Nuclear Envelope Reformation Research Articles

Vps60 initiates alternative ESCRT-III filaments.

Endosomal sorting complex required for transport-III (ESCRT-III) participates in essential cellular functions, from cell division to endosome maturation. The remarkable increase of its subunit diversity through evolution may have enabled the acquisition of novel functions. Here, we characterize a novel ESCRT-III copolymer initiated by Vps60. Membrane-bound Vps60 polymers recruit Vps2, Vps24, Did2, and Ist1, as previously shown for Snf7. Snf7- and Vps60-based filaments can coexist on membranes without interacting as their polymerization and recruitment of downstream subunits remain spatially and biochemically separated. In fibroblasts, Vps60/CHMP5 and Snf7/CHMP4 are both recruited during endosomal functions and cytokinesis, but their localization is segregated and their recruitment dynamics are different. Contrary to Snf7/CHMP4, Vps60/CHMP5 is not recruited during nuclear envelope reformation. Taken together, our results show that Vps60 and Snf7 form functionally distinct ESCRT-III polymers, supporting the notion that diversification of ESCRT-III subunits through evolution is linked to the acquisition of new cellular functions.

Identification of PP2A-B55 targets uncovers regulation of emerin during nuclear envelope reassembly in Drosophila.

Mitotic exit requires the dephosphorylation of many proteins whose phosphorylation was needed for mitosis. Protein phosphatase 2A with its B55 regulatory subunit (PP2A-B55) promotes this transition. However, the events and substrates that it regulates are incompletely understood. We used proteomic approaches in Drosophila to identify proteins that interact with and are dephosphorylated by PP2A-B55. Among several candidates, we identified emerin (otefin in Drosophila). Emerin resides in the inner nuclear membrane and interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) via a LEM domain. We found that the phosphorylation of emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, lamin and additional emerin in mitosis. We show that dephosphorylation of emerin at these sites by PP2A-B55 determines the timing of nuclear envelope reformation. Genetic experiments indicate that this regulation is required during embryonic development. Phosphoregulation of the emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely conserved across species.

The large GTPase AtGBPL3 links nuclear envelope formation and morphogenesis to transcriptional repression.

Guanylate binding proteins (GBPs) are prominent regulators of immunity not known to be required for nuclear envelope formation and morphogenesis. Here we identify the Arabidopsis GBP orthologue AtGBPL3 as a lamina component with essential functions in mitotic nuclear envelope reformation, nuclear morphogenesis and transcriptional repression during interphase. AtGBPL3 is preferentially expressed in mitotically active root tips, accumulates at the nuclear envelope and interacts with centromeric chromatin as well as with lamina components transcriptionally repressing pericentromeric chromatin. Reduced expression of AtGBPL3 or associated lamina components similarly altered nuclear morphology and caused overlapping transcriptional deregulation. Investigating the dynamics of AtGBPL3-GFP and other nuclear markers during mitosis (1) revealed that AtGBPL3 accumulation on the surface of daughter nuclei precedes nuclear envelope reformation and (2) uncovered defects in this process in roots of AtGBPL3 mutants, which cause programmed cell death and impair growth. AtGBPL3 functions established by these observations are unique among dynamin-family large GTPases.

Maintaining soluble protein homeostasis between nuclear and cytoplasmic compartments across mitosis.

The nuclear envelope (NE) is central to the architecture of eukaryotic cells, both as a physical barrier separating the nucleus from the cytoplasm and as gatekeeper of selective transport between them. However, in open mitosis, the NE fragments to allow for spindle formation and segregation of chromosomes, resulting in intermixing of nuclear and cytoplasmic soluble fractions. Recent studies have shed new light on the mechanisms driving reinstatement of soluble proteome homeostasis following NE reformation in daughter cells. Here, we provide an overview of how mitotic cells confront this challenge to ensure continuity of basic cellular functions across generations and elaborate on the implications for the proteasome - a macromolecular machine that functions in both cytoplasmic and nuclear compartments.

Oligomeric CHMP7 mediates three-way ER junctions and ER-mitochondria interactions.

In metazoans the endoplasmic reticulum (ER) undergoes extensive remodeling during the cell cycle. The endosomal sorting complexes required for transport (ESCRT) protein CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation during mitotic exit. However, potential roles of ER-associated CHMP7 at non-mitotic stages remain unclear. Here we discovered a new role of CHMP7 in mediating three-way ER and ER-mitochondrial membrane contact sites (MCSs). We showed that CHMP7 localizes to multiple cellular membranes including the ER, mitochondrial-associated membranes (MAMs) and the outer mitochondrial membrane (OMM) via its N-terminal membrane-binding domain. CHMP7 undergoes dynamic assembly at three-way ER junctions and ER-mitochondrial MCSs through hydrophobic interactions among α helix-1 and α helix-2 of the C-terminal CHMP-like domain, which was required for tethering different organelles in vivo. Furthermore, CHMP7 mediates the formation of three-way ER junctions in parallel with Atlastins (ATLs). Importantly, CHMP7 also regulates ER-mitochondrial interactions and its depletion affects mitochondrial division independently of ESCRT complex. Taken together, our results suggest a direct role of CHMP7 in the formation of the ER contacts in interphase.

Phase Separation in the Nucleus and at the Nuclear Periphery during Post-Mitotic Nuclear Envelope Reformation.

Membrane-enclosed organelle compartmentalization is not the only way by which cell processes are spatially organized. Phase separation is emerging as a new driver in the organization of membrane-less compartments and biological processes. Liquid–liquid phase separation has been indicated as a new way to control the kinetics of molecular reactions and is based on weak multivalent interactions affecting the stoichiometry of the molecules involved. In the nucleus, liquid–liquid phase separation may represent an ancestral means of controlling genomic activity by forming discrete chromatin regions, regulating transcriptional activity, contributing to the assembly of DNA damage response foci, and controlling the organization of chromosomes. Liquid–liquid phase separation also contributes to chromatin function through its role in the reorganization of the nuclear periphery in the post-mitotic phase. Herein, we describe the basic principles regulating liquid–liquid phase separation, analyze examples of phase separation occurring in the nucleus, and dedicate attention to the implication of liquid–liquid phase separation in the reorganization of the nuclear periphery by the endosomal sorting complexes required for transport (ESCRT) machinery. Although some caution is warranted, current scientific knowledge allows for the hypothesis that many factors and processes in the cell are yet to be discovered which are functionally associated with phase separation.

Repo-Man/protein phosphatase 1 SUMOylation mediates binding to lamin A and serine 22 dephosphorylation.

Lamin A phosphorylation/de-phosphorylation is an important process during cells division as it allows for nuclear envelope (NE) disassembly at mitotic entry and its re-assembly during mitotic exit. Several kinases have been identified as responsible for these phosphorylations, but no protein phosphatase has been implicated in their reversal. One of the mitotic phosphosites in lamin A responsible for its dynamic behaviour is serine 22 (S22) which is de-phosphorylated during mitotic exit. Recent evidence has also linked the nuclear pool of lamin A S22ph in interphase to gene expression regulation. Previous work suggested that the phosphatase responsible for lamin A S22 de-phosphorylation is chromatin bound and interacts with lamin A via SUMO-SIM motives. We have previously reported that Repo-Man/protein phosphatase 1 (PP1) is a chromatin-associated phosphatase that regulates NE reformation. Here we propose that Repo-Man/PP1 phosphatase mediates lamin A S22 de-phosphorylation. We indeed show that depletion of Repo-Man leads to NE defects, causes hyperphosphorylation of lamin A S22 that can be rescued by a wild-type but not a SUMOylation-deficient mutant. Lamin A and Repo-Man interact in vivo and in vitro, and the interaction is mediated by SUMOylation. Moreover, the localization of Repo-Man/PP1 to the chromatin is essential for lamin A S22 de-phosphorylation.

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

The ESCRT machinery directs quality control over inner nuclear membrane architecture.

SUMMARYThe late-acting endosomal sorting complex required for transport (ESCRT) machinery has been implicated in facilitating the resealing of the nuclear envelope (NE) after mitosis, enabling compartmentalization of the genome away from the cytoplasm. Here, we leverage the stereotypic first division of the C. elegans embryo to identify additional functions of the ESCRT machinery in maintaining the structure of the inner nuclear membrane. Specifically, impaired ESCRT function results in a defect in the pruning of inner nuclear membrane invaginations, which arise normally during NE reformation and expansion. Additionally, in combination with a hypomorphic mutation that interferes with assembly of the underlying nuclear lamina, inhibition of ESCRT function significantly perturbs NE architecture and increases chromosome segregation defects, resulting in penetrant embryonic lethality. Our findings highlight links between ESCRT-mediated inner nuclear membrane remodeling, maintenance of nuclear envelope morphology, and the preservation of the genome during early development.

Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation.

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

The ESCRT Machinery Directs Quality Control Over Inner Nuclear Membrane Architecture

The late-acting endosomal sorting complex required for transport (ESCRT) machinery has been implicated in facilitating the resealing of the nuclear envelope (NE) after mitosis, enabling compartmentalization of the genome away from the cytoplasm. Here, we leverage the stereotypic first division of the C. elegans embryo to identify additional functions of the ESCRT machinery in maintaining the structure of the inner nuclear membrane. Specifically, impaired ESCRT function results in a defect in the pruning of inner nuclear membrane invaginations, which arise normally during NE reformation and expansion. Additionally, in combination with a hypomorphic mutation that interferes with assembly of the underlying nuclear lamina, inhibition of ESCRT function significantly perturbs NE architecture and increases chromosome segregation defects, resulting in penetrant embryonic lethality. Our findings provide new links between ESCRT-mediated inner nuclear membrane remodeling, maintenance of nuclear envelope morphology, and the preservation of the genome during early development.

ESCRT Is a Great Sealer: Non-Endosomal Function of the ESCRT Machinery in Membrane Repair and Autophagy.

Components of the endosomal sorting complex required for transport (ESCRTs) were first identified in a genetic screen in budding yeast as factors interfering with vacuolar protein sorting. In the last three decades, intensive studies have revealed the subunit composition of ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, their structure, the assembling mechanisms and their molecular and physiological functions. In plants, ESCRTs are essential for development, growth and stress responses. ESCRTs are best known for their function in endosomal trafficking, during which they are required for sorting ubiquitylated membrane proteins into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs). The formation of ILVs requires the function of ESCRT-III, which has been shown to mediate the membrane scission. Although the function of plant ESCRTs has been predominantly discussed in the context of endosomal trafficking, recent studies in other model organisms revealed a versatile role of ESCRTs in diverse cellular events with broad physiological implications. The non-endosomal functions of ESCRTs include cytokinesis, viral budding, autophagy, nuclear envelope reformation and membrane repair, although many of these have not yet been studied in plants. In this review, recent findings on non-endosomal ESCRT functions in plant, yeast and animals are highlighted and discussed.

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection.

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.

Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity.

Nucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153-Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.

Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly.

Complex gene regulation in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, animal and plant cells disassemble the nuclear envelope, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. However, these pores have a size limit of 39 nm4–7, raising the question of how larger cytoplasmic components are cleared from the nucleus. Here, we show that large cytoplasmic components are displaced prior to nuclear envelope assembly by movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to reestablish the compartmentalization of eukaryotic cells after open mitosis.

Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2.

Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.

Host AAA+ ATPase TER94 Plays Critical Roles in Building the Baculovirus Viral Replication Factory and Virion Morphogenesis.

TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.

Bridging Microtubules Promote Centering of the Kinetochores by Length-Dependent Pulling Forces

Chromosome positioning to the equatorial plane of the mitotic spindle is necessary to prevent lagging chromosomes and abnormal nuclear envelope reformation. It has been proposed that two centering mechanisms play a key role here, microtubule (MT) catastrophe promoted by kinesin-8 motors and pushing forces exerted by chromokinesins. Surprisingly, our experiments suggest that removal of PRC1 molecules from the antiparallel overlaps of the bridging MTs disrupts alignment of the kinetochores. Here we show, by introducing a theoretical model, that kinetochore MTs cross-linked by bridging MTs exert centering pulling forces. Our model also shows that length-dependent catastrophe and rescue regulated by motor proteins and passive cross-linkers are necessary for well defined length of MTs and their antiparallel overlap, respectively. We predict that stable antiparallel overlaps subsequently navigate positioning of the kinetochores in the center of the metaphase plate. These predictions were confirmed in experiments with overexpression of PRC1 proteins and silencing of Kif18A motors.

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk.

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation.

Postmitotic annulate lamellae assembly contributes to nuclear envelope reconstitution in daughter cells

In higher eukaryotic cells, the nuclear envelope (NE) is composed of double nuclear membranes studded with nuclear pore complexes (NPCs) and undergoes dynamic disassembly and reassembly during the cell cycle. However, how the NE and NPC reassemble remains largely unclear. Here, using HeLa, HEK293, and Drosophila cells, along with immunofluorescence microscopy and transmission EM methods, we found that postmitotic annulate lamellae (AL) assembly contributes to NE and NPC assembly. We observed that the AL are parallel membrane-pair stacks and possess regularly spaced AL pore complexes (ALPCs) that are morphologically similar to the NPCs. We found that the AL assemble in the cytoplasm during mitotic exit simultaneously with NE re-formation in daughter cells. Then, the assembled AL either bound the decondensing chromatin to directly transform into the NE or bound and fused with the outer nuclear membrane to join the assembling NE. The AL did not colocalize with sheet and tubular endoplasmic reticulum (ER) marker proteins on the ER or the lamin B receptor-localized membrane in the cytoplasm, suggesting that postmitotic AL assembly occurs independently of the chromatin and ER. Collectively, our results indicate that postmitotic AL assembly is a common cellular event and an intermediate step in NE and NPC assembly and in NE expansion in higher eukaryotic cells.