MITD1 is recruited to midbodies by ESCRT-III and participates in cytokinesis

Diverse cellular processes, including multivesicular body formation, cytokinesis, and viral budding, require the sequential functions of endosomal sorting complexes required for transport (ESCRTs) 0 to III. Of these multiprotein complexes, ESCRT-III in particular plays a key role in mediating membrane fission events by forming large, ring-like helical arrays. A number of proteins playing key effector roles, most notably the ATPase associated with diverse cellular activities protein VPS4, harbor present in microtubule-interacting and trafficking molecules (MIT) domains comprising asymmetric three-helical bundles, which interact with helical MIT-interacting motifs in ESCRT-III subunits. Here we assess comprehensively the ESCRT-III interactions of the MIT-domain family member MITD1 and identify strong interactions with charged multivesicular body protein 1B (CHMP1B), CHMP2A, and increased sodium tolerance-1 (IST1). We show that these ESCRT-III subunits are important for the recruitment of MITD1 to the midbody and that MITD1 participates in the abscission phase of cytokinesis. MITD1 also dimerizes through its C-terminal domain. Both types of interactions appear important for the role of MITD1 in negatively regulating the interaction of IST1 with VPS4. Because IST1 binding in turn regulates VPS4, MITD1 may function through downstream effects on the activity of VPS4, which plays a critical role in the processing and remodeling of ESCRT filaments in abscission.

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The Chlamydia trachomatis Extrusion Exit Mechanism Is Regulated by Host Abscission Proteins

Microorganisms ◽

10.3390/microorganisms7050149 ◽

2019 ◽

Vol 7 (5) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

Meghan Zuck ◽

Kevin Hybiske

Keyword(s):

Host Cell ◽

Intracellular Pathogens ◽

Body Protein ◽

Functional Roles ◽

Endosomal Sorting ◽

Exit Mechanism ◽

Viral Budding ◽

Membrane Fission ◽

Exit Strategies ◽

Vacuolar Protein

The cellular exit strategies of intracellular pathogens have a direct impact on microbial dissemination, transmission, and engagement of immune responses of the host. Chlamydia exit their host via a budding mechanism called extrusion, which offers protective benefits to Chlamydia as they navigate their extracellular environment. Many intracellular pathogens co-opt cellular abscission machinery to facilitate cell exit, which is utilized to perform scission of two newly formed daughter cells following mitosis. Similar to viral budding exit strategies, we hypothesize that an abscission-like mechanism is required to physically sever the chlamydial extrusion from the host cell, co-opting the membrane fission activities of the endosomal sorting complex required for transport (ESCRT) family of proteins that are necessary for cellular scission events, including abscission. To test this, C. trachomatis L2-infected HeLa cells were depleted of key abscission machinery proteins charged multivesicle body protein 4b (CHMP4B), ALIX, centrosome protein 55 (CEP55), or vacuolar protein sorting-associated protein 4A (VPS4A), using RNA interference (RNAi). Over 50% reduction in extrusion formation was achieved by depletion of CHMP4B, VPS4A, and ALIX, but no effect on extrusion was observed with CEP55 depletion. These results demonstrate a role for abscission machinery in C. trachomatis extrusion from the host cell, with ALIX, VPS4A and CHMP4B playing key functional roles in optimal extrusion release.

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A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805504115 ◽

2018 ◽

Vol 115 (38) ◽

pp. E8900-E8908 ◽

Cited By ~ 19

Author(s):

Jessica B. A. Sadler ◽

Dawn M. Wenzel ◽

Lauren K. Williams ◽

Marta Guindo-Martínez ◽

Steven L. Alam ◽

...

Keyword(s):

Cancer Susceptibility ◽

Genome Instability ◽

Multivesicular Body ◽

Mitotic Checkpoint ◽

Body Protein ◽

Endosomal Sorting ◽

Daughter Cells ◽

Dna Replication Stress ◽

Human Polymorphism ◽

Mitotic Stress

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4CT232) increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: The CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome missegregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.

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VPS4 triggers constriction and cleavage of ESCRT-III helical filaments

Science Advances ◽

10.1126/sciadv.aau7198 ◽

2019 ◽

Vol 5 (4) ◽

pp. eaau7198 ◽

Cited By ~ 26

Author(s):

Sourav Maity ◽

Christophe Caillat ◽

Nolwenn Miguet ◽

Guidenn Sulbaran ◽

Gregory Effantin ◽

...

Keyword(s):

High Speed ◽

Membrane Structures ◽

Vesicle Budding ◽

Endosomal Sorting ◽

Force Microscopy ◽

Cellular Processes ◽

Membrane Fission ◽

End Caps

Many cellular processes such as endosomal vesicle budding, virus budding, and cytokinesis require extensive membrane remodeling by the endosomal sorting complex required for transport III (ESCRT-III). ESCRT-III protein family members form spirals with variable diameters in vitro and in vivo inside tubular membrane structures, which need to be constricted to proceed to membrane fission. Here, we show, using high-speed atomic force microscopy and electron microscopy, that the AAA-type adenosine triphosphatase VPS4 constricts and cleaves ESCRT-III CHMP2A-CHMP3 helical filaments in vitro. Constriction starts asymmetrically and progressively decreases the diameter of CHMP2A-CHMP3 tubular structure, thereby coiling up the CHMP2A-CHMP3 filaments into dome-like end caps. Our results demonstrate that VPS4 actively constricts ESCRT-III filaments and cleaves them before their complete disassembly. We propose that the formation of ESCRT-III dome-like end caps by VPS4 within a membrane neck structure constricts the membrane to set the stage for membrane fission.

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

The Journal of Cell Biology ◽

10.1083/jcb.200707031 ◽

2008 ◽

Vol 180 (2) ◽

pp. 389-402 ◽

Cited By ~ 314

Author(s):

Phyllis I. Hanson ◽

Robyn Roth ◽

Yuan Lin ◽

John E. Heuser

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Negative Curvature ◽

Multivesicular Body ◽

Multivesicular Bodies ◽

Deficient Mutant ◽

Membrane Deformation ◽

Endosomal Sorting ◽

Viral Budding ◽

Circular Arrays

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.

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Structure and function of ESCRT-III

Biochemical Society Transactions ◽

10.1042/bst0370156 ◽

2009 ◽

Vol 37 (1) ◽

pp. 156-160 ◽

Cited By ~ 48

Author(s):

Suman Lata ◽

Guy Schoehn ◽

Julianna Solomons ◽

Ricardo Pires ◽

Heinrich G. Göttlinger ◽

...

Keyword(s):

Multivesicular Body ◽

Multivesicular Bodies ◽

Tubular Structures ◽

Body Protein ◽

Enveloped Viruses ◽

Endosomal Sorting ◽

Vacuolar Protein ◽

And Function ◽

Endosomal Vesicles

ESCRT-III (endosomal sorting complex required for transport III) is required for the formation and abscission of intraluminal endosomal vesicles, which gives rise to multivesicular bodies, budding of some enveloped viruses and cytokinesis. ESCRT-III is composed of 11 members in humans, which, except for one, correspond to the six ESCRT-III-like proteins in yeast. At least CHMP (charged multivesicular body protein) 2A and CHMP3 assemble into helical tubular structures that provide a platform for membrane interaction and VPS (vacuolar protein sorting) 4-catalysed effects leading to disassembly of ESCRT-III CHMP2A–CHMP3 polymers in vitro. Progress towards the understanding of the structures and function of ESCRT-III, its activation, its regulation by accessory factors and its role in abscission of membrane enveloped structures in concert with VPS4 are discussed.

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ESCRTs and human disease

Biochemical Society Transactions ◽

10.1042/bst0370167 ◽

2009 ◽

Vol 37 (1) ◽

pp. 167-172 ◽

Cited By ~ 65

Author(s):

Suraj Saksena ◽

Scott D. Emr

Keyword(s):

Human Disease ◽

Protein Sorting ◽

Critical Role ◽

Multivesicular Body ◽

Human Diseases ◽

Down Regulation ◽

Endosomal Sorting ◽

Retroviral Budding

The ESCRT (endosomal sorting complex required for transport) machinery plays a critical role in receptor down-regulation, retroviral budding, and other normal and pathological processes. The ESCRT components are conserved in all five major subgroups of eukaryotes. This review summarizes the growing number of links identified between ESCRT-mediated protein sorting in the MVB (multivesicular body) pathway and various human diseases.

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The CHMP4b- and Src-docking sites in the Bro1 domain are autoinhibited in the native state of Alix

Biochemical Journal ◽

10.1042/bj20081388 ◽

2009 ◽

Vol 418 (2) ◽

pp. 277-284 ◽

Cited By ~ 22

Author(s):

Xi Zhou ◽

Shujuan Pan ◽

Le Sun ◽

Joe Corvera ◽

Yu-Chen Lee ◽

...

Keyword(s):

Multivesicular Body ◽

Human Embryonic Kidney ◽

Native State ◽

Interacting Protein ◽

Body Protein ◽

Linked Gene ◽

Endosomal Sorting ◽

Protein X ◽

Membrane Bound ◽

Docking Sites

The Bro1 domain of Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], which plays important roles in endosomal sorting and multiple ESCRT (endosomal sorting complex required for transport)-linked processes, contains the docking sites for the ESCRT-III component CHMP4b (charged multivesicular body protein 4b) and the regulatory tyrosine kinase, Src. Although the structural bases for these docking sites have been defined by crystallography studies, it has not been determined whether these sites are available in the native state of Alix. In the present study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK (human embryonic kidney)-293 cell lysates, these two docking sites are not available in cytosolic Alix, but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix's involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain.

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Essential Role of hIST1 in Cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0474 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1374-1387 ◽

Cited By ~ 96

Author(s):

Monica Agromayor ◽

Jez G. Carlton ◽

John P. Phelan ◽

Daniel R. Matthews ◽

Leo M. Carlin ◽

...

Keyword(s):

Mammalian Cells ◽

Multivesicular Body ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Membrane Fission ◽

Immunodeficiency Virus ◽

Positive Modulator ◽

Essential Function ◽

Hiv 1

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.

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A dominant-negative ESCRT-III protein perturbs cytokinesis and trafficking to lysosomes

Biochemical Journal ◽

10.1042/bj20071296 ◽

2008 ◽

Vol 411 (2) ◽

pp. 233-239 ◽

Cited By ~ 29

Author(s):

Joseph D. Dukes ◽

Judith D. Richardson ◽

Ruth Simmons ◽

Paul Whitley

Keyword(s):

Membrane Trafficking ◽

Multivesicular Body ◽

Dominant Negative ◽

Eukaryotic Cells ◽

Body Protein ◽

Recycling Endosomes ◽

Protein Subunits ◽

Autoinhibitory Domain ◽

Endosomal Sorting ◽

A Subunit

In eukaryotic cells, the completion of cytokinesis is dependent on membrane trafficking events to deliver membrane to the site of abscission. Golgi and recycling endosomal-derived proteins are required for the terminal stages of cytokinesis. Recently, protein subunits of the ESCRT (endosomal sorting complexes required for transport) that are normally involved in late endosome to lysosome trafficking have also been implicated in abscission. Here, we report that a subunit, CHMP3 (charged multivesicular body protein-3), of ESCRT-III localizes at the midbody. Deletion of the C-terminal autoinhibitory domain of CHMP3 inhibits cytokinesis. At the midbody, CHMP3 does not co-localize with Rab11, suggesting that it is not present on recycling endosomes. These results combined provide compelling evidence that proteins involved in late endosomal function are necessary for the end stages of cytokinesis.

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