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PubMed1994

The spindle apparatus in early embryonic divisions of Ephestia kuehniella Z. (Pyralidae, Lepidoptera) is formed by alignment of minispindles.

Spindles were isolated from deposited eggs of the Mediterranean mealmoth, Ephestia kuehniella. Their structure and development were studied using anti-tubulin immunofluorescence. The microtubules were labelled with three different monoclonal antibodies. These were directed against beta-tubulin, tyrosinated alpha-tubulin and acetylated alpha-tubulin. Significant differences in the staining behaviour were not detected with the three antibodies. An unusual mode of spindle formation was observed during the first mitotic division after fusion of the pronuclei. Several of the ensuing embryonic divisions may show the same phenomenon. Prophase of these divisions was characterised by an irregular arrangement of microtubules in the nuclear area. The microtubular mass in the nuclear area increased concomitantly with chromosome condensation. Microtubular foci, comparable to the forming asters of canonical spindles, were not detected. The formation of an orderly pattern in the microtubule mass was signalled by the appearance of minispindles apparently developing around individual chromosomes. Several minispindles subsequently aligned and formed metaphase-like entities within the nuclear area. The metaphase-like entities, in turn, aligned with one another and gave rise to a conventional bipolar metaphase spindle with small asters. The further development of the spindle was conventional. The chromosomes migrated towards the spindle poles and finally daughter nuclei formed. The anaphase and telophase spindles possessed both a prominent array of interzone microtubules and asters. The events in prophase of early embryonic mitosis of E. kuehniella may represent a rare case of chromosome-induced spindle formation.

Animals↗
PubMedDate not supplied

Manipulation of the oocyte: possible damage to the spindle apparatus.

Oocytes are structured, polarized cells. For high developmental potential, it is essential that the distribution of organelles and molecules, and the function of meiotic spindles remain intact during handling of oocytes in assisted reproduction. Spindles are dynamic cell organelles. Spindle formation depends on activity of motor proteins, energy supply and temperature. Disturbances in spindle function may predispose oocytes to aneuploidy or maturation arrest. Thus, perturbation of the cytoskeletal integrity of oocytes may critically influence the fate of the embryo. Recently, enhanced polarizing microscopy has been developed for non-invasive analysis of spindle morphology in living mammalian oocytes. Chemically induced dynamic alterations have been characterized in the spindle in individual mouse oocytes and it has been shown that spindle aberrations are predictive of risks for non-disjunction. Spindle imaging identified adverse, irreversible effects of handling in living human oocytes (for instance, the extreme susceptibility of human oocytes to cooling). Also, oocyte immaturity may be detected. Selection of metaphase II oocytes and an injection site for intracytoplasmic sperm injection (ICSI) that avoids spindle damage may increase the yield of euploid embryos. The detection of genetic, environmentally induced, or treatment-related defects in oocyte maturation by non-invasive spindle imaging can improve quality control and assist in the selection of morphologically normal oocytes for assisted reproduction.

Aneuploidy↗
PubMed1996

Two major autoantigen-antibody systems of the mitotic spindle apparatus.

OBJECTIVE: To characterize human autoantigen-antibody systems related to the mitotic poles and spindles. METHODS: Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians. RESULTS: Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, anti-NuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with anti-NuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sjögren's syndrome. NuMA-2 antibodies were found in the sera of 7 patients. Interphase cells showed no nuclear or cytoplasmic staining, but mitotic cells had brightly stained poles and spindles. At anaphase/telophase, staining shifted to the midbody and the intercellular bridge. Anti-NuMA-2 sera immunoprecipitated a protein of 116 kd. This group of patients was more heterogeneous and had both systemic and organ-specific autoimmune diseases. CONCLUSIONS: NuMA protein (here called NuMA-1) and a 116-kd protein (here called NuMA-2) are the major targets of the autoimmune response in the mitotic apparatus, since most of the selected sera (based on IIF staining of the mitotic spindles and poles) recognized 1 of these 2 antigens.

Adult↗
PubMed1995

Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation.

The present study was conducted to determine if the cryopreservation of immature human oocytes has a deleterious effect on the meiotic spindle following maturation in vitro. Oocytes were obtained in excess from in-vitro fertilization patients and divided into four groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immature oocytes cryopreserved before or after maturation in vitro respectively. Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controls and included oocytes matured in vitro and in vivo respectively. The meiotic spindle was identified after incubation in anti-tubulin monoclonal antibody (1 h, 37 degrees C) and fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomes were counterstained with 4',6'-diamidino-2-phenylindole. Following cryopreservation, group 1 oocytes demonstrated a 63% survival rate and 68% maturation rate in vitro. In all, 58% of the oocytes in group 2 survived the thaw. The number of oocytes with normal spindles in group 1 (81.0%) was not significantly different from control groups 3 (83.8%) and 4 (88.9%), while the number of group 2 oocytes with normal structures (43.5%) was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002), and 4 (P = 0.025). These results suggest that cryopreservation of the prophase I human oocyte does not significantly increase abnormalities in the resulting meiotic spindle.

Cell Survival↗
PubMed1995

Loss of spatial control of the mitotic spindle apparatus in a Chlamydomonas reinhardtii mutant strain lacking basal bodies.

The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in > 99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cells is disrupted in bld2-1 cells. The positions of the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild-type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtubules found in green algae known as the rootlet microtubules. We propose that the rootlet microtubules perform the functions of astral microtubules and that functional centrioles are necessary for the organization of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas.

Actins↗
PubMed2006

The mushroom body defect gene product is an essential component of the meiosis II spindle apparatus in Drosophila oocytes.

In addition to their well-known effects on the development of the mushroom body, mud mutants are also female sterile. Here we show that, although the early steps of ovary development are grossly normal, a defect becomes apparent in meiosis II when the two component spindles fail to cohere and align properly. The products of meiosis are consequently mispositioned within the egg and, with or without fertilization, soon undergo asynchronous and spatially disorganized replication. In wild-type eggs, Mud is found associated with the central spindle pole body that lies between the two spindles of meiosis II. The mutant defect thus implies that Mud should be added to the short list of components that are required for the formation and/or stability of this structure. Mud protein is also normally found in association with other structures during egg development: at the spindle poles of meiosis I, at the spindle poles of early cleavage and syncytial embryos, in the rosettes formed from the unfertilized products of meiosis, with the fusomes and spectrosomes that anchor the spindles of dividing cystoblasts, and at the nuclear rim of the developing oocyte. In contrast to its important role at the central spindle pole body, in none of these cases is it clear that Mud plays an essential role. But the commonalities in its location suggest potential roles for the protein in development of other tissues.

Amino Acid Sequence↗
PubMed1986

A gene required for the separation of chromosomes on the spindle apparatus in yeast.

We describe the phenotypes caused by a cold-sensitive lethal mutation (ndc1-1) that defines the NDC1 gene of yeast. Incubation of ndc1-1 at a nonpermissive temperature causes failure of chromosome separation in mitosis but does not block the cell cycle. This defect results in an asymmetric cell division in which one daughter cell doubles in ploidy and the other inherits no chromosomes. The spindle poles are properly segregated to the two daughter cells. The primary visible defect is that the chromosomes remain associated with only one pole, and are thus delivered to one daughter cell. Meiosis II, but not meiosis I, is sensitive to the ndc1-1 defect, suggesting that NDC1 is required for some feature common to mitosis and meiosis II. ndc1-1 appears to define a new class of cell cycle gene required for the attachment of chromosomes to the spindle pole.

Aneuploidy↗
PubMed2005

The Drosophila gamma-tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus.

Gamma-tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of gamma-tubulin partners in metazoans. For the first time in a multicellular organism, we have investigated the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae gamma-tubulin-associated protein Spc97p. Mutant analysis shows that Dgrip84 is essential for viability. Its depletion promotes a moderate increase in the mitotic index, correlated with the appearance of monopolar or unpolarized spindles, impairment of centrosome maturation, and increase of polyploid nuclei. This in vivo study is strengthened by an RNA interference approach in cultured S2 cells. Electron microscopy analysis suggests that monopolar spindles might result from a failure of centrosome separation and an unusual microtubule assembly pathway via centriolar triplets. Moreover, we point to an involvement of Dgrip84 in the spindle checkpoint regulation and in the maintenance of interphase microtubule dynamics. Dgrip84 also seems essential for male meiosis, ensuring spindle bipolarity and correct completion of cytokinesis. These data sustain that Dgrip84 is required in some aspects of microtubule dynamics and organization both in interphase and mitosis. The nature of a minimal gamma-tubulin complex necessary for proper microtubule organization in the metazoans is discussed.

Animals↗
PubMed2006

Dynamic localization of the human papillomavirus type 11 origin binding protein E2 through mitosis while in association with the spindle apparatus.

Papillomaviral DNA replicates as extrachromosomal plasmids in squamous epithelium. Viral DNA must segregate equitably into daughter cells to persist in dividing basal/parabasal cells. We have previously reported that the viral origin binding protein E2 of human papillomavirus types 11 (HPV-11), 16, and 18 colocalized with the mitotic spindles. In this study, we show the localization of the HPV-11 E2 protein to be dynamic. It colocalized with the mitotic spindles during prophase and metaphase. At anaphase, it began to migrate to the central spindle microtubules, where it remained through telophase and cytokinesis. It was additionally observed in the midbody at cytokinesis. A peptide spanning residues 285 to 308 in the carboxyl-terminal domain of HPV-11 E2 (E2C) is necessary and sufficient to confer localization on the mitotic spindles. This region is conserved in HPV-11, -16, and -18 and bovine papillomavirus type 4 (BPV-4) E2 and is also required for the respective E2C to colocalize with the mitotic spindles. The E2 protein of bovine papillomavirus type 1 is tethered to the mitotic chromosomes via the cellular protein Brd4. However, the HPV-11 E2 protein did not associate with Brd4 during mitosis. Lastly, a chimeric BPV-1 E2C containing the spindle localization domain from HPV-11 E2C gained the ability to localize to the mitotic spindles, whereas the reciprocal chimera lost the ability. We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections.

Animals↗
PubMed1995

Evaluation of the meiotic spindle apparatus in oocytes undergoing intracytoplasmic sperm injection.

OBJECTIVE: To determine whether intracytoplasmic sperm injection has an adverse effect on the meiotic spindle of oocytes after injection of a sperm into the ooplasm. DESIGN: Hamster oocytes were injected with human sperm (Test group) and evaluated for meiotic spindle and chromosome morphology using immunofluorescent staining. Results were compared with control uninjected oocytes exposed to the microscope environment (CS group) and untreated oocytes remaining in the incubator (CI group). SETTING: Basic research center at a medical school. RESULTS: No significant differences were noted in spindle appearance and chromosome alignment between Test (13 abnormal/68 normal) and both control groups (CS 9/73 and CI 12/71; P = 0.602). CONCLUSION: Our results demonstrate that injection of human sperm into the cytoplasm of hamster oocytes may not result in a significant increase in damage to the meiotic spindle provided care is taken to orient the polar body away from the site of injection.

Animals↗
PubMed1982

Quantitative studies on the arrangement of human metaphase chromosomes. IX. Arrangement of chromosomes with and without spindle apparatus.

The spatial relationships in human male metaphase cells treated with and without colcemide were compared with each other. The following results were obtained: (1) In normal male metaphases the overall distributions of chromosomal distances regardless of chromosome identification numbers did not show normal distribution, neither in the colcemid-free sample nor in the colcemide-treated sample. (2) In both samples larger chromosomes showed a more peripheral position, and smaller chromosomes showed a more central position. This finding was statistically significant. (3) No differences between the two samples could be observed concerning the following parameters: overall distributions of the centromere-centromere distances, distributions of the distances between the homologous chromosomes (except the small acrocentric chromosomes), rank positions of the mean distances between homologous chromosomes, and rank positions of the mean distances of the different chromosomes from the center of the mitosis (except few chromosomes). (4) Visible, but not statistically accessible, differences appeared between the two samples in respect to rank positions of the mean distances of all possible acrocentric pairing groups, rank positions of the mean distances of the homologous acrocentric chromosomes from the center of the mitosis, and distances of the X chromosome from the center of the mitosis. (5) Statistically significant differences appeared between the two samples with respect to distance distributions of the small acrocentric chromosomes and positions of the chromosomes 1, 16, 18, Y, and 21, 22 in relation to the center of the mitosis.

Chromosome Mapping↗