Open Mitosis Research Articles

Life-cycle-coupled evolution of mitosis in close relatives of animals

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope during cell division: disassembling the nuclear envelope in an open mitosis or constructing an intranuclear spindle in a closed mitosis1,2. Both classes of mitotic remodelling involve key differences in the core division machinery but the evolutionary reasons for adopting a specific mechanism are unclear. Here we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species in this clade have diverged towards either a fungal-like closed mitosis or an animal-like open mitosis, probably to support distinct multinucleated or uninucleated states. Our results indicate that multinucleated life cycles favour the evolution of closed mitosis.

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.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex

SUMMARYNuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.

We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an "analog-sensitive" Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.

Visualizing Nuclear Pore Complex Assembly In Situ in Human Cells at Nanometer Resolution by Correlating Live Imaging with Electron Microscopy.

In eukaryotic cells that undergo open mitosis, nuclear pore complex assembly proceeds via two distinct pathways: postmitotic and interphase assembly. Studying both assembly processes is challenging because postmitotic assembly is fast, interphase assembly is rare and sporadic, and assembly intermediates in both pathways are very small with a diameter below 100nm. Here, we present a protocol for studying nuclear pore complex biogenesis in situ in cultured human cells in a spatiotemporally resolved and quantitative manner by combining live imaging with three-dimensional electron microscopy. The method described here can alsobe applied for studying other cell cycle-associated events with high spatiotemporal resolution.

A CTDNEP 1-Lipin 1-mTOR Regulatory Network Restricts ER Membrane Biogenesis to Enable Chromosome Motions Necessary for Mitotic Fidelity

The endoplasmic reticulum (ER) dramatically restructures in open mitosis to become excluded from the mitotic spindle; however, the significance of ER reorganization to mitotic progression is not known. Here, we demonstrate that limiting ER membrane biogenesis enables mitotic chromosome movements necessary for chromosome biorientation and prevention of micronuclei formation. Aberrantly expanded ER membranes increase the effective viscosity of the mitotic cytoplasm to physically restrict chromosome dynamics – slowed chromosome motions impede correction of mitotic errors induced by transient disassembly, leading to severe micronucleation. We define the mechanistic link between regulation of ER membrane biogenesis and mitotic fidelity by demonstrating that a CTDNEP1-lipin 1-mTOR regulatory network limits ER lipid synthesis to prevent chromosome missegregation. Together, this work shows that ER membranes reorganize in mitosis to enable chromosome movements necessary for mitotic error correction and reveal dysregulated lipid metabolism as a potential source of aneuploidy in cancer cells. 

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.

Closed mitosis requires local disassembly of the nuclear envelope.

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Attenuation of cGAS/STING activity during mitosis.

The innate immune system recognizes cytosolic DNA associated with microbial infections and cellular stress via the cGAS/STING pathway, leading to activation of phospho-IRF3 and downstream IFN-I and senescence responses. To prevent hyperactivation, cGAS/STING is presumed to be nonresponsive to chromosomal self-DNA during open mitosis, although specific regulatory mechanisms are lacking. Given a role for the Golgi in STING activation, we investigated the state of the cGAS/STING pathway in interphase cells with artificially vesiculated Golgi and in cells arrested in mitosis. We find that whereas cGAS activity is impaired through interaction with mitotic chromosomes, Golgi integrity has little effect on the enzyme's production of cGAMP. In contrast, STING activation in response to either foreign DNA (cGAS-dependent) or exogenous cGAMP is impaired by a vesiculated Golgi. Overall, our data suggest a secondary means for cells to limit potentially harmful cGAS/STING responses during open mitosis via natural Golgi vesiculation.

REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis.

The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.

Abstract 1459: A mechanism counteracting micronucleation for maintenance of genomic integrity

Abstract A hallmark of eukaryotic cells is that they store all their chromosomes in a single nucleus. This is important for the maintenance of genomic integrity, as individual chromosomes packaged into separate micronuclei are prone to massive DNA damage. Micronuclei are characteristic for cancer cells and thought to drive the evolution of cancer genomes. However, the mechanisms normally promoting the packaging of all chromosomes into a single nucleus have remained largely elusive. Animal cells undergo an open mitosis, in which the cell disassembles its nucleus to release a set of individualized chromosomes. At the end of mitosis, cells reassemble a single nucleus around a complete set of chromosomes, utilizing endoplasmic reticulum-derived membranes. How cells restrict nuclear envelope assembly to the surface around the set of anaphase chromosomes is unclear. It has been proposed that the mitotic spindle may help anaphase chromosomes form a continuous surface by bringing them into proximity. Using live cell imaging, we found that the mitotic spindle is not required to form a single nucleus in human cells. We then performed an RNAi screen for spindle-independent factors that are required to shape a single nucleus and identified the protein barrier-to-autointegration factor (BAF). BAF has been previously suggested to contribute to nuclear assembly by linking chromatin to nuclear envelope membranes containing “LEM” domain (Lap2/Emerin/Man1) proteins. However, by RNAi and mutational analysis, we unexpectedly discovered that BAF's LEM-binding activity was dispensable for nuclear assembly in human cells. Instead, we found that BAF's ability to dimerize and cross-bridge distant DNA segments was essential to shape a single nucleus. By probing purified chromatin and recombinant BAF proteins with atomic force microscopy, we found that BAF forms a rigid shell at the chromatin surface, depending on its DNA-DNA cross-bridging activity. In cells, BAF restricted membranes to the surface formed by the set of anaphase chromosomes, thereby preventing the formation of micronuclei. Thus, BAF regulates chromosome mechanics to shape a single nucleus during mitotic exit. Citation Format: Matthias Samwer, Maximilian W. Schneider, Rudolf Hoefler, Philipp Schmalhorst, Julian Jude, Johannes Zuber, Daniel W. Gerlich. A mechanism counteracting micronucleation for maintenance of genomic integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1459.

Importin α and vNEBD Control Meiotic Spindle Disassembly in Fission Yeast

In metazoans, the nuclear envelope (NE) breakdown (NEBD) occurs during "open" mitosis and meiosis. Inthe fission yeast Schizosaccharomyces pombe, the mitosis and the first meiotic division (MI) are "closed," during which the NE is maintained. Intriguingly, during the second meiotic division (MII), the NE is also maintained, but nuclear and cytoplasmic molecules are mixed similarly to open mitosis, a phenomenon of unknown biological significance called "virtual" NEBD (vNEBD). Here, we show that importin-α-dependent nucleocytoplasmic transport regulates spindle disassembly late in anaphase B at MI, as previously reported for mitosis. At MII, however, spindle dissolution is triggered by vNEBD early in anaphase B, a mechanism that short-circuits the nucleocytoplasmic transport system. We demonstrate that the sequential action of these two spindle disassembly systems regulates the spatiotemporal order and ploidy of the meiotic products.

Importin and vNEBD Control Meiotic Spindle Disassembly in Fission Yeast

In metazoans, the nuclear envelope (NE) breaks down (NEBD) during “open” mitosis and meiosis. In the fission yeast Schizosaccharomyces pombe, the mitosis and the first meiotic division (meiosis I) are “closed”, during which the NE is maintained. Intriguingly, during the second meiotic division (meiosis II) the NE is also maintained, but nuclear and cytoplasmic molecules are mixed similar to open mitosis, a phenomenon of unknown biological significance called “virtual” NEBD (vNEBD). Here we show that importin α-dependent nucleocytoplasmic transport regulates spindle disassembly late in anaphase B at meiosis I, as previously reported for mitosis. At meiosis II, however, spindle dissolution is triggered by vNEBD early in anaphase B, a mechanism that short-circuits the nucleocytoplasmic transport system. We demonstrate that the sequential action of these two spindle disassembly systems regulates the spatiotemporal order and ploidy of the meiotic products.

Mechanisms of nuclear pore complex assembly - two different ways of building one molecular machine.

The nuclear pore complex (NPC) mediates all macromolecular transport across the nuclear envelope. In higher eukaryotes that have an open mitosis, NPCs assemble at two points in the cell cycle: during nuclear assembly in late mitosis and during nuclear growth in interphase. How the NPC, the largest nonpolymeric protein complex in eukaryotic cells, self‐assembles inside cells remained unclear. Recent studies have started to uncover the assembly process, and evidence has been accumulating that postmitotic and interphase NPC assembly use fundamentally different mechanisms; the duration, structural intermediates, and regulation by molecular players are different and different types of membrane deformation are involved. In this Review, we summarize the current understanding of these two modes of NPC assembly and discuss the structural and regulatory steps that might drive the assembly processes. We furthermore integrate understanding of NPC assembly with the mechanisms for rapid nuclear growth in embryos and, finally, speculate on the evolutionary origin of the NPC implied by the presence of two distinct assembly mechanisms.

Mechanisms and functions of nuclear envelope remodelling.

As a compartment border, the nuclear envelope (NE) needs to serve as both a protective membrane shell for the genome and a versatile communication interface between the nucleus and the cytoplasm. Despite its important structural role in sheltering the genome, the NE is a dynamic and highly adaptable boundary that changes composition during differentiation, deforms in response to mechanical challenges, can be repaired upon rupture and even rapidly disassembles and reforms during open mitosis. NE remodelling is fundamentally involved in cell growth, division and differentiation, and if perturbed can lead to devastating diseases such as muscular dystrophies or premature ageing.

A Nucleoporin Docks Protein Phosphatase 1 to Direct Meiotic Chromosome Segregation and Nuclear Assembly

During M-phase entry in metazoans with open mitosis, the concerted action of mitotic kinases disassembles nuclei and promotes assembly of kinetochores-the primary microtubule attachment sites on chromosomes. At M-phase exit, these major changes in cellular architecture must be reversed. Here, we show that the conserved kinetochore-localized nucleoporin MEL-28/ELYS docks the catalytic subunit of protein phosphatase 1 (PP1c) to direct kinetochore disassembly-dependent chromosome segregation during oocyte meiosis I and nuclear assembly during the transition from M phase to interphase. During oocyte meiosis I, MEL-28-PP1c disassembles kinetochores in a timely manner to promote elongation of the acentrosomal spindles that segregate homologous chromosomes. During nuclear assembly, MEL-28 recruits PP1c to the periphery of decondensed chromatin, where it directs formation of a functional nuclear compartment. Thus, a pool of phosphatase activity associated with a kinetochore-localized nucleoporin contributes to two key events that occur during M-phase exit in metazoans: kinetochore disassembly and nuclear reassembly.

Bistability of mitotic entry and exit switches during open mitosis in mammalian cells.

Mitotic entry and exit are switch-like transitions that are driven by the activation and inactivation of Cdk1 and mitotic cyclins. This simple on/off reaction turns out to be a complex interplay of various reversible reactions, feedback loops, and thresholds that involve both the direct regulators of Cdk1 and its counteracting phosphatases. In this review, we summarize the interplay of the major components of the system and discuss how they work together to generate robustness, bistability, and irreversibility. We propose that it may be beneficial to regard the entry and exit reactions as two separate reversible switches that are distinguished by differences in the state of phosphatase activity, mitotic proteolysis, and a dramatic rearrangement of cellular components after nuclear envelope breakdown, and discuss how the major Cdk1 activity thresholds could be determined for these transitions.

An open and closed case

Cell Division One of the first recognized stages of an animal cell's entry into mitosis is the breakdown of the nuclear envelope (NE), a double-membraned structure that contains a cell's chromosomes. However, certain yeasts, including the lab workhorse Saccharomyces cerevisiae , keep their NE intact throughout mitosis; so-called closed mitosis. Makarova et al. studied two yeasts, one of which, Schizosaccharomyces japonicus , performs open mitosis, breaking down its NE during chromosome partitioning; and one of which, S. pombe , performs closed mitosis. They found that S. pombe –like mitotic regulation of a single lipid-modifying enzyme allowed S. japonicus to expand its NE and thus perform closed mitosis. Curr. Biol. 26 , 237 (2016)

Virtual Nuclear Envelope Breakdown and Its Regulators in Fission Yeast Meiosis.

Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called “open mitosis.” In contrast, many fungi undergo a process termed “closed mitosis” in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called “anaphase II”) when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This “virtual” nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis.

Structure and biological functions of mammalian LEM-domain proteins

During the process of open mitosis in higher eukaryotic cells, the nuclear envelope (NE) is disassembled and reassembled with highly organized and periodical dynamic morphological changes. Recent studies demonstrated that LEM-domain protein family mediates interactions among inner nuclear membrane, nuclear lamina protein and chromatin by interacting with barrier-to-autointegration-factor (BAF). The structure and function of the ternary complex formed by LEM-domain protein, nuclear lamina protein and BAF are dependent on each other. Moreover, the network formation based on this structure and function is critical for the development of basic biological processes of nuclear, and it plays important roles in chromatin separation in late metaphase and anaphase, NE reassembly after mitosis, morphological maintenance of nuclear and NE in interphase, regulation of DNA replication and DNA damage repair, regulation of gene expression and signaling pathway, and infection of retrovirus. Mutations in genes encoding LEM family proteins have important impacts on development and progression of laminopathic diseases and tumorigenesis. This review provides a detailed summary of structural and functional studies of the LEM family proteins.