Abnormal Spindle Morphology Research Articles

Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function

Poly(ADP-ribose) (PAR) is a large, negatively charged post-translational modification that is produced by polymerization of NAD+ by PAR polymerases (PARPs). There are at least 18 PARPs in the human genome, several of which have functions that are unknown. PAR modifications are dynamic; PAR structure depends on the balance between synthesis and hydrolysis by PAR glycohydrolase2. We previously found that PAR is enriched in vertebrate somatic-cell mitotic spindles and demonstrated a requirement for PAR in the assembly of Xenopus egg extract spindles. Here, we knockdown all characterized PARPs using RNA interference (RNAi), and identify tankyrase-1 as the PARP that is required for mitosis. Tankyrase-1 localizes to mitotic spindle poles, to telomeres and to the Golgi apparatus. Tankyrase-1 RNAi was recently shown to result in mitotic arrest, with abnormal chromosome distributions and spindle morphology observed--data that is interpreted as evidence of post-anaphase arrest induced by failure of telomere separation6. We show that tankyrase-1 RNAi results in pre-anaphase arrest, with intact sister-chromatid cohesion. We also demonstrate a requirement for tankyrase-1 in the assembly of bipolar spindles, and identify the spindle-pole protein NuMA as a substrate for covalent modification by tankyrase-1.

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor.

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

Depletion of Drad21/Scc1 in Drosophila Cells Leads to Instability of the Cohesin Complex and Disruption of Mitotic Progression

Background: The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. In this study, we examine the consequences of depleting Drad21 and SA, two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells.Results: We have shown that a bona fide cohesin complex exists in Drosophila embryos. Strikingly, the Drad21/Scc1 and SA/Scc3 non-SMC subunits associate more intimately with one another than they do with the SMCs. We have observed defects in mitotic progression in cells from which Drad21 has been depleted: cells delay in prometaphase with normally condensed, but prematurely separated, sister chromatids and with abnormal spindle morphology. Much milder defects are observed when SA is depleted from cells. The dynamics of the chromosome passenger protein, INCENP, are affected after Drad21 depletion. We have also made the surprising observation that SA is unstable in the absence of Drad21; however, we have shown that the converse is not true. Interference with Drad21 in living Drosophila embryos also has deleterious effects on mitotic progression.Conclusions: We conclude that Drad21, as a member of a cohesin complex, is required in Drosophila cultured cells and embryos for proper mitotic progression. The protein is required in cultured cells for chromosome cohesion, spindle morphology, dynamics of a chromosome passenger protein, and stability of the cohesin complex, but apparently not for normal chromosome condensation. The observation of SA instability in the absence of Drad21 implies that the expression of cohesin subunits and assembly of the cohesin complex will be tightly regulated.

Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy

Objective: Meiotic spindles are crucial for normal chromosome alignment and separation of chromosomes during meiosis I and meiosis II. Traditional evaluation of the integrity spindles has been the immunofluorescent staining of tubulin. Recently, Polscope has emerged as a tool to view spindles in living oocytes. In this study we assess the spindle status of thawed oocytes after vitrification using Polscope and investigate their feasibility to replace immunofluorescent staining. Design: The Polscope was used to image spindles in living mouse oocytes before vitification. After thawing, the spindles of oocytes were imaged again and classified according to the spindles images. The results were compared with those by immunofluorescent staining of tubulin. Materials/Methods: The oocyte-cumulus complexes were collected from ICR mice superovulated by PMSG and hCG injections. The cumulus cells were removed by hyaluronidase. The MII oocytes were imaged by Polscope. Only oocytes with good spindle morphology were subsequently undergoing vitrification thawing protocol. The oocytes were vitrified with 5.5 M Ethylene Glycol- and 1 M sucrose-containing Dulbecco phosphate buffered saline. They were loaded into thin walled pulled straw and were stored at liquid nitrogen till thawing. After thawing, the oocytes were evaluated by Polscope again. The spindleimages were saved in computer for later analysis. The survived thawed oocytes were then fixed and incubated with anti-tubulin monoclonal antibody. Tubulin was stained by fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG and chromatin was stained with Hoechst 33258. Results: Spindle images by Polscope were calssified as three groups (Wang et al. ASRM, 2001). Group I oocytes had normal spindles. Group II oocytes had abnormal spindles. Group III oocytes had no image of spindles. Spindle morphology by immunofluorescent staining was classified as normal, reduced or disrupted. Totally, 100 MII oocytes were vitrified. After thawing, 79 oocytes were morphologically intact. Images by Polscope were classified as 26 in group I, 28 in group II and 25 in group III. With immunofluorescent staining, 25 were classified as having normal spindle morphology, 30 as having abnormal spindle morphology and 24 as having disrupted morphology. The chromosome pattern was compact in 50 oocytes, dispersed in 24 oocytes and absent in 5 oocytes. Conclusions: The spindles of frozen thawed oocytes can be assessed using Polscope and the image is well correlated to the results by traditional immunofluorescent staining. After vitrification and thawing, only 26 (33%) of 79 oocytes appear good and have good morphology of spindles. A portion (32%) of oocytes has relative good appearance but absent spindle images. Without Polscope or immunofluorescent stain to microtubulin, these cannot be selected out. It reflected the unavoidable cryo-injury to meiotic spindle and the imperative improvement of the protocol. Whether the spindles of the thawed oocytes can recover after some period of incubation will need further investigation. Supported by: This study was supported by grants from Chang Gung Memorial Hospital CMRP 891 and CMRP 949.

Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster.

The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphase-anaphase transition but absent once cells center anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.

Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans

In A. nidulans, the temperature-sensitive cell cycle mutation bimC4 causes an elevated mitotic index at restrictive temperature. Under restrictive conditions the mutation interferes with separation of the spindle pole bodies, causes abnormal spindle morphology, and prevents nuclear division. We have cloned and sequenced the wild-type bimC gene. The predicted protein product has homology to Drosophila kinesin heavy chain. We conclude that this kinesin-like protein has an important role in nuclear division in Aspergillus.

Chapter 4 Permeabilized Cell Models for Studying Chromosome Movement in Dividing PtK1 Cell

This chapter discusses the permeabilized cell models for studying chromosome movement in dividing PtK cells. PtK cells are utilized for making permeabilized cell models because their size and morphology is well suited for studying mitosis. These cells are large for tissue culture cells, and have metaphase spindles that are over 10 μm long. It is found that although the cells change shape and thicken as they enter mitosis, dividing cells do not round up to the same extent as most other cells. It is observed that although the PtK, cell line is considered to be stable, a significant proportion of these cells appear to be polyploid or have abnormal mitotic spindles. The easiest way to recognize abnormal spindle morphology is to examine dividing cells with polarization optics. The phase and Nomarski appearance of the cells after lysis are not greatly altered. Immediately after lysis, the rod-shaped mitochondria round up and appear to fragment. The density of staining of the ground substance is similar in lysed and unlysed cells, and microfilaments, intermediate filaments, and microtubules are present in both classes of cells. The other membranous organelles in the permeabilized cell do not appear to be noticeably disrupted, although the mitochondria are swollen and the cristae distorted.