ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication.

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

LAP1 supports nuclear adaptability during constrained melanoma cell migration and invasion.

Metastasis involves dissemination of cancer cells away from a primary tumour and colonization at distal sites. During this process, the mechanical properties of the nucleus must be tuned since they pose a challenge to the negotiation of physical constraints imposed by the microenvironment and tissue structure. We discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells, at the invasive front of human primary melanoma tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. Here, using in vitro and in vivo models that recapitulate human melanoma progression, we found that expression of the shorter isoform, LAP1C, supports nuclear envelope blebbing, constrained migration and invasion by allowing a weaker coupling between the nuclear envelope and the nuclear lamina. We propose that LAP1 renders the nucleus highly adaptable and contributes to melanoma aggressiveness.

AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody.

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.

CDK1 controls CHMP7-dependent nuclear envelope reformation.

Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic (M) exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during M-exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon M-entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.

Membrane and organelle dynamics during cell division.

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated.

The ESCRT-machinery: closing holes and expanding roles.

The ESCRT machinery is an ancient, evolutionarily conserved membrane remodelling complex that is deployed by cells to perform a diverse collection of physiological and pathophysiological processes. ESCRT proteins are needed for multivesicular body biogenesis, release of enveloped retroviruses, reformation of the nuclear envelope and cytokinetic abscission during mitotic exit. These events all share the requirement for a topologically equivalent membrane remodelling for their completion that is thought to be performed by ESCRT-III. More recently, ESCRTs have been shown to play essential roles in repairing damaged cellular membranes, so preserving cellular viability and organellar function. Here, we will examine new advances in our understanding of the cell biology of this fascinating cellular machinery.

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.

ALIX Regulates Tumor-Mediated Immunosuppression by Controlling EGFR Activity and PD-L1 Presentation.

The immunosuppressive transmembrane protein PD-L1 was shown to traffic via the multivesicular body (MVB) and to be released on exosomes. A high-content siRNA screen identified the endosomal sorting complexes required for transport (ESCRT)-associated protein ALIX as a regulator of both EGFR activity and PD-L1 surface presentation in basal-like breast cancer (BLBC) cells. ALIX depletion results in prolonged and enhanced stimulation-induced EGFR activity as well as defective PD-L1 trafficking through the MVB, reduced exosomal secretion, and its redistribution to the cell surface. Increased surface PD-L1 expression confers an EGFR-dependent immunosuppressive phenotype on ALIX-depleted cells. An inverse association between ALIX and PD-L1 expression was observed in human breast cancer tissues, while an immunocompetent mouse model of breast cancer revealed that ALIX-deficient tumors are larger and show an increased immunosuppressive environment. Our data suggest that ALIX modulates immunosuppression through regulation of PD-L1 and EGFR and may, therefore, present a diagnostic and therapeutic target for BLBC.

Capping protein regulates actin dynamics during cytokinetic midbody maturation.

During cytokinesis, a cleavage furrow generated by actomyosin ring contraction is restructured into the midbody, a platform for the assembly of the abscission machinery that controls the final separation of daughter cells. The polymerization state of F-actin is important during assembly, ingression, disassembly, and closure of the contractile ring and for the cytoskeletal remodeling that accompanies midbody formation and progression to abscission. Actin filaments must be cleared from the abscission sites before the final cut can take place. Although many conserved proteins interact with and influence the polymerization state of actin filaments, it is poorly understood how they regulate cytokinesis in higher eukaryotes. We report here that the actin capping protein (CP), a barbed end actin binding protein, participates in the control of actin polymerization during later stages of cytokinesis in human cells. Cells depleted of CP furrow and form early midbodies, but they fail cytokinesis. Appropriate recruitment of the ESCRT-III abscission machinery to the midbody is impaired, preventing the cell from progressing to the abscission stage. To generate actin filaments of optimal length, different actin nucleators, such as formins, balance CP's activity. Loss of actin capping activity leads to excessive accumulation of formin-based linear actin filaments. Depletion of the formin FHOD1 results in partial rescue of CP-induced cytokinesis failure, suggesting that it can antagonize CP activity during midbody maturation. Our work suggests that the actin cytoskeleton is remodeled in a stepwise manner during cytokinesis, with different regulators at different stages required for successful progression to abscission.

Membrane Binding by CHMP7 Coordinates ESCRT-III-Dependent Nuclear Envelope Reformation.

In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2, 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4, 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7's N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.

ESCRT-III controls nuclear envelope reformation.

During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents. NE reformation requires the coating of chromatin by membrane derived from the endoplasmic reticulum, and a subsequent annular fusion step to ensure that the formed envelope is sealed. How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis. Here we show that the endosomal sorting complex required for transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the forming NE in human cells, and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged multivesicular body protein 2A (CHMP2A) is directed to the forming NE through binding to CHMP4B, and provides an activity essential for NE reformation. Localization also requires the p97 complex member ubiquitin fusion and degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodelling events.

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.

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.

Phosphoinositides in the mammalian endo-lysosomal network.

The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform.

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 ESCRT machinery: new functions in viral and cellular biology.

The ESCRT (endosomal sorting complex required for transport) machinery consists of a number of cytosolic proteins that make up three functional subcomplexes: ESCRT-I, ESCRT-II and ESCRT-III. These proteins function in multivesicular body formation and cell division and are co-opted by enveloped retroviruses to facilitate viral egress. Analysis of these functions may help illuminate conserved mechanisms of ESCRT function.

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport.

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.