Detection of the 125-kDa nuclear protein mitotin in centrosomes, the poles of the mitotic spindle, and the midbody.

Mitotin is a nuclear protein detectable in all proliferating cells investigated so far, including human and plant cells. In interphase cells the protein is localized mainly in the nucleoplasm. In G2/M phase it displays a characteristic redistribution and a marked increase which initiated the name mitotin. This study presents the precise localization of mitotin in cytoplasmic structures in two cell types, the potoroo rat kangaroo PtK2 cell and the human lung cancer EPLC 65 cell. In addition to its nuclear localization the antigen is detectable in centrosomes, in the poles of the mitotic spindle, and along spindle fibers. During the last mitotic stages, cytokinesis and reconstitution of nuclei, mitotin displays a rapid decrease and another redistribution. A significant amount of the antigen is retained in the bridge connecting the dividing cells, the midbody.     

The Golgi protein GM130 regulates centrosome morphology and function

The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNA interference-mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase, and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. Although GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase.     

Stathmin Prevents the Transition from a Normal to an Endomitotic Cell Cycle during Megakaryocytic Differentiation

Physiological polyploidy is a characteristic of several cell types including themegakaryocytes (MK) that give rise to circulating blood platelets. MK achieve polyploidy byswitching from a normal to an endomitotic cell cycle characterized by the absence of late mitoticstages. During an endomitotic cycle, the cells enter into mitosis and proceed normally throughmetaphase and early anaphase. However, late anaphase, telophase and cytokinesis are aborted. Thisabortive mitosis is associated with atypical multipolar mitotic spindles and limited chromosomesegregation. Stathmin is a microtubule-depolymerizing protein that is important for the regulation ofthe mitotic spindle and interfering with its expression disrupts the normal mitotic spindle and leadsto aberrant mitotic exit. As cells enter mitosis, the microtubule depolymerizing-activity of stathminis switched-off, allowing microtubules to polymerize and assemble into a mitotic spindle.Reactivation of stathmin in the later stages of mitosis is necessary for the disassembly of the mitoticspindle and the exit from mitosis. Previous studies had shown that stathmin expression isdownregulated as MK become polyploid and inhibition of its expression in K562 cells increasestheir propensity to become polyploid. In this report, we describe our studies of the mechanism bywhich stathmin plays its role in MK polyploidization. We show that stathmin overexpressionprevents the transition from a mitotic cycle to an endomitotic cycle as determined by a decrease inthe number of multipolar mitotic spindles. These observations support a model in whichdownregulation of stathmin expression in megakaryocytes and other polyploid cells may be acritically important factor in endomitosis and polyploidy.     

Lamins position the nuclear pores and centrosomes by modulating dynein

Lamins, the type V nuclear intermediate filament proteins, are reported to function in both interphase and mitosis. For example, lamin deletion in various cell types can lead to an uneven distribution of the nuclear pore complexes (NPCs) in the interphase nuclear envelope, whereas deletion of B-type lamins results in spindle orientation defects in mitotic neural progenitor cells. How lamins regulate these functions is unknown. Using mouse cells deleted of different combinations or all lamins, we show that lamins are required to prevent the aggregation of NPCs in the nuclear envelope near centrosomes in late G2 and prophase. This asymmetric NPC distribution in the absence of lamins is caused by dynein forces acting on NPCs via the dynein adaptor BICD2. We further show that asymmetric NPC distribution upon lamin depletion disrupts the distribution of BICD2 and p150 dynactin on the nuclear envelope at prophase, which results in inefficient dynein-driven centrosome separation during prophase. Therefore lamins regulate microtubule-based motor forces in vivo to ensure proper NPC distribution in interphase and centrosome separation in the mitotic prophase.     

Enolase is present at the centrosome of HeLa cells.

Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.     

A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies.

Six monoclonal antibodies identify a 210 kDa polypeptide which shows a cell cycle specific redistribution from the nucleus to the mitotic spindle. In interphase cells this polypeptide was localized in the nucleus and behaved during differential cell extraction as a component of the nuclear matrix. It accumulated in the centrosome region at prophase, in the pole regions of the mitotic spindle at metaphase and in crescents at the poles in anaphase, and reassociated with the nuclei as they reformed in telophase. Due to its staining pattern we call the protein the Spindle Pole-Nucleus (SPN) antigen. The localization of SPN antigen during mitosis was dependent on the integrity of the spindle since treatment of cells with nocodazole resulted in the dispersal of SPN antigen into many small foci which acted as microtubule organizing centres when the drug was removed. The SPN antigen was present in nuclei and mitotic spindles of all human and mammalian cell lines and tissues so far tested. When microinjected into the cytoplasm or nuclei of HeLa cells, one antibody caused a block in mitosis. Total cell number remained constant or decreased slightly after 24 h. At this time, about half the cells were arrested in a prometaphase-like state and revealed aberrant spindles. Many other cells were multinucleate. These results show that the SPN antigen is a protein associated with mitotic spindle microtubules which has to function correctly for the cell to complete mitosis.     

Separate to operate: control of centrosome positioning and separation

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.     

Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle

The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes. However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity. Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome. Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared. In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle. Nucleating activity did not change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle.     

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.     

Occludin localizes to centrosomes and modifies mitotic entry

Proper control of cell cycle progression and barrier function are essential processes to the maintenance of epithelial cell homeostasis. The contribution of tight junction proteins to barrier function is well established, whereas their contribution to cell cycle control is only beginning to be understood. Centrosomes are the principal microtubule organizing centers in eukaryotic cells and centrosome duplication and separation are linked to the cell cycle and mitotic entry. Here we demonstrate that occludin localizes with centrosomes in Madin-Darby canine kidney cells. Immunocytochemistry and biochemical fractionation studies reveal occludin localizes with centrosomes during interphase and occludin Ser-490 phosphorylation at centrosomes increases with mitotic entry. Stable expression of aspartic acid phosphomimetic (S490D) results in centrosomal localization of occludin and increases cell numbers. Furthermore, we provide evidence that occludin regulates centrosome separation and mitotic entry as the nonphosphorylatable alanine mutation (S490A) impedes centrosome separation, delays mitotic entry, and reduces proliferation. Collectively, these studies demonstrate a novel location and function for occludin in centrosome separation and mitosis.     

Centrobin/NIP2 Is a Microtubule Stabilizer Whose Activity Is Enhanced by PLK1 Phosphorylation during Mitosis

Centrobin/NIP2 is a centrosomal protein that is required for centrosome duplication. It is also critical for microtubule organization in both interphase and mitotic cells. In the present study, we observed that centrobin is phosphorylated in a cell cycle stage-specific manner, reaching its maximum at M phase. PLK1 is a kinase that is responsible for M phase-specific phosphorylation of centrobin. The microtubule forming activity of centrobin was enhanced by PLK1 phosphorylation. Furthermore, mitotic spindles were not assembled properly with the phospho-resistant mutant of centrobin. Based on these results, we propose that centrobin functions as a microtubule stabilizing factor and PLK1 enhances centrobin activity for proper spindle formation during mitosis.     

Importin β2 Mediates the Spatio-temporal Regulation of Anillin through a Noncanonical Nuclear Localization Signal

The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin β2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin β2 binding does not regulate anillin''s function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin''s function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.     

Structural alterations of the mitotic apparatus induced by the heat shock response in Drosophila cells

The general architecture of the mitotic apparatus was studied at the ultrastructural level in Drosophila cultured cells. Its two main characteristics are a very polarized spindle and a strong compartmentalization, ensured by large remnants of the nuclear envelope. Such compartmentalization has previously been reported for the rapid syncytial divisions of the early embryo; a similar finding in these cells with a long cycle strongly suggests that this organization constitutes a general mechanism for mitosis in Drosophila. We followed the modifications of these structures after a heat shock of 20, 50 or 120 min at 37°C. Contrary to interphase cells, mitotic cells appear very sensitive to hyperthermia. This stress treatment induced a disruption of the mitotic spindle, a reappearance and an extension of the Golgi apparatus, an inactivation of microtubule nucleation and a disorganization of the centrosome. This organelle seems the first to be affected by the heat shock response. The centrosome is not only inactivated, but also is structurally affected. During the recovery phase after heat stress, the mitotic cells presented a remarkable ring-shaped accumulation of electrondense material around the centrioles. We conclude that in Drosophila cells the mitotic phase, and more specifically the centrosome, are targets of the stress response.     

Intracellular localization of terminal transferase during the cell cycle.

Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure.     

Interphase nucleo-cytoplasmic shuttling and localization of SIRT2 during mitosis

The human NAD+-dependent protein deacetylase SIRT2 resides predominantly in the cytoplasm where it functions as a tubulin deacetylase. Here we report that SIRT2 maintains a largely cytoplasmic localization during interphase by active nuclear export in a Crm1-dependent manner. We identified a functional, leptomycin B-sensitive, nuclear export signal sequence within SIRT2. During the cell cycle, SIRT2 becomes enriched in the nucleus and is associated with mitotic structures, beginning with the centrosome during prophase, the mitotic spindle during metaphase, and the midbody during cytokinesis. Cells overexpressing wild-type or a catalytically inactive SIRT2 exhibit an increase in multinucleated cells. The findings suggest a novel mechanism of regulating SIRT2 function by nucleo-cytoplasmic shuttling, as well as a role for SIRT2 in the nucleus during interphase and throughout mitosis.     

A developmental mutation (npfL1) resulting in cell death in Physarum polycephalum.

In Physarum, microscopic uninucleate amoebae develop into macroscopic multinucleate plasmodia. In the mutant strain, RA614, plasmodium development is blocked. RA614 carries a recessive mutation (npfL1) in a gene that functions in sexual as well as apogamic development. In npfL+ apogamic development, binucleate cells arise from uninucleate cells by mitosis without cytokinesis at the end of an extended cell cycle. In npfL1 cultures, apogamic development became abnormal at the end of the extended cell cycle. The cells developed a characteristic rounded, vacuolated appearance, nuclear fusion and vigorous cytoplasmic motion occurred, and the cells eventually died. Nuclei were not visible by phase-contrast microscopy in most of the abnormally developing cells, but fluorescence microscopy after DAPI staining revealed intensely staining, condensed nuclei without nucleoli. Studies of tubulin organization during npfL1 development indicated a high frequency of abnormal mitotic spindles and, in some interphase cells, abnormally thick microtubules. Some of these features were observed at low frequency in the parental npfL+ strain and may represent a pathway of cell death, resembling apoptosis, that may be triggered in more than one way. Nuclear fusion occurred during interphase and mitosis in npfL1 cells, and multipolar spindles were also observed. None of these features were observed in npfL+ cells, suggesting that a specific effect of the npfL1 mutation may be an incomplete alteration of nuclear structure from the amoebal to the plasmodial state.     

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.     

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.