Formation of the Nuclear Envelope Permeability Barrier Studied by Sequential Photoswitching and Flux Analysis

In higher eukaryotes, the nuclear envelope breaks down during mitosis. It reforms during telophase, and nuclear import is reestablished within <10 min after anaphase onset. It is widely assumed that import functionality simultaneously leads to the exclusion of bulk cytoplasmic proteins. However, nuclear pore complex assembly is not fully completed when import capacity is regained, which raises the question of whether the transport and permeability barrier functions of the nuclear envelope are indeed coupled. In this study, we therefore analyzed the reestablishment of the permeability barrier of the nuclear envelope after mitosis in living cells by monitoring the flux of the reversibly photoswitchable fluorescent protein Dronpa from the cytoplasm into the nucleus after photoactivation. We performed many consecutive flux measurements in the same cell to directly monitor changes in nuclear envelope permeability. Our measurements at different time points after mitosis in individual cells show that contrary to the general view and despite the rapid reestablishment of facilitated nuclear import, the nuclear envelope remains relatively permeable for passive diffusion for the first 2 h after mitosis. Our data demonstrate that reformation of the permeability barrier of nuclear pore complexes occurs only gradually and is uncoupled from regaining active import functionality.     

Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.     

Unusual pattern of mitosis in the free-living flagellateDimastigella mimosa (Kinetoplastida)

Summary The three-dimensional ultrastructural organization of the mitotic apparatus ofDimastigella mimosa was studied by computer-aided, serial-section reconstruction. The nuclear envelope remains intact during nuclear division. During mitosis, chromosomes do not condense, whereas intranuclear microtubules are found in close association with six pairs of kinetochores. No discrete microtubule-organizing centers, except kinetochore pairs, could be found within the nucleus. The intranuclear microtubules form six separate bundles oriented at different angles to each other. Each bundle contains up to 8 tightly packed microtubules which push the daughter kinetochores apart. At late anaphase only, midzones of these bundles align along an extended interzonal spindle within the narrow isthmus between segregating progeny nuclei. The nuclear division inD. mimosa can be described as closed intranuclear mitosis with acentric and separate microtubular bundles and weakly condensed chromosomes.Abbreviation MTOC microtubule-organizing center     

Virtual breakdown of the nuclear envelope in fission yeast meiosis

Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.     

Phosphorylation of NuSAP by Cdk1 regulates its interaction with microtubules in mitosis

Cell division in eukaryotes depends on a fine control of the dynamic changes of microtubules. Nucleolar and spindle-associated protein (NuSAP) is a microtubule-binding and -bundling protein essential for the integrity of the anaphase spindle and cell division. NuSAP contains two consensus cdk phosphorylation sites in its microtubule-binding domain. Here we show NuSAP is phosphorylated by cdk1 in early mitosis. This phosphorylation inhibits the binding of NuSAP to microtubules. During metaphase-to anaphase transition, NuSAP is dephosphorylated to promote spindle midzone formation and cell cycle progression. Expression of cdk1 phosphorylation-null mutant causes extensive bundling of microtubules in the prometaphase spindle. Our results suggest that phosphorylation and dephosphorylation of NuSAP during progression of mitosis regulate spindle organization through modulation of the dynamics of microtubules.     

CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.     

Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.     

The spindle matrix through the cell cycle in Drosophila

A spindle matrix has long been proposed to provide structural support for counterbalancing force production and a substrate for essential mitotic factors. For years the molecular identity of such a structure remained elusive. Recently a complex of nuclear proteins that reorganize into a spindle-like structure during prophase through metaphase that shows characteristics of a spindle matrix has been identified in Drosophila. We review how these results support the concept of a spindle matrix and discuss its possible function(s) during mitosis. Importantly, these molecules also appear to play critical roles during interphase in nuclear organization and function. Given that during cell division the entire nucleus undergoes a dynamic and tightly orchestrated reorganization, the reorganization of spindle matrix components during mitosis may comprise one phase of a continuum of "nuclear architectural remodeling events" that can be considered to extend throughout the entire cell cycle, even in the absence of a defined nucleus.     

Immobility,inheritance and plasticity of shape of the yeast nucleus

Background  

Since S. cerevisiae undergoes closed mitosis, the nuclear envelope of the daughter nucleus is continuous with that of the maternal nucleus at anaphase. Nevertheless, several constitutents of the maternal nucleus are not present in the daughter nucleus. The present study aims to identify proteins which impact the shape of the yeast nucleus and to learn whether modifications of shape are passed on to the next mitotic generation. The Esc1p protein of S. cerevisiae localizes to the periphery of the nucleoplasm, can anchor chromatin, and has been implicated in targeted silencing both at telomeres and at HMR.     

Mutations in the ATP-binding domain affect the subcellular distribution of mitotic centromere-associated kinesin (MCAK)

Mitotic centromere-associated kinesin (MCAK) is important for anaphase chromosome segregation. MCAK is diffusely localized to both the cytoplasm and the nucleus during interphase. At prophase MCAK is recruited to mitotic centromeres. It is associated with centromeres throughout mitosis and then returns to exhibiting a diffuse nuclear and cytoplasmic localization during interphase. MCAK has several predicted nuclear localization sequences. The subcellular distribution of expressed deletion constructs of GFP-MCAK suggest that the nucleocytoplasmic ratio of MCAK protein is dependent on a balance between several predicted nuclear localization sequences (NLS) and a putative nuclear exclusion sequence (NES) in the amino-terminal region of MCAK. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, which, in turn, influences the degree of centromere binding.     

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)–containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.     

Divergent strategies for controlling the nuclear membrane satisfy geometric constraints during nuclear division

Eukaryotes segregate chromosomes in "open" or "closed" mitosis, depending on whether their nuclear envelopes (NEs) break down or remain intact. Here we show that the control of the nuclear surface area may determine the choice between these two modes. The dividing nucleus does not expand its surface in the fission yeast Schizosaccharomyces japonicus, confining the mitotic spindle and causing it to?buckle. The NE ruptures in anaphase, releasing the compressive stress and allowing chromosome segregation.?Blocking the NE expansion in the related species Schizosaccharomyces pombe that undergoes closed mitosis induces spindle buckling and collapse in the absence of an intrinsic NE rupture mechanism. We propose that scaling considerations could have shaped the evolution of eukaryotic mitosis by necessitating either nuclear surface expansion or the NE breakdown.     

Nuclear compartmentalization is abolished during fission yeast meiosis

In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells. It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle. Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.     

Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells

Summary. The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.Correspondence and reprints: Department of Biology, 221 Morrill Science Center II, University of Massachusetts, Amherst, MA 01003, U.S.A.Received September 30, 2002; accepted February 12, 2003 Published online September 23, 2003     

Thrombopoietin-induced Polyploidization of Bone Marrow Megakaryocytes Is Due to a Unique Regulatory Mechanism in Late Mitosis

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.     

Nuclear membrane: nuclear envelope PORosity in fission yeast meiosis

The fission yeast Schizosaccharomyces pombe undergoes closed mitosis but 'virtual nuclear envelope breakdown' at anaphase of meiosis II, in which the nuclear envelope is structurally closed but functionally open.