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PubMed1995

Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.

We have isolated a human homolog of Xenopus Eg5, a kinesin-related motor protein implicated in the assembly and dynamics of the mitotic spindle. We report that microinjection of antibodies against human Eg5 (HsEg5) blocks centrosome migration and causes HeLa cells to arrest in mitosis with monoastral microtubule arrays. Furthermore, an evolutionarily conserved cdc2 phosphorylation site (Thr-927) in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and by p34cdc2/cyclin B in vitro. Mutation of Thr-927 to nonphosphorylatable residues prevents HsEg5 from binding to centrosomes, indicating that phosphorylation controls the association of this motor with the spindle apparatus. These results indicate that HsEg5 is required for establishing a bipolar spindle and that p34cdc2 protein kinase directly regulates its localization.

Amino Acid Sequence↗
PubMed1999

Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation.

Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.

Animals↗
PubMed1977

Genetic damage induced by ethyl alcohol in Aspergillus nidulans.

Heterozygous diploid conidia of Aspergillus nidulans were treated during germination with ethyl alcohol in concentrations ranging from 0.25% to 20% (v/v). The diploid strain carried three recessive conidial color mutations, in addition to genetic markers on all eight pairs of linkage groups. It was thereby possible to detect events of crossing over, non-disjunction, and mutation. An increase in the dose of ethanol was associated with a decrease in conidial viability and an increase in the relative and absolute frequencies of formation of (a) normal colonies which produced colored sectors and (b) phenotypically abnormal colonies, the majority of which (83.1%) produced normal sectors. At a concentration of 5% (v/v) ethanol, the survivors included 17.59% of the former and 44.7% of the latter colonies. Genetic analysis of the various segregants suggested that the frequencies of both mitotic crossing over and non-disjunction or the misdistribution of chromosomes were increased by ethanol. Among 133 abnormal colonies which segregated normal clones, 79 (59.4%) were associated with one of these genetic events. A total of 297 haploids and 130 diploids arose as normal segregants from the abnormal colonies. There were 31 recognizable events of non-disjunction and 14 crossing over in linkage groups I and II, where these events could be distinguished. These data suggested that the predominant effect of ethanol was a disruption of chromosome distribution. A cytological examination of ethanol-treated, germinating conidia revealed an interference with the mitotic spindle apparatus. The frequency of detectable spindles decreased more than 3-fold after 8 h exposure to 5% (v/v) ethanol. This finding supported the conclusion that ethanol disrupted chromosome distribution, and suggested the mechanism by which it does so. Human clinical data on alcohol consumption were examined in light of these findings.

Aspergillus nidulans↗
PubMed1992

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.

Animals↗
PubMed2004

The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation.

The centromere defines where on a chromosome the kinetochores assemble. Kinetochores, large protein structures, mediate chromosome segregation during mitosis and meiosis by performing three key functions. Firstly, kinetochores attach chromosomes to the microtubule spindle apparatus. Secondly, kinetochores co-ordinate microtubule dynamics to allow chromosomes to move along the spindle. Lastly, kinetochores generate the 'wait' signal which prevents anaphase onset until all the chromosomes are correctly aligned on the spindle. This signal forms part of the spindle checkpoint mechanism, a highly conserved cell cycle checkpoint which maintains the accuracy of the chromosome segregation process. This article provides a brief historical overview before focusing on some of the outstanding issues and more recent developments in the field.

Amino Acid Sequence↗
PubMed2006

Asymmetrization of first cleavage by transient disassembly of one spindle pole aster in the leech Helobdella robusta.

Unequal first cleavage is characteristic of a diverse group of protostome animals. In the nematode Caenorhabditis elegans, unequal first cleavage is achieved through the interaction of an apparently symmetric mitotic spindle apparatus with a clearly polarized cell cortex. In the clitellate annelid Tubifex tubifex, by contrast, the spindle is monastral and contains only one gamma-tubulin-reactive centrosome; this monastral spindle is inherently asymmetric throughout mitosis. Here, we have used immunostaining for beta- and gamma-tubulin to follow spindle dynamics during the unequal first cleavage in another clitellate annelid, the leech Helobdella robusta. We find that the mitotic spindle is diastral and symmetric through early metaphase, then becomes asymmetric following the transient down-regulation of one centrosome, as judged by gamma-tubulin immunofluorescence. Low levels of drugs that affect microtubule dynamics can symmetrize the first cleavage without affecting the gamma-tubulin dynamics. Our results provide a striking example of the evolvability of cellular mechanisms underlying an unambiguously homologous developmental process.

Animals↗
PubMed2005

Dynamics and unexpected localization of the plakin binding protein, kazrin, in mouse eggs and early embryos.

The cell uses the cytoskeleton in virtually every aspect of cell survival and function. One primary function of the cytoskeleton is to connect to and stabilize intercellular junctions. To accomplish this task, microtubules, actin filaments, and intermediate filaments utilize cytolinker proteins, which physically bind the cytoskeletal filament to the core proteins of the adhesion junction. The plakin family of linker proteins have been in the spotlight recently as critical components for embryo survival and, when mutated, the cause of diseases such as muscular dystrophy and cardiomyopathies. Here, we reveal the dynamics of a recently discovered plakin binding protein, kazrin (kaz), during early mouse development. Kaz was originally found in adult tissues, primarily epidermis, linking periplakin to the plasma membrane and colocalizing with desmoplakin in desmosomes. Using reverse transcriptase-polymerase chain reaction, Western blots, and confocal microscopy, we found kaz in unfertilized eggs associated with the spindle apparatus and cytoskeletal sheets. As quickly as 5 min after egg activation, kaz relocates to a diffuse peri-spindle position, followed 20-30 min later by clear localization to the presumptive cytokinetic ring. Before the blastocyst stage of development, kaz associates with the nuclear matrix in a cell cycle-dependent manner, and also associates with the cytoplasmic actin cytoskeleton. After blastocyst formation, kaz localization and potential function(s) become highly complex as it is found associating with cell-cell junctions, the cytoskeleton, and nucleus. Postimplantation stages of development reveal that kaz retains a multifunctional, tissue-specific role as it is detected at diverse locations in various embryonic tissue types.

Animals↗
PubMed2004

Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5.

Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes. The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation. To gain further insights into lin-5 function, we screened for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5(ev571ts ), and isolated 68 suppressing mutations. Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5(ev571ts ). These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain. This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens. Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5(ev571ts ) mutation and restored by a representative intragenic suppressor mutation. Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo. The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex. A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions. Taken together, our results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5.

Amino Acid Sequence↗
PubMed1998

Parathyroid hormone regulates the expression of the nuclear mitotic apparatus protein in the osteoblast-like cells, ROS 17/2.8.

The parathyroid hormone (PTH) signaling pathways that effect changes in osteoblast gene expression also alter the organization of the cytoskeletal proteins. PTH regulates the expression of nucleoskeletal proteins, such as nuclear mitotic apparatus protein (NuMA) and topoisomerase II-alpha. NuMA is a structural component of the interphase nucleus and organizes the microtubules of the mitotic spindle during mitogenesis. We propose that PTH-induced alterations in osteoblast cytoarchitecture are accompanied by changes in osteoblast nuclear structure that contribute to changes in gene expression. We used immunofluorescence and confocal microscopy to determine the effect of PTH on the expression and nuclear distribution of NuMA in the rat osteosarcoma cell line, ROS 17/2.8. Cells were treated with PTH or vehicle, then fixed and stained with NuMA antibody. Optical sections of interphase naive cells revealed a diffuse distribution of NuMA, interspersed with speckles, in the central nuclear planes but not in nucleoli. During the metaphase and anaphase, NuMA localized at the mitotic spindle apparatus. The percentage of NuMA-immunopositive ROS 17/2.8 cells decreased with increasing confluence, but serum starvation did not attenuate NuMA expression. Cell density-dependent changes in cytoskeletal organization were observed in these cells. PTH treatment induced changes in cytoskeletal organization and increased the percentage of NuMA-immunopositive ROS 17/2.8 cells. These data suggest that PTH effects changes in osteoblast nuclear architecture by regulating NuMA, and that these alterations may be coupled to cytoskeletal organization.

Animals↗
PubMed1994

NuMA, a nuclear protein involved in mitosis and nuclear reformation.

NuMA, a nuclear protein that associates with the mitotic apparatus, was identified in 1980 as a high molecular weight component of the nuclear matrix with the unusual property of associating with the microtubules of the spindle apparatus during mitosis. Over the past two years, a burst of interest in this intriguing protein has led to the clear documentation of its cell cycle redistribution, determination of its primary sequence, elucidation of its cell cycle dependent targeting domains, as well as disruption of its function through antibody microinjection and expression of dominant-negative mutants. Together, these data support a central role for NuMA in both mitotic-spindle dynamics and the reformation of the daughter cell nuclei at the end of mitosis.

Animals↗
PubMed2006

The mitotic spindle matrix: a fibro-membranous lamin connection.

The mitotic spindle apparatus has attracted the attention of cell biologists for decades. Whereas the main function of this microtubule-based system is to segregate chromosomes, spindle morphogenesis and chromosome segregation must also coordinate with the segregation of the whole cell. The finding that RanGTPase stimulates the assembly of a lamin B-containing membranous matrix in mitosis(1) may provide a connection between the segregation of mitotic chromosomes and the partitioning of membrane systems during cell division.

Chromosome Segregation↗
PubMed1990

A study of mitotic division fidelity and numerical chromosome changes in ageing Syrian hamster dermal cells.

Events associated with culture ageing in Syrian hamster dermal cells have been studied from the time of culture isolation during continuous passage until they senesced and died. Microscopic examination of mitotic cells using differential staining of chromosome and spindle apparatus assessed the faithfulness of cell division. Other indicators of the quality of cell division were obtained from chromosome counts, micronucleus frequencies and incidences of binucleate cells. A loss of spindle fidelity and an increase in aneuploidy corresponded to the period of culture senescence. The data presented indicate that the loss of division fidelity and chromosome number instability is an important indicator of the progression of a mammalian culture to senescence under in vitro conditions. Such information may provide the basis of a model for the study of factors which modify mitotic fidelity and senescence and provide a methodology for monitoring the suitability of mammalian cultures for commercial usage.

Animals↗
PubMed1986

Resinless section electron microscopy of HeLa cell mitotic architecture.

The use of resinless sections extends embedment-free electron microscopy to the cytoskeleton of thick specimens. Here we examine HeLa cells rounded at mitosis. Extraction of mitotic HeLa cells with Triton X-100 removes lipids and soluble proteins, leaving the cytoskeletal framework and spindle apparatus. After fixation, the samples are embedded and sectioned, and the temporary embedding resin is removed for direct visualization in the electron microscope. The micrographs show that the cytoskeletal framework, chromosomes, spindle, and centrioles form an interconnected entity. The pericentriolar region, indistinct in conventional micrographs, appears composed of distinct fibers interconnecting the spindle microtubules and centriole. The resinless sections also reveal characteristic lacunae at late anaphase/early telophase. These probably result from reformation of the interphase cytoskeleton lagging reassembly of the nucleus.

Centrioles↗
PubMed2001

A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.

In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Anaphase↗
PubMed2003

Recognizing chromosomes in trouble: association of the spindle checkpoint protein Bub3p with altered kinetochores and a unique defective centromere.

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G(2)/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested a model in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of our model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6(Delta31)], we determined that Bub3-GFP can associate with a single defective kinetochore. Our studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae.

Base Sequence↗
PubMed1986

Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis.

The fission yeast top2 locus is defined by five temperature-sensitive mutations that cause heat-labile activity of type II DNA topoisomerase in the cell extracts. We show that the top2 locus is a structural gene for type II topoisomerase by cloning a genomic DNA fragment that complements top2. The top2 mutants at restrictive temperature produce abnormal chromosomes at the time of mitosis; these are transiently extended into filamentous structures along with the elongating mitotic spindle but are not separated. A primary defect in top2 appears to be the formation of aberrant mitotic chromosomes inseparable by the force generated by the spindle apparatus. Consistently, the top2 cells that become lethal during mitosis contain a catenated dimer of an ARS plasmid. DNA and RNA continue to be synthesized if cytokinesis is blocked. Uncoordinated mitosis, that is the occurrence of spindle dynamics without chromosome separation, is revealed in top2, and is discussed in relation to mitotic regulation. Different phenotypes between top2 and top1-top2 described in the present paper can be explained by a previously proposed hypothesis that type II topoisomerase has dual in vivo functions: one that decatenates and unknots duplex DNAs is essential in mitosis, whereas the other which relaxes supercoils is required throughout the cell cycle if type I topoisomerase is absent.

Blotting, Southern↗
PubMed1999

[Analysis of micronuclei induced under hyperthermic conditions in human lymphocyte culture by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) methods].

The spectral karyotyping (SKY) method is a novel molecular cytogenetic technique which simultaneously discerns entire chromosomes. In order to elucidate the origins of micronuclei induced under hyperthermic conditions in human lymphocyte culture, peripheral blood cells were cultured at 40 degrees C or 42 degrees C for 3-24 h, using the cytokinesis-block method with cytochalasin B. The induced micronuclei were identified by the fluorescence in situ hybridization (FISH) and SKY methods. At 42 degrees C for more than 6 h, the frequency of occurrence of micronuclei in binucleated cells rose with increasing incubation time. By the FISH method, 83.3% of micronuclei induced in 24 h culture at 42 degrees C were shown to be positive for the human centromeric probes. By the SKY method, each micronucleus induced under the hyperthermic conditions was identified unequivocally and shown to contain a specific chromosome. These results suggest that the micronuclei induced under the hyperthermic conditions in human lymphocyte culture contain chromosomes which do not migrate to the poles at the anaphase of the cell cycle because of the breakdown of the spindle apparatus.

Cells, Cultured↗
PubMed1992

CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae.

A genetic screen was devised to identify genes important for spindle pole body (SPB) and/or microtubule functions. Four mutants defective in both nuclear fusion (karyogamy) and chromosome maintenance were isolated; these mutants termed cik (for chromosome instability and karyogamy) define three complementation groups. The CIK1 gene was cloned and characterized. Sequence analysis of the CIK1 gene predicts that the CIK1 protein is 594 amino acids in length and possesses a central 300-amino-acid coiled-coil domain. Two different CIK1-beta-galactosidase fusions localize to the SPB region in vegetative cells, and antibodies against the authentic protein detect CIK1 in the SPB region of alpha-factor-treated cells. Evaluation of cells deleted for CIK1 (cik1-delta) indicates that CIK1 is important for the formation or maintenance of a spindle apparatus. Longer and slightly more microtubule bundles are visible in cik1-delta strains than in wild type. Thus, CIK1 encodes a SPB-associated component that is important for proper organization of microtubule arrays and the establishment of a spindle during vegetative growth. Furthermore, the CIK1 gene is essential for karyogamy, and the level of the CIK1 protein at the SPB appears to be dramatically induced by alpha-factor treatment. These results indicate that molecular changes occur at the microtubule-organizing center (MTOC) as the yeast cell prepares for karyogamy and imply that specialization of the MTOC or its associated microtubules occurs in preparation for particular microtubule functions in the yeast life cycle.

Amino Acid Sequence↗