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

2008 ◽  
Vol 411 (2) ◽  
pp. 233-239 ◽  
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.

The role of CHMP2B in frontotemporal dementia

2009 ◽  
Vol 37 (1) ◽  
pp. 208-212 ◽  
Author(s):  
Hazel Urwin ◽  
Shabnam Ghazi-Noori ◽  
John Collinge ◽  
Adrian Isaacs
Keyword(s):  
Frontotemporal Dementia ◽  
Neuroblastoma Cells ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Endosomal Trafficking ◽  
Human Neuroblastoma ◽  
Body Protein ◽  
Endosomal Sorting ◽  
Negative Effect

Mutations in the CHMP2B (charged multivesicular body protein 2B) gene that lead to C-terminal truncations of the protein can cause frontotemporal dementia. CHMP2B is a member of ESCRT-III (endosomal sorting complex required for transport III), which is required for formation of the multivesicular body, a late endosomal structure that fuses with the lysosome to degrade endocytosed proteins. Overexpression of mutant C-terminally truncated CHMP2B proteins produces an enlarged endosomal phenotype in PC12 and human neuroblastoma cells, which is likely to be due to a dominant-negative effect on endosomal function. Disruption of normal endosomal trafficking is likely to affect the transport of neuronal growth factors and autophagic clearance of proteins, both of which could contribute to neurodegeneration in frontotemporal dementia.

scholarly journals Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion

2018 ◽  
Vol 115 (19) ◽  
pp. E4396-E4405 ◽  
Author(s):  
Sebastian Bänfer ◽  
Dominik Schneider ◽  
Jenny Dewes ◽  
Maximilian T. Strauss ◽  
Sven-A. Freibert ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Interaction ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Amino Terminus ◽  
Multivesicular Bodies ◽  
Dramatic Decrease ◽  
Endosomal Sorting ◽  
Galectin 3 ◽  
Binding Lectin

The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

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

2018 ◽  
Vol 115 (38) ◽  
pp. E8900-E8908 ◽  
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.

scholarly journals Mvb12 Is a Novel Member of ESCRT-I Involved in Cargo Selection by the Multivesicular Body Pathway

2007 ◽  
Vol 18 (2) ◽  
pp. 646-657 ◽  
Author(s):  
Andrea J. Oestreich ◽  
Brian A. Davies ◽  
Johanna A. Payne ◽  
David J. Katzmann
Keyword(s):  
Normal Cell ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Eukaryotic Cells ◽  
Cell Physiology ◽  
Cellular Functions ◽  
Protein Loss ◽  
Endosomal Sorting ◽  
Vacuolar Protein ◽  
Selection Of

The multivesicular body (MVB) sorting pathway impacts a variety of cellular functions in eukaryotic cells. Perhaps the best understood role for the MVB pathway is the degradation of transmembrane proteins within the lysosome. Regulation of cargo selection by this pathway is critically important for normal cell physiology, and recent advances in our understanding of this process have highlighted the endosomal sorting complexes required for transport (ESCRTs) as pivotal players in this reaction. To better understand the mechanisms of cargo selection during MVB sorting, we performed a genetic screen to identify novel factors required for cargo-specific selection by this pathway and identified the Mvb12 protein. Loss of Mvb12 function results in differential defects in the selection of MVB cargoes. A variety of analyses indicate that Mvb12 is a stable member of ESCRT-I, a heterologous complex involved in cargo selection by the MVB pathway. Phenotypes displayed upon loss of Mvb12 are distinct from those displayed by the previously described ESCRT-I subunits (vacuolar protein sorting 23, -28, and -37), suggesting a distinct function than these core subunits. These data support a model in which Mvb12 impacts the selection of MVB cargoes by modulating the cargo recognition capabilities of ESCRT-I.

Structure and function of ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 156-160 ◽  
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.

Identification of novel downstream targets of platelet glycoprotein VI activation by differential proteome analysis: implications for thrombus formation

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4102-4110 ◽  
Author(s):  
Christian Schulz ◽  
Nina V. Leuschen ◽  
Thomas Fröhlich ◽  
Michael Lorenz ◽  
Susanne Pfeiler ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Membrane Trafficking ◽  
Thrombus Formation ◽  
Multivesicular Body ◽  
Platelet Glycoprotein ◽  
Body Protein ◽  
Glycoprotein Vi ◽  
Differentially Abundant Proteins ◽  
Platelet Proteins

Abstract Platelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.

scholarly journals Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress

2020 ◽  
Author(s):  
Moona Huttunen ◽  
Artur Yakimovich ◽  
Ian J. White ◽  
Janos Kriston-Vizi ◽  
Juan Martin-Serrano ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Multivesicular Body ◽  
Cellular Membrane ◽  
Double Membrane ◽  
Endosomal Sorting ◽  
A Cell ◽  
Infected Cells ◽  
Membrane Bound ◽  
Virus Egress

Unlike most enveloped viruses, poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple membrane-bound particles, termed intracellular enveloped virions (IEVs), are then released from infected cells by fusion. While the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify novel cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to a high-throughput RNAi screen directed against cellular membrane trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of these MVB-IEVs showed that they account for half of IEV production indicating that MVBs serve as a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

Abstract 3734: Differential Platelet Proteome Analysis Following Specific Activation Of Glycoprotein VI

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Christian Schulz ◽  
Nina V Leuschen ◽  
Thomas Fröhlich ◽  
Michael Lorenz ◽  
Georg J Arnold ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Membrane Trafficking ◽  
Proteome Analysis ◽  
Multivesicular Body ◽  
Healthy Human ◽  
Body Protein ◽  
2D Dige ◽  
Glycoprotein Vi ◽  
Platelet Proteome ◽  
Differentially Abundant Proteins

Background: Platelets play a key role in haemostasis and in the pathophysiology of various diseases including arterial thrombosis. Glycoprotein (GP)VI, the major platelet collagen receptor, mediates platelet activation and firm adhesion to collagen structures exposed at sites of vascular injury. Here, we determined the effects of specific activation of GPVI on the human platelet proteome by two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Methods and Results: Platelets from healthy human donors were isolated and purified. Platelets were stimulated with an activating monoclonal antibody specific for GPVI, or control IgG. Platelet proteins were subjected to 2D-DIGE with extensive software-assisted image analysis. Subsequent identification of the differentially abundant proteins was carried out by MALDI TOF/TOF MS and LC ESI-MS/MS. We identified 8 differentially regulated proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included pleckstrin, beta-centractin, src substrate cortactin, charged multivesicular body protein 3, aldose reductase and protein disulfide isomerase-associated 3 precursor. Conclusion: Specific platelet activation via GPVI results in the differential regulation of several proteins. The results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor, which may help to identify novel pharmacological targets.

scholarly journals Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation

2009 ◽  
Vol 83 (20) ◽  
pp. 10797-10807 ◽  
Author(s):  
Ritesh Tandon ◽  
David P. AuCoin ◽  
Edward S. Mocarski
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Body Protein ◽  
Escrt Machinery ◽  
Hcmv Replication ◽  
Viral Maturation ◽  
Vacuolar Protein

ABSTRACT The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

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

2009 ◽  
Vol 418 (2) ◽  
pp. 277-284 ◽  
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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