may also affect local membrane topology. Here, we focus on the role of phosphoinositides in membrane trafficking and underscore the significant role that yeast has played in the field.

Association of the endosomal sorting complex ESCRT-II with the Vps20 subunit of ESCRT-III generates a curvature-sensitive complex capable of nucleating ESCRT-III filaments.

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.

TFG-1 function in protein secretion and oncogenesis.

Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.

ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes simultaneously.

The ESCRT machinery consists of multiple protein complexes that collectively participate in the biogenesis of multivesicular endosomes (MVEs). The ESCRT-0 complex is composed of two subunits, Hrs and STAM, both of which can engage ubiquitinylated substrates destined for lysosomal degradation. Here, we conduct a comprehensive analysis of ESCRT-0:ubiquitin interactions using isothermal titration calorimetry and define the affinity of each ubiquitin-binding domain (UBD) within the intact ESCRT-0 complex. Our data demonstrate that ubiquitin binding is non-cooperative between the ESCRT-0 UBDs. Additionally, our findings show that the affinity of the Hrs double ubiquitin interacting motif (DUIM) for ubiquitin is more than 2-fold greater than that of UBDs found in STAM, suggesting that Hrs functions as the major ubiquitin-binding protein in ESCRT-0. In vivo, Hrs and STAM localize to endosomal membranes. To study recombinant ESCRT-0 assembly on lipid bilayers, we used atomic force microscopy. Our data show that ESCRT-0 forms mostly heterodimers and heterotetramers of Hrs and STAM when analyzed in the presence of membranes. Consistent with these findings, hydrodynamic analysis of endogenous ESCRT-0 indicates that it exists largely as a heterotetrameric complex of its two subunits. Based on these data, we present a revised model for ESCRT-0 function in cargo recruitment and concentration at the endosome.