Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.     

Charged multivesicular body protein 2B (CHMP2B) of the endosomal sorting complex required for transport-III (ESCRT-III) polymerizes into helical structures deforming the plasma membrane

The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.     

Novel interactions of ESCRT-III with LIP5 and VPS4 and their implications for ESCRT-III disassembly

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.     

Biochemical Analyses of Human IST1 and Its Function in Cytokinesis

The newly described yeast endosomal sorting complexes required for transport (ESCRT) protein increased sodium tolerance-1 (Ist1p) binds the late-acting ESCRT proteins Did2p/charged MVB protein (CHMP) 1 and Vps4p and exhibits synthetic vacuolar protein sorting defects when combined with mutations in the Vta1p/LIP5–Vps60p/CHMP5 complex. Here, we report that human IST1 also functions in the ESCRT pathway and is required for efficient abscission during HeLa cell cytokinesis. IST1 binding interactions with VPS4, CHMP1, LIP5, and ESCRT-I were characterized, and the IST1–VPS4 interaction was investigated in detail. Mutational and NMR spectroscopic studies revealed that the IST1 terminus contains two distinct MIT interacting motifs (MIM1 and MIM2) that wrap around and bind in different groves of the MIT helical bundle. IST1, CHMP1, and VPS4 were recruited to the midbodies of dividing cells, and depleting either IST1 or CHMP1 proteins blocked VPS4 recruitment and abscission. In contrast, IST1 depletion did not inhibit human immunodeficiency virus-1 budding. Thus, IST1 and CHMP1 act together to recruit and modulate specific VPS4 activities required during the final stages of cell division.     

Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.     

Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis

TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.     

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.     

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

VPS4 proteins are AAA⁺ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA⁺ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His₆-VPS4A on liposomes containing Ni²⁺-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.     

Structural basis for ESCRT-III CHMP3 recruitment of AMSH

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90?? long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.     

Essential Role of hIST1 in Cytokinesis

The last steps of multivesicular body (MVB) formation, human immunodeficiency virus (HIV)-1 budding and cytokinesis require a functional endosomal sorting complex required for transport (ESCRT) machinery to facilitate topologically equivalent membrane fission events. Increased sodium tolerance (IST) 1, a new positive modulator of the ESCRT pathway, has been described recently, but an essential function of this highly conserved protein has not been identified. Here, we describe the previously uncharacterized KIAA0174 as the human homologue of IST1 (hIST1), and we report its conserved interaction with VPS4, CHMP1A/B, and LIP5. We also identify a microtubule interacting and transport (MIT) domain interacting motif (MIM) in hIST1 that is necessary for its interaction with VPS4, LIP5 and other MIT domain-containing proteins, namely, MITD1, AMSH, UBPY, and Spastin. Importantly, hIST1 is essential for cytokinesis in mammalian cells but not for HIV-1 budding, thus providing a novel mechanism of functional diversification of the ESCRT machinery. Last, we show that the hIST1 MIM activity is essential for cytokinesis, suggesting possible mechanisms to explain the role of hIST1 in the last step of mammalian cell division.     

Structural basis for autoinhibition of ESCRT-III CHMP3

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.     

Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used "deep-etch" electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis-deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.     

Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins - hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/CHMP6, and hSnf7-1/CHMP4a - to each other, studying the effects of deleting predicted alpha-helical domains on their behavior in transfected cells. Surprisingly, removing approximately 40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA+ adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default 'closed' state and an activated 'open' state under control of sequences at their C-terminus and associated factors.     

ESCRT machinery and cytokinesis: the road to daughter cell separation

The endosomal sorting complex required for transport (ESCRT) machinery is a set of cellular protein complexes required for at least three topologically equivalent membrane scission events, namely multivesicular body (MVB) formation, retroviral particle release and midbody abscission during cytokinesis. Recently, several studies have explored the mechanism by which the core ESCRT-III subunits mediate membrane scission and might be differentially required according to the functions of the pathway. In this review, we discuss the links between the ESCRT machinery and cytokinesis, with special focus on abscission initiation and regulation.     

UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting

Endosomal sorting complexes required for transport (ESCRTs) regulate several events involving membrane invagination, including multivesicular body (MVB) biogenesis, viral budding, and cytokinesis. In each case, upstream ESCRTs combine with additional factors, such as Bro1 proteins, to recruit ESCRT-III and the ATPase VPS4 in order to drive membrane scission. A clue to understanding how such diverse cellular processes might be controlled independently of each other has been the identification of ESCRT isoforms. Mammalian ESCRT-I comprises TSG101, VPS28, VPS37A-D, and MVB12A/B. These could generate several ESCRT-I complexes, each targeted to a different compartment and able to recruit distinct ESCRT-III proteins. Here we identify a novel ESCRT-I component, ubiquitin-associated protein 1 (UBAP1), which contains a region conserved in MVB12. UBAP1 binds the endosomal Bro1 protein His domain protein tyrosine phosphatase (HDPTP), but not Alix, a Bro1 protein involved in cytokinesis. UBAP1 is required for sorting EGFR to the MVB and for endosomal ubiquitin homeostasis, but not for cytokinesis. UBAP1 is part of a complex that contains a fraction of total cellular TSG101 and that also contains VPS37A but not VPS37C. Hence, the presence of UBAP1, in combination with VPS37A, defines an endosome-specific ESCRT-I complex.     

Mechanism of inhibition of retrovirus release from cells by interferon-induced gene ISG15

Budding of retroviruses from cell membranes requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including endosome sorting complex required for transport III (ESCRT-III) protein complex and vacuolar protein sorting 4 (VPS4) and its ATPase. In response to infection, a cellular mechanism has evolved that blocks virus replication early and late in the budding process through expression of interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin. Interferon treatment of DF-1 cells blocks avian sarcoma/leukosis virus release, demonstrating that this mechanism is functional under physiological conditions. The late block to release is caused in part by a loss in interaction between VPS4 and its coactivator protein LIP5, which is required to promote the formation of the ESCRT III-VPS4 double-hexamer complex to activate its ATPase. ISG15 is conjugated to two different LIP5-ESCRT-III-binding charged multivesicular body proteins, CHMP2A and CHMP5. Upon ISGylation of each, interaction with LIP5 is no longer detected. Two other ESCRT-III proteins, CHMP4B and CHMP6, are also conjugated to ISG15. ISGylation of CHMP2A, CHMP4B, and CHMP6 weakens their binding directly to VPS4, thereby facilitating the release of this protein from the membrane into the cytosol. The remaining budding complex fails to release particles from the cell membrane. Introducing a mutant of ISG15 into cells that cannot be conjugated to proteins prevents the ISG15-dependent mechanism from blocking virus release. CHMP5 is the primary switch to initiate the antiviral mechanism, because removal of CHMP5 from cells prevents ISGylation of CHMP2A and CHMP6.     

Interaction of AMSH with ESCRT-III and deubiquitination of endosomal cargo

The "class E" vacuolar protein sorting (VPS) pathway mediates sorting of ubiquitinated cargo into the forming vesicles of the multivesicular bodies (MVB), and it is essential for down-regulation of signaling by growth factors and budding of enveloped viruses such as Ebola and HIV-1. Work in yeast has identified DOA4 as a gene that is recruited by the class E machinery to remove ubiquitin from the endosomal cargo before it is incorporated into MVB vesicles, but the identity of the mammalian counterpart is unclear. Here we report the interaction of AMSH (associated molecule with the SH3 domain of STAM), an endosomal deubiquitinating enzyme, with the endodomal sorting complex required for transport (ESCRT-III) subunits CHMP1A, CHMP1B, CHMP2A, and CHMP3. We also show that a catalytically inactive AMSH inhibits retroviral budding in a dominant-negative manner and induces the accumulation of ubiquitinated forms of an endosomal cargo, namely murine leukemia virus Gag. Finally, VPS4 and AMSH compete for binding to the C-terminal regions of CHMP1A and CHMP1B, revealing a coordinated interaction with ESCRT-III. Taken together, these results are consistent with a role of AMSH in the deubiquitination of the endosomal cargo preceding lysosomal degradation.     

Burning cellular bridges: Two pathways to the big breakup

During cytokinetic abscission, the endosomal sorting complex required for transport (ESCRT) proteins are recruited to the midbody and direct the severing of the intercellular bridge. In this issue, Christ et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507009) demonstrate that two separate but redundant pathways exist to recruit ESCRT-III proteins to the midbody.Over the past 140 years, eukaryotic cell division has been extensively studied and is now understood to be an elaborate, tightly regulated set of events that culminates in the formation of two distinct daughter cells. The M phase of animal cells is characterized by a profound structural reorganization, regulated by a cohort of mitotic kinases and performed by mitosis-specific cytoskeletal structures, including the spindle apparatus and the cytokinetic midbody (Scholey et al., 2003). The completion of cytokinesis, called abscission, involves the severing of the intercellular bridge on both sides of the midbody. In 2007, two landmark studies demonstrated that several ESCRT proteins localize to the midbody and are required for the completion of cytokinesis (Carlton and Martin-Serrano, 2007; Morita et al., 2007). Aside from abscission, the ESCRTs participate in the formation of multivesicular endosomes (MVEs), function in plasma membrane repair, and participate in numerous other cellular processes (Katzmann et al., 2002; Morita and Sundquist, 2004; Hurley, 2015).The canonical model for ESCRT function at MVEs involves the hierarchical recruitment of ESCRT proteins in four unique complexes: ESCRT-0 through ESCRT-III. The ESCRTs cluster cargos and deform membrane, and current models suggest that ESCRT-III subunits polymerize to form filaments that spiral down into the neck of a nascent intralumenal vesicle (Schuh and Audhya, 2014). With the assistance of the VPS4 AAA ATPase, ESCRT filaments are remodeled to facilitate vesicle fission (Shen et al., 2014). Though MVE maturation utilizes all four ESCRT complexes, cytokinetic abscission has been previously thought to require only ESCRT-I, ESCRT-III, and the ESCRT-associated ALG2-interacting factor ALIX (Morita et al., 2010). ALIX interacts with both the ESCRT-I protein TSG101 and all three ESCRT-III CHMP4 isoforms and has been postulated to act as an ESCRT-II bypass for linking ESCRT-I and ESCRT-III in abscission (Schuh and Audhya, 2014). However, the precise mechanism underlying the recruitment of the ESCRT-III complex to the midbody during cytokinesis has remained ambiguous.In this issue, Christ et al. address how the ESCRT-III component CHMP4B (Vps32 in other metazoan systems) is recruited to the midbody and demonstrate the necessity of the ESCRT-II complex in this process. They observed that recruitment of CHMP4B to the midbody was abrogated when they codepleted ALIX and the ESCRT-I component TSG101 in cultured HeLa cells, but that CHMP4B did accumulate when only one of these components was depleted. These data indicate that CHMP4B can be recruited to the midbody via TSG101 or ALIX, but that the two proteins are unlikely to perform this function as a complex, suggesting that CHMP4B recruitment to the midbody involves two independent pathways.After immunofluorescence staining of fixed cells, Christ et al. (2016) found that the endogenous ESCRT-III protein CHMP6 and the ESCRT-II protein EAP20 (VPS20 and VPS25 in other systems, respectively) localize to the midbody, consistent with a previous overexpression study (Thoresen et al., 2014). They additionally performed several depletion experiments to establish that ESCRT-II recruits CHMP6 without affecting TSG101 localization, demonstrating that CHMP6 acts downstream of ESCRT-I and ESCRT-II. This shows that ESCRT-I recruits ESCRT-III to the cytokinetic midbody the same way it does at the MVE.Christ et al. (2016) also show that CHMP4B can still be recruited normally when the ESCRT-II component EAP30 (VPS22 in other systems) is depleted, but not when EAP30 is codepleted with ALIX, strongly suggesting that ALIX-dependent accumulation of CHMP4B does not involve CHMP6 and, more generally, that there are two pathways that can each recruit CHMP4B to the midbody: an ESCRT-I–ESCRT-II–CHMP6 pathway and an ALIX-dependent pathway. It will be important for future work to consider the partial redundancy between these two pathways when assaying the dispensability of early acting ESCRT complexes in cellular processes.In addition, Christ et al. (2016) observed that ALIX depletion led to furrow regression and binucleation in dividing cells with chromatin spanning the intercellular bridge, the same phenotype observed in cells expressing a CHMP4C construct lacking the ALIX interaction domain. Further, they showed that CHMP4C localization to the midbody is abrogated after ALIX depletion but is unaffected by TSG101 knockdowns, strongly implicating ALIX in CHMP4C recruitment independently of ESCRT-I.Our overall understanding of the regulation of abscission still remains elementary (Fig. 1). In addition to the roles of the ESCRT machinery, the chromosomal passenger complex (CPC) regulates the timing of cytokinesis and abscission via interactions with the Polo-like kinase PLK1, the mitotic kinesin-like protein MKLP1, and CEP55, a key component of the midbody that associates directly with both ESCRT-I and ALIX (Schuh and Audhya, 2014). One current model is that the CPC promotes the formation of a ternary complex consisting of CHMP4C, ANCHR, and VPS4 and prevents premature action by VPS4 in response to chromatin trapped in the midbody (Thoresen et al., 2014). It has also been suggested that CHMP4C phosphorylation by the enzymatic core of the CPC, the Aurora B kinase, directs CHMP4C localization to the midbody and its retention of VPS4 (Carlton et al., 2012). With the new findings by Christ et al. (2016), the relationship between Aurora B–mediated phosphorylation of CHMP4C and its ability to bind ALIX must now be further explored. Additionally, because ALIX appears to be the primary factor that recruits CHMP4C to the midbody, it may represent a novel therapeutic target for activation or bypass of the NoCut abscission checkpoint.Open in a separate windowFigure 1.Model for the recruitment of CHMP4B and CHMP4C to the midbody and their roles in regulating the timing of abscission. PLK-1 phosphorylation of CEP55 inhibits its binding to MKLP1. At the end of anaphase, PLK1 is degraded and MKLP1 recruits CEP55 to the midbody. CEP55 recruits TSG101 and ALIX to the midbody, and Christ et al. (2016) demonstrate that there are two pathways that lead to the subsequent recruitment of CHMP4B: one through ESCRT-I–ESCRT-II–CHMP6 and the second directly through ALIX. ALIX also recruits CHMP4C, which, upon phosphorylation by the CPC, is hypothesized to form a ternary complex with ANCHR and VPS4. Formation of this complex prevents VPS4 from facilitating the completion of abscission until all chromatin is cleared from the intercellular bridge.In contrast to the necessity of ALIX during cytokinetic abscission, its role during MVE formation and ubiquitin-dependent cargo degradation remains debatable. Depletion studies suggest that ALIX is dispensable for the lysosomal sorting of several cargoes (Bowers et al., 2006). However, ALIX is capable of targeting to late endosomal membranes through its interaction with lysobisphosphatidic acid, and some data suggest that ALIX can promote ESCRT-III filament assembly at MVEs (Matsuo et al., 2004; Pires et al., 2009; Bissig and Gruenberg, 2014). In the future, it will be essential to elucidate the mechanisms by which ALIX and CHMP6 direct the nucleation of CHMP4B/ESCRT-III spiral filaments and to determine whether the membrane landscapes of the MVE and the cytokinetic bridge differ in a manner that promotes one pathway over the other. As cryoelectron microscopy–based approaches in cells and reconstituted systems advance, the answer to these questions may become more accessible.     

Divergent pathways lead to ESCRT-III-catalyzed membrane fission

Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission.     

ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters in cytokinetic abscission

Cytokinetic abscission, the final stage of cell division where the two daughter cells are separated, is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. The ESCRT-III subunit CHMP4B is a key effector in abscission, whereas its paralogue, CHMP4C, is a component in the abscission checkpoint that delays abscission until chromatin is cleared from the intercellular bridge. How recruitment of these components is mediated during cytokinesis remains poorly understood, although the ESCRT-binding protein ALIX has been implicated. Here, we show that ESCRT-II and the ESCRT-II–binding ESCRT-III subunit CHMP6 cooperate with ESCRT-I to recruit CHMP4B, with ALIX providing a parallel recruitment arm. In contrast to CHMP4B, we find that recruitment of CHMP4C relies predominantly on ALIX. Accordingly, ALIX depletion leads to furrow regression in cells with chromosome bridges, a phenotype associated with abscission checkpoint signaling failure. Collectively, our work reveals a two-pronged recruitment of ESCRT-III to the cytokinetic bridge and implicates ALIX in abscission checkpoint signaling.