UMAD1 contributes to ESCRT-III dynamic subunit turnover during cytokinetic abscission.

Abscission is the final stage of cytokinesis whereby the midbody, a thin intercellular bridge, is resolved to separate the daughter cells. Cytokinetic abscission is mediated by the endosomal sorting complex required for transport (ESCRT), a conserved membrane remodelling machinery. The midbody organiser CEP55 recruits early acting ESCRT factors such as ESCRT-I and ALIX (also known as PDCD6IP), which subsequently initiate the formation of ESCRT-III polymers that sever the midbody. We now identify UMAD1 as an ESCRT-I subunit that facilitates abscission. UMAD1 selectively associates with VPS37C and VPS37B, supporting the formation of cytokinesis-specific ESCRT-I assemblies. TSG101 recruits UMAD1 to the site of midbody abscission, to stabilise the CEP55-ESCRT-I interaction. We further demonstrate that the UMAD1-ESCRT-I interaction facilitates the final step of cytokinesis. Paradoxically, UMAD1 and ALIX co-depletion has synergistic effects on abscission, whereas ESCRT-III recruitment to the midbody is not inhibited. Importantly, we find that both UMAD1 and ALIX are required for the dynamic exchange of ESCRT-III subunits at the midbody. Therefore, UMAD1 reveals a key functional connection between ESCRT-I and ESCRT-III that is required for cytokinesis.

The ESCRT machinery counteracts Nesprin-2G-mediated mechanical forces during nuclear envelope repair.

Transient nuclear envelope ruptures during interphase (NERDI) occur due to cytoskeletal compressive forces at sites of weakened lamina, and delayed NERDI repair results in genomic instability. Nuclear envelope (NE) sealing is completed by endosomal sorting complex required for transport (ESCRT) machinery. A key unanswered question is how local compressive forces are counteracted to allow efficient membrane resealing. Here, we identify the ESCRT-associated protein BROX as a crucial factor required to accelerate repair of the NE. Critically, BROX binds Nesprin-2G, a component of the linker of nucleoskeleton and cytoskeleton complex (LINC). This interaction promotes Nesprin-2G ubiquitination and facilitates the relaxation of mechanical stress imposed by compressive actin fibers at the rupture site. Thus, BROX rebalances excessive cytoskeletal forces in cells experiencing NE instability to promote effective NERDI repair. Our results demonstrate that BROX coordinates mechanoregulation with membrane remodeling to ensure the maintenance of nuclear-cytoplasmic compartmentalization and genomic stability.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress.

Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas 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 cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an 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 MVB-IEVs showed that they account for half of IEV production indicating that MVBs are 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.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna ® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .

Midbody Remnant Inheritance Is Regulated by the ESCRT Subunit CHMP4C.

The inheritance of the midbody remnant (MBR) breaks the symmetry of the two daughter cells, with functional consequences for lumen and primary cilium formation by polarized epithelial cells, and also for development and differentiation. However, despite its importance, neither the relationship between the plasma membrane and the inherited MBR nor the mechanism of MBR inheritance is well known. Here, the analysis by correlative light and ultra-high-resolution scanning electron microscopy reveals a membranous stalk that physically connects the MBR to the apical membrane of epithelial cells. The stalk, which derives from the uncleaved side of the midbody, concentrates the ESCRT machinery. The ESCRT CHMP4C subunit enables MBR inheritance, and its depletion dramatically reduces the percentage of ciliated cells. We demonstrate (1) that MBRs are physically connected to the plasma membrane, (2) how CHMP4C helps maintain the integrity of the connection, and (3) the functional importance of the connection.

Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics.

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.

CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope.

The coordinated reformation of the nuclear envelope (NE) after mitosis re-establishes the structural integrity and the functionality of the nuclear compartment. The endosomal sorting complex required for transport (ESCRT) machinery, a membrane remodeling pathway that is highly conserved in eukaryotes, has been recently involved in NE resealing by mediating the annular fusion of the nuclear membrane (NM). We show here that CC2D1B, a regulator of ESCRT polymerization, is required to re-establish the nuclear compartmentalization by coordinating endoplasmic reticulum (ER) membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE. Accordingly, CC2D1B determines the spatiotemporal distribution of the CHMP7-ESCRT-III axis during NE reformation. Crucially, in CC2D1B-depleted cells, ESCRT activity is uncoupled from Spastin-mediated severing of spindle microtubules, resulting in persisting microtubules that compromise nuclear morphology. Therefore, we reveal CC2D1B as an essential regulatory factor that licenses the formation of ESCRT-III polymers to ensure the orderly reformation of the NE.

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

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.

Growing functions of the ESCRT machinery in cell biology and viral replication.

The vast expansion in recent years of the cellular processes promoted by the endosomal sorting complex required for transport (ESCRT) machinery has reinforced its identity as a modular system that uses multiple adaptors to recruit the core membrane remodelling activity at different intracellular sites and facilitate membrane scission. Functional connections to processes such as the aurora B-dependent abscission checkpoint also highlight the importance of the spatiotemporal regulation of the ESCRT machinery. Here, we summarise the role of ESCRTs in viral budding, and what we have learned about the ESCRT pathway from studying this process. These advances are discussed in the context of areas of cell biology that have been transformed by research in the ESCRT field, including cytokinetic abscission, nuclear envelope resealing and plasma membrane repair.

ESCRT machinery: Damage control at the nuclear membrane.

Rupture of the nuclear envelope (NE) during interphase is thought to be an infrequent event in healthy cells. Two papers recently published in Science describe the transient disruption of the NE continuity in cells migrating through confined spaces, and uncover an essential role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the resealing of these nuclear discontinuities.

ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins.

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.

An ESCRT module is required for neuron pruning.

Neural circuits are refined by both functional and structural changes. Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. Here, we reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. Our data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore we show that this ESCRT module requires a direct, in vivo, interaction between Shrub/CHMP4B and the accessory protein Myopic/HD-PTP.

SNX15 links clathrin endocytosis to the PtdIns3P early endosome independently of the APPL1 endosome.

Sorting nexins (SNXs) are key regulators of the endosomal network. In designing an RNAi-mediated loss-of-function screen, we establish that of 30 human SNXs only SNX3, SNX5, SNX9, SNX15 and SNX21 appear to regulate EGF receptor degradative sorting. Suppression of SNX15 results in a delay in receptor degradation arising from a defect in movement of newly internalised EGF-receptor-labelled vesicles into early endosomes. Besides a phosphatidylinositol 3-phosphate- and PX-domain-dependent association to early endosomes, SNX15 also associates with clathrin-coated pits and clathrin-coated vesicles by direct binding to clathrin through a non-canonical clathrin-binding box. From live-cell imaging, it was identified that the activated EGF receptor enters distinct sub-populations of SNX15- and APPL1-labelled peripheral endocytic vesicles, which do not undergo heterotypic fusion. The SNX15-decorated receptor-containing sub-population does, however, undergo direct fusion with the Rab5-labelled early endosome. Our data are consistent with a model in which the EGF receptor enters the early endosome following clathrin-mediated endocytosis through at least two parallel pathways: maturation through an APPL1-intermediate compartment and an alternative more direct fusion between SNX15-decorated endocytic vesicles and the Rab5-positive early endosome.

Knowing when to cut and run: mechanisms that control cytokinetic abscission.

Abscission, the final step of cytokinesis, mediates the severing of the membrane tether, or midbody, that connects two daughter cells. It is now recognized that abscission is a complex process requiring tight spatiotemporal regulation of its machinery to ensure equal chromosome segregation and cytoplasm content distribution between daughter cells. Failure to coordinate these events results in genetic damage. Here, we review recent evidence suggesting that proper abscission timing is coordinated by cytoskeletal rearrangements and recruitment of regulators of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery such as CEP55 and MIT-domain-containing protein 1 (MITD1) to the abscission site. Additionally, we discuss the surveillance mechanism known as the Aurora B-mediated abscission checkpoint (NoCut), which prevents genetic damage by ensuring proper abscission delay when chromatin is trapped at the midbody.

ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD fold that binds membranes.

The endosomal sorting complexes required for transport (ESCRT) proteins have a critical function in abscission, the final separation of the daughter cells during cytokinesis. Here, we describe the structure and function of a previously uncharacterized ESCRT-III interacting protein, MIT-domain containing protein 1 (MITD1). Crystal structures of MITD1 reveal a dimer, with a microtubule-interacting and trafficking (MIT) domain at the N terminus and a unique, unanticipated phospholipase D-like (PLD) domain at the C terminus that binds membranes. We show that the MIT domain binds to a subset of ESCRT-III subunits and that this interaction mediates MITD1 recruitment to the midbody during cytokinesis. Depletion of MITD1 causes a distinct cytokinetic phenotype consistent with destabilization of the midbody and abscission failure. These results suggest a model whereby MITD1 coordinates the activity of ESCRT-III during abscission with earlier events in the final stages of cell division.

ESCRT-III governs the Aurora B-mediated abscission checkpoint through CHMP4C.

The endosomal sorting complex required for transport (ESCRT) machinery plays an evolutionarily conserved role in cytokinetic abscission, the final step of cell division where daughter cells are physically separated. Here, we show that charged multivesicular body (MVB) protein 4C (CHMP4C), a human ESCRT-III subunit, is involved in abscission timing. This function correlated with its differential spatiotemporal distribution during late stages of cytokinesis. Accordingly, CHMP4C functioned in the Aurora B-dependent abscission checkpoint to prevent both premature resolution of intercellular chromosome bridges and accumulation of DNA damage. CHMP4C engaged the chromosomal passenger complex (CPC) via interaction with Borealin, which suggested a model whereby CHMP4C inhibits abscission upon phosphorylation by Aurora B. Thus, the ESCRT machinery may protect against genetic damage by coordinating midbody resolution with the abscission checkpoint.

The UBAP1 subunit of ESCRT-I interacts with ubiquitin via a SOUBA domain.

The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.

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.

Host factors involved in retroviral budding and release.

The plasma membrane is the final barrier that enveloped viruses must cross during their egress from the infected cell. Here, we review recent insights into the cell biology of retroviral assembly and release; these insights have driven a new understanding of the host proteins, such as the ESCRT machinery, that are used by retroviruses to promote their final separation from the host cell. We also review antiviral host factors such as tetherin, which can directly inhibit the release of retroviral particles. These studies have illuminated the role of the lipid bilayer as the unexpected target for virus restriction by the innate immune response.