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At least 19 recordsLinked to original sources
PubMed1992

The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus.

The formation and maintenance of the bipolar mitotic spindle apparatus require a complex and balanced interplay of several mechanisms, including the stabilization and separation of polar microtubules and the action of various microtubule motors. Nonmicrotubule elements are also present throughout the spindle apparatus and have been proposed to provide a structural support for the spindle. The Nuclear-Mitotic Apparatus protein (NuMA) is an abundant 240 kD protein that is present in the nucleus of interphase cells and concentrates in the polar regions of the spindle apparatus during mitosis. Sequence analysis indicates that NuMA possesses an unusually long alpha-helical central region characteristic of many filament forming proteins. In this report we demonstrate that microinjection of anti-NuMA antibodies into interphase and prophase cells results in a failure to form a mitotic spindle apparatus. Furthermore, injection of metaphase cells results in the collapse of the spindle apparatus into a monopolar microtubule array. These results identify for the first time a nontubulin component important for both the establishment and stabilization of the mitotic spindle apparatus in multicellular organisms. We suggest that nonmicrotubule structural components may be important for these processes.

Anaphase↗
PubMed2003

Microtubules are the only structural constituent of the spindle apparatus required for induction of cell cleavage.

Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced "cells" containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.

Animals↗
PubMed2002

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus.

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.

A Kinase Anchor Proteins↗
PubMed2000

LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans.

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.

Amino Acid Sequence↗
PubMed1996

A new possible parameter for the detection of aneuploidy inducing substances: the analysis of qualitative and quantitative abnormalities of the spindle apparatus.

In the present study, compared to other cytogenetic methods, we measured the number of aneuploid cells directly by analyzing anomalies of the mitotic spindle. Qualitative and quantitative abnormalities of the mitotic spindle apparatus in transformed and non-transformed cell lines in vitro were classified. We treated the different cell lines with well known aneugenic agents as Benomyl and Griseofulvin and investigated the mitotic spindle under different experimental conditions. The spindle apparatus was stained by indirect immunofluorescence and the chromatin was counterstained by fluorescent dyes. The mitotic spindle showed a great sensitivity to the aneuploidy-inducing substances used in our experiments. The spindle-disturbing effect of the tested substances was demonstrated to be dose- dependent. The morphological alterations appeared to be independent of the aneuploidy-inducing test substance used, but showed a relation to the dose and length of treatment. Thus, the analysis of the mitotic spindle may be a useful screening parameter for the detection of aneuploidy-inducing substances and further investigations will provide additional results to specific parameters.

Aneuploidy↗
PubMed1988

Autoantibodies to the mitotic spindle apparatus in Mycoplasma pneumoniae disease.

Antibodies to the mitotic spindle apparatus (MS) have been detected in only a very few patient sera studied for antinuclear antibodies by routine screening (G. A. McCarty, D. W. Velencia, and M. J. Fritzler, J. Rheumatol. 11:213-218, 1984). We found anti-MS antibodies to be quite common in sera from patients with Mycoplasma pneumoniae, especially in those who developed cold agglutinins (71%). Absorption experiments indicated that the formation of anti-MS antibodies is not triggered by epitopes on the M. pneumoniae surface. These antibodies were distinct from cold agglutinins, antibrain antibodies, and smooth-muscle antibodies. All patients previously reported to harbor anti-MS had clinical features compatible with a diagnosis of systemic lupus erythematosus, rheumatoid arthritis, or an evolving connective-tissue disorder. Such systemic disorders might develop in a small proportion of patients who have had M. pneumoniae illness.

Autoantibodies↗
PubMed1986

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells.

A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus.

Animals↗
PubMed1986

Naturally occurring masked antibodies in murine sera recognize a component of the mitotic spindle apparatus.

Antibody activities previously masked in autoimmune MRL and normal Balb/c mice were expressed by briefly subjecting their sera to acidic (pH 2.0) or alkaline (pH 12.0) environments. An enzyme-linked immunosorbent assay (ELISA) revealed that these pH-expressable immunoglobulins reacted with specific nucleotide-BSA antigens (primarily 5'-AMP, -GMP, -TMP) but not with single (ss)- or double (ds)-stranded DNAs or with unconjugated BSA. ELISA analysis of pH-expressed antibodies purified via GMP-BSA/Sepharose indicated that they bound not only to the homologous hapten (GMP) but to AMP and TMP as well, i.e., anti-pAGT antibodies. Further, indirect immunofluorescent assays (IIF) with fixed HEp-2 cells demonstrated that purified anti-pAGT antibodies recognized an epitope within the mitotic spindle apparatus. These results document the existence of a previously undefined masked antibody population in murine sera with specificities directed toward certain nucleotides and a component of the mitotic spindle apparatus. Last, these anti-pAGT (or anti-mitotic spindle) antibodies are not restricted to murine systems inasmuch as they have been detected in every human serum (greater than 100 samples) examined thus far.

Animals↗
PubMed2000

Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus.

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.

3T3 Cells↗
PubMed1998

The Tomaj mutant alleles of alpha Tubulin67C reveal a requirement for the encoded maternal specific tubulin isoform in the sperm aster, the cleavage spindle apparatus and neurogenesis during embryonic development in Drosophila.

The three dominant TomajD and their eleven revertant (TomajR) alleles have been localized to the alpha Tubulin67C gene of Drosophila melanogaster. Although the meiotic divisions are normally completed in eggs laid by TomajD/+, TomajD/-, TomajR/- females, embryogenesis arrests prior to the gonomeric division. The arrest is caused by: (1) the failure of prominent sperm aster formation; and (2) a consequent lack of female pronuclear migration towards the male pronucleus. Concomitant with the sperm aster defect, the four female meiotic products fuse (tetra-fusion), similar to what is seen in eggs of wild-type virgin females. In eggs of females heterozygous for weaker TomajR alleles, embryogenesis comes to a cessation before or shortly after cortical migration of cleavage nuclei. The apparent source of embryonic defect is the cleavage spindle apparatus. One of the three TomajD alleles is cold-sensitive and its cold-sensitive period coincides with the completion of female meiosis and pronuclear migration. Disorganized central and peripheral nervous systems are also characteristic of embryos derived from the temperature-sensitive TomajD/+ females. The Tomaj mutant phenotypes indicate an involvement of the normal alpha Tubulin67C gene product in: (1) the formation of the sperm aster; (2) cleavage spindle apparatus formation/function; and (3) the differentiation of the embryonic nervous system. The TomajD alleles encode a normal-sized alpha Tubulin67C isotype. Sequence analyses of the TomajD alleles revealed the replacement in different positions of a single negatively charged or neutral amino acid with a positively charged one. These residues presumably identify important functional sites.

Alleles↗
PubMed2001

Evaluation of the meiotic spindle apparatus in metaphase II human oocytes following cytoplasmic donation.

PURPOSE: To determine if the removal of cytoplasm from metaphase II human donor oocytes damages the meiotic spindle apparatus. MATERIALS AND METHODS: Cryopreservation of metaphase II human oocytes was performed using a fast-freeze, fast-thaw protocol. Upon thaw, oocytes were incubated for 3-4 h and then used for cytoplasmic donation (test oocytes). Oocytes thawed but not used for donation served as controls. Test and control oocytes were fixed using a microtubule-stabilizing buffer. Tubulin was localized using antitubulin monoclonal antibody. Chromosomes were identified by counterstaining with DAPI. RESULTS: Forty-four oocytes had cytoplasm removed (test group) while 12 were not used for the procedure (controls). Twenty-three oocytes survived the donation procedure. Rates of normal spindle structure for the control and test groups were 21/23 (91.3%) and 12/12 (100%), respectively. CONCLUSION: The removal of cytoplasm from a metaphase II human donor oocyte does not appear to significantly increase the damage to chromosome alignment or to the spindle structure.

Adult↗
PubMed1991

Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes.

A 3-h exposure of V79 Chinese hamster cells with the sensory irritant 2-chlorobenzylidene malonitrile (CS) caused apolar mitoses in a dose-dependent manner. With a preparation and staining technique that allows for the visualization of the spindle apparatus and the chromosomes it was found that unlike in Colcemid-induced c-metaphases residual spindle fibers or microtubule material were present in the majority of CS-induced c-metaphases. The observation suggests different mechanisms for the induction of the c-mitotic effect by the two spindle poisons.

Animals↗
PubMed1990

Mitotic spindle damage induced by 3,4-dichloroaniline in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes.

3,4-Dichloroaniline (3,4-DCA), an intermediate in various chemical syntheses, has been detected as an environmental contaminant in surface waters and in the effluents from dye manufacturing plants. Previously we showed that the compound caused mitotic spindle damage. In the present study a preparation and staining technique has been used that allows a direct examination of the spindle apparatus and chromosome behaviour of 3,4-DCA-exposed V79 Chinese hamster cells. After a 3 h exposure the compound caused monopolar mitoses and metaphase arrest in a dose-dependent manner, most probably by interfering with centriole separation. At a concentration of 1 mM, increased levels of aneuploid cells could be observed in cells exposed for two cell division cycles.

Aneuploidy↗
PubMed1998

The Des-1 protein, required for central spindle assembly and cytokinesis, is associated with mitochondria along the meiotic spindle apparatus and with the contractile ring during male meiosis in Drosophila melanogaster.

Spermatogenesis in Drosophila melanogaster serves as an excellent model system for the isolation and analysis of genes required in the control of chromosome segregation and cytokinesis. We report here the isolation and molecular characterization of a novel P-element induced allele of the des-1 gene, which leads to male sterility as a consequence of the failure of central spindle assembly in meiotic spermatocytes and the formation of aberrant meiotic end products characteristic of cytokinesis failure. We have raised affinity-purified antibodies against a Des-1 fusion protein, and localized the Des-1 protein in Drosophila spermatocytes. We show that the Des- protein is colocalized with mitochondria throughout male meiosis, becoming intimately associated with mitochondria along the spindle apparatus during anaphase and telophase, and with the Nebenkern, or mitochondrial derivative, of the meiotic end products. In addition, a significant association of Des-1 with the contractile ring is observed during anaphase and telophase of meiosis. These observations, together with the presence of six potential transmembrane domains in the Des-1 protein, raise the possibility that Des-1 may act as part of an anchoring mechanism that links membrane-bounded cellular compartments to components of the cytoskeleton.

Animals↗
PubMed2004

Functional analysis of bacterial artificial chromosomes in mammalian cells: mouse Cdc6 is associated with the mitotic spindle apparatus.

Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the functional organization of genes and other large chromosomal domains. To facilitate functional studies in cell cultures, we have developed a simple approach for generating stable cell lines with variable copy numbers of any BAC. Here we describe hamster cell lines with BAC transgenes that express mouse Cdc6 at levels that correlate with BAC copy number; show that mouse Cdc6 is regulated normally during the cell cycle, binds chromatin, and is degraded during apoptosis; and report a novel fraction of Cdc6 that associates with the spindle apparatus during mitosis. With RNA interference to assess genetic complementation by BAC alleles, this system will facilitate functional studies on large chromosomal domains at variable copy number in mammalian cell models.

Animals↗
PubMed1984

Antibody to the mitotic spindle apparatus: immunologic characteristics and cytologic studies.

A naturally occurring autoantibody directed at the mitotic spindle polar apparatus (MSA) was detected in sera from 18 patients with defined or evolving connective tissue diseases by routine indirect immunofluorescence on a tissue culture cell. This IgG antibody stained the mitotic spindle poles of dividing tissue culture and tissue section substrates, with staining most prominent on substrates of human origin. With some sera, and cell lines, interphase cells showed isolated nuclear staining. Cytoplasmic staining was not apparent in any sera on any substrate. This specificity was shown to be distinct from tubulin by double fluorescence labeling studies. Patient sera exhibiting only this specificity did not precipitate antigens in rabbit or calf thymus nuclear extracts. However, several patients with systemic lupus erythematosus exhibited anti-MSA in combination with other autoantibodies. This autoantibody may be a useful probe for spindle pole-related structures.

Arthritis, Rheumatoid↗
PubMed2003

Effect of the dithiocarbamate pesticide zineb and its commercial formulation, the azzurro. V. Abnormalities induced in the spindle apparatus of transformed and non-transformed mammalian cell lines.

Abnormalities induced in the mitotic spindle by zineb and azzurro (1.0-25.0 micro g/ml, 24h) were evaluated in Chinese hamster ovary (CHO) and HeLa cells, and in non-transformed human fibroblasts (NTHF). Spindles were stained with FITC-conjugated anti-beta tubulin. Treatment with 10.0 micro g/ml of zineb induced complete inhibition of cell viability in NTHF cells while 10.0 micro g/ml of azzurro decreased cell growth down to 62%. Higher doses of both compounds induced cell death. In HeLa and CHO cells, 15.0 micro g/ml of zineb and 10.0-15.0 micro g/ml of azzurro decreased viability, whereas 25.0 micro g/ml of both compounds was cytotoxic. A significantly decreased mitotic index (MI) was observed in NTHF treated with 5.0 micro g/ml zineb or azzurro, whereas 10.0 micro g/ml of both chemicals were necessary to induce the same phenomenon in HeLa and CHO cells. Treatment with 1.0-5.0 micro g/ml of zineb or azzurro induced a dose-dependent increase of degenerated spindles in NTHF and the number of degenerated or multipolar spindles in HeLa and CHO cells increased in a dose-dependent manner with 1.0-10.0 micro g/ml zineb and azzurro. Although zineb and azzurro were able to induce mitotic spindle abnormalities in all cell types, non-transformed cells were less resistant than immortalized cells.

Animals↗