Premature assembly of nucleolus-derived foci induced by a reversible hypotonic shock in metaphase CV1 and HeLa cells

The assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells is an early stage of nucleolus reassembly during mitosis. In current literature, significant attention is paid to the molecular composition of NDF and their participation in reassembly of the mature nucleolus. However, very little is known about mechanisms controlling the NDF formation. The authors have demonstrated for the first time that a reversible action of low ionic strength buffers (lypotonic shock treatment) on living mitotic human HeLa and green monkey CV1 cells triggers a premature assembly of NDF at metaphase. Like the true NDF, i. e., those assembled in telophase mitosis, NDF prematurally induced at metaphase contain RNA and proteins required for rRNA processing (fibrillarin, B23/nucliophosmin, C23/nucleolin), but lack UBF, an auxiliary factor of RNA polymerase I. We have assumed that a reversible action of hypotonic shock on metaphase cells may result in temporal increase in intracellular [Ca2+](i) that, in its turn, may induce a premature assembly of NDF under isotonic conditions. The structural integrity of the mitotic spindle apparently plays an essential role in the response of metaphase cells to hypotonic shock treatments.     

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase

The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1–2 μm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp 140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis. Edited by: S. A. Gerbi     

Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.     

Myosin light chain kinases and phosphatase in mitosis and cytokinesis

At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.     

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.     

Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of LIMK2, and a subcellular localization of LIMK1 and LIMK2. The activity of LIMK2, distinct from LIMK1, toward cofilin phosphorylation did not change in the normal cell division cycle. In contrast, LIMK2 was hyperphosphorylated and its activity was markedly increased when HeLa cells were synchronized at mitosis with nocodazole treatment. Immunofluorescence analysis showed that LIMK1 was localized at cell-cell adhesion sites in interphase and prophase, redistributed to the spindle poles during prometaphase to anaphase, and accumulated at the cleavage furrow in telophase. In contrast, LIMK2 was diffusely localized in the cytoplasm during interphase, redistributed to the mitotic spindle, and finally to the spindle midzone during anaphase to telophase. These findings suggest that LIMK2 is activated in response to microtubule disruption, and that LIMK1 and LIMK2 may play different roles in regulating for the mitotic spindle organization, chromosome segregation, and cytokinesis during the cell division cycle.     

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).     

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Background

The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.

Results

Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.

Conclusions

The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.     

Dyskerin Localizes to the Mitotic Apparatus and Is Required for Orderly Mitosis in Human Cells

Dyskerin is a highly conserved, nucleolar RNA-binding protein with established roles in small nuclear ribonucleoprotein biogenesis, telomerase and telomere maintenance and precursor rRNA processing. Telomerase is functional during S phase and the bulk of rRNA maturation occurs during G₁ and S phases; both processes are inactivated during mitosis. Yet, we show that during the course of cell cycle progression, human dyskerin expression peaks during G₂/M in parallel with the upregulation of pro-mitotic factors. Dyskerin redistributed from the nucleolus in interphase cells to the perichromosomal region during prometaphase, metaphase and anaphase. With continued anaphase progression, dyskerin also localized to the cytoplasm within the mid-pole region. Loss of dyskerin function via siRNA-mediated depletion promoted G₂/M accumulation and this was accompanied by an increased mitotic index and activation of the spindle assembly checkpoint. Live cell imaging further revealed an array of mitotic defects including delayed prometaphase progression, a significantly increased incidence of multi-polar spindles, and anaphase bridges culminating in micronucleus formation. Together, these findings suggest that dyskerin is a highly dynamic protein throughout the cell cycle and increases the repertoire of fundamental cellular processes that are disrupted by absence of its normal function.     

A chromosome separation checkpoint

Here we discuss a “chromosome separation checkpoint” that might regulate the anaphase‐telophase transition. The concept of cell cycle checkpoints was originally proposed to account for extrinsic control mechanisms that ensure the order of cell cycle events. Several checkpoints have been shown to regulate major cell cycle transitions, namely at G1‐S and G2‐M. At the onset of mitosis, the prophase‐prometaphase transition is controlled by several potential checkpoints, including the antephase checkpoint, while the spindle assembly checkpoint guards the metaphase‐anaphase transition. Our hypothesis is based on the recently uncovered feedback control mechanism that delays chromosome decondensation and nuclear envelope reassembly until effective separation of sister chromatids during anaphase is achieved. A central player in this potential checkpoint is the establishment of a constitutive, midzone‐based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis. We propose that this surveillance mechanism represents an additional step towards ensuring mitotic fidelity.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

esiRNA分析人源驱动蛋白MKLP1在胞质分裂中的功能

为探讨人源驱动蛋白MKLP1在有丝分裂和胞质分裂中的作用,以E.coliRNaseⅢ制备MKLP1的3′UTResiRNA转染HeLa细胞,通过定量RTPCR、Western印迹检测MKLP1esiRNA对MKLP1基因的沉默效率.再利用FACS分析、免疫荧光染色和活细胞成像分析检测MKLP1表达缺失后在有丝分裂和胞质分裂不同时期的细胞形态学、细胞分裂指数、细胞百分数,动态观察有丝分裂和胞质分裂期间的表型改变,以系统分析MKLP1的功能.最后通过挽救实验验证MKLP1esiRNA的作用特异性.实验显示MKLP1esiRNA转染HeLa细胞能够有效地特异性消除MKLP1的表达,并被异位表达的MKLP1所挽救.MKLP1蛋白在有丝分裂后期和末期前期位于纺锤体中间带,在末期后期和胞质分裂的最后阶段集中于中间体的中心处.MKLP1表达缺失使中间体正确形成和胞质分裂的完成受到严重抑制,造成大量双多核细胞堆积.结果表明,MKLP1在胞质分裂中间体形成和有丝分裂末期前期向后期过渡过程中起关键作用,是纺锤体中间体中间带相关蛋白,为胞质分裂所必需.     

THE DISTRIBUTION OF SPINDLE MICROTUBULES DURING MITOSIS IN CULTURED HUMAN CELLS

WI-38 and HeLa cells in mitosis have been selected from fixed monolayer cultures and serially sectioned for electron microscopy. Sections perpendicular to the spindle axis permit counting of the number of microtubules at each position on the spindle axis and hence the preparation of tubule distribution profiles. Errors intrinsic to this method are discussed. The changes in the tubule distributions from one mitotic stage to another provide evidence concerning the behavior of the spindle tubules during mitosis. The ratio of the number of tubules passing the chromosomes on the metaphase plate to the maximum number in each half spindle is about 1/2. This ratio changes little in early anaphase, and then decreases in late anaphase at about the same time that a zone of increased tubule number develops at the middle of the interzone. The region where the stem bodies form contains about 3/2 the number of tubules seen elsewhere in the interzone. This ratio is almost constant as the mid-body forms in telophase and then increases to 2/1 in early interphase before the final stages of cytokinesis occur.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.     

Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore-enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover.     

Localization of nucleolar phosphoproteins B23 and C23 during mitosis

Nucleolar phosphoproteins B23 and C23 were simultaneously localized in unsynchronized male rat-kangaroo PtK2 cells during mitosis using a mouse monoclonal antibody against protein B23 and a rabbit antibody against protein C23. The distribution of proteins B23 and C23 during mitosis was compared with the distribution of the silver staining protein. During interphase, proteins B23 and C23 were both localized to the nucleolus. As the nucleolus disappeared in prophase, the distribution of protein B23 became nucleoplasmic, whereas most of protein C23 remained associated with the disappearing nucleolus. Throughout metaphase and anaphase protein B23 was found associated with the chromosomes, whereas protein C23 seemed to disappear. When the nucleolus reformed during telophase, protein C23 appeared first in ‘prenucleolar bodies’ and then in the nucleolus, whereas protein B23 did not appear in the nucleolus until late telophase or early G1 phase. Silver staining during mitosis closely paralleled the distribution of protein C23, supporting previous conclusions that protein C23 is a silver staining nucleolus organizer region (NOR) protein [19, 20].     

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.