Aurora A Phosphorylation of YY1 during Mitosis Inactivates its DNA Binding Activity.

Successful execution of mitotic cell division requires the tight synchronisation of numerous biochemical pathways. The underlying mechanisms that govern chromosome segregation have been thoroughly investigated. However, the mechanisms that regulate transcription factors in coordination with mitotic progression remain poorly understood. In this report, we identify the transcription factor YY1 as a novel mitotic substrate for the Aurora A kinase, a key regulator of critical mitotic events, like centrosome maturation and spindle formation. Using in vitro kinase assays, we show that Aurora A directly phosphorylates YY1 at serine 365 in the DNA-binding domain. Using a new phospho-specific antibody, we show that YY1 phosphorylation at serine 365 occurs during mitosis, and that this phosphorylation is significantly reduced upon inhibition of Aurora A. Furthermore, we show, using electrophoretic mobility shift and chromatin immunoprecipitation assays, that phosphorylation of YY1 at this site abolishes its DNA binding activity in vitro and in vivo. In conformity with this loss of binding activity, phosphorylated YY1 also loses its transctivation ability as demonstrated by a luciferase reporter assay. These results uncover a novel mechanism that implicates Aurora A in the mitotic inactivation of transcription factors.

l-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes.

STUDY QUESTION: How does l-carnitine (LC) supplementation during vitrification and in vitro maturation (IVM) of germinal vesicle stage (GV)-oocytes improve the developmental competence of the resultant metaphase II (MII) oocytes? SUMMARY ANSWER: LC supplementation during both vitrification of GV-oocytes and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. WHAT IS KNOWN ALREADY: Vitrification of GV-oocytes results in a lower success rate of blastocyst development compared with non-vitrified oocytes. LC supplementation during both vitrification and IVM of mouse GV-oocytes significantly improves embryonic development after IVF. STUDY DESIGN, SIZE, DURATION: GV-oocytes were collected from (B6.DBA)F1 and B6 mouse strains and subjected to vitrification and warming with or without 3.72 mM LC supplementation. After IVM with or without LC supplementation, the rate of nuclear maturation and the quality of MII oocytes were evaluated. At least 20 oocytes/group were examined, and each experiment was repeated at least three times. All experiments were conducted during 2013-2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Extrusion of the first polar body in IVM oocytes was observed as an indication of nuclear maturation. Spindle assembly and chromosomal alignment were examined by immunostaining of α-tubulin and nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Mitochondrial distribution and oxidative activity were measured by staining with Mitotracker Green Fluorescence Mitochondria (Mitotracker Green FM) and chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos), respectively. ATP levels were determined by using the Bioluminescent Somatic Cell Assay Kit. MAIN RESULTS AND THE ROLE OF CHANCE: LC supplementation during both vitrification and IVM of GV-oocytes significantly increased the proportions of oocytes with normal MII spindles to the levels comparable with those of non-vitrified oocytes in both mouse strains. While vitrification of GV-oocytes lowered the proportions of MII oocytes with peripherally concentrated mitochondrial distribution compared with non-vitrified oocytes, LC supplementation significantly increased the proportion of such oocytes in the (B6.DBA)F1 strain. LC supplementation decreased the proportion of oocytes with mitochondrial aggregates in both vitrified and non-vitrified oocytes in the B6 strain. The oxidative activity of mitochondria was mildly decreased by vitrification and drastically increased by LC supplementation irrespective of vitrification in both mouse strains. No change was found in ATP levels irrespective of vitrification or LC supplementation. Results were considered to be statistically significant at P < 0.05 by either χ(2)- or t-test. LIMITATIONS, REASONS FOR CAUTION: It remains to be tested whether beneficial effect of LC supplementation during vitrification and IVM of GV-oocytes leads to fetal development and birth of healthy offspring after embryo transfer to surrogate females. WIDER IMPLICATIONS OF THE FINDINGS: This protocol has the potential to improve the quality of vitrified human oocytes and embryos during assisted reproduction treatment. STUDY FUNDING/COMPETING INTEREST: Partially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and Mitacs Elevate Postdoctoral Fellowship, Canada.

Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis.

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.

Oxidative stress and tumor necrosis factor-alpha-induced alterations in metaphase II mouse oocyte spindle structure.

OBJECTIVE(S): To examine the effect of exogenous exposure to hydrogen peroxide (H(2)O(2)) and tumor necrosis factor (TNF)-alpha on mouse metaphase II (MII) oocyte spindle structure and to examine the potential benefits of supplementing the culture media with vitamin C. DESIGN: Prospective study. SETTING: Research laboratory in a tertiary hospital. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microtubule changes and alterations in chromosomal alignment. RESULT(S): Both concentration- and time-dependent alterations were seen in spindle structure after exposure to H(2)O(2). An H(2)O(2) concentration as low as 12.5 microM increased the odds of an oocyte with altered microtubule and chromosome alignment (score >or=3) by 93%. Significantly increased damage was seen with increasing period of incubation. Higher scores were seen after exposure to both TNF-alpha alone and in combination with H(2)O(2) compared with controls. Changes in chromosomal alignment were comparable among the three groups. Oocytes coincubated with H(2)O(2) and vitamin C at 200 microM demonstrated less damage compared with those with H(2)O(2) alone. CONCLUSION(S): Oxidative stress results in concentration and time-dependent alterations in the spindle structure and augments the effects induced by TNF-alpha. Proper oocyte handling in vitro may help reduce oxidative insult, thus improving the oocyte quality. Antioxidants may have a protective effect and need to be further evaluated.

Duplicated insertion mutation in the microtubule-associated protein Spag5 (astrin/MAP126) and defective proliferation of immature Sertoli cells in rat hypogonadic (hgn/hgn) testes.

Male rats with hypogonadism (hgn/hgn) experience sterility from testicular dysplasia, which is controlled by a single recessive gene, hgn. The postnatal growth of the seminiferous tubules was severely affected. In this study, we localized the hgn locus to a 320 kb region on rat chromosome 10 and detected the insertion of a 25 bp duplication into the sixth exon of the sperm-associated antigen 5 (Spag5/astrin/MAP126) gene, which codes for a microtubule-associated protein. This mutation results in a truncated Spag5 protein lacking the primary spindle-targeting domain at the C terminus. Immunological staining with antibodies to markers for Sertoli and germ cells during the early postnatal period indicated that the abnormal mitosis with dispersed chromosomes in hgn/hgn testes occurs in proliferating Sertoli cells. Therefore, apoptotic Sertoli cell death would result from the disorganization of the spindle apparatus caused by defective Spag5. These findings suggested that the Spag5 is essential for testis development in rats and that the hgn/hgn rat is a unique animal model for studying the function of Spag5.

[Dynamics of microtubular cytoskeleton in higher plant meiosis. VI. Mechanisms of the successive cytokinesis].

Intracellular morphological processes of successive cytokinesis in cereal pollen mother cells during normal and abnormal meiosis were studied. It was shown that the central spindle fiber system transforms into a phragmoplast at telophase. A model of centrifugal movement of the phragmoplast as a modification of B-anaphase has been proposed.

[Polyarchal spindles in higher plant meiosis].

Morphogenesis and functioning of polyarchal spindles in higher plant meiosis were studied. A mechanism of anastral spindle formation in discussed.

A computerized cellular imaging system for high content analysis in Monastrol suppressor screens.

In this paper, we describe a new bioimage informatics system developed for high content screening (HCS) applications with the goal to extract and analyze phenotypic features of hundreds of thousands of mitotic cells simultaneously. The system introduces the algorithm of multi-phenotypic mitotic analysis (MMA) and integrates that with algorithms of correlation analysis and compound clustering used in gene microarray studies. The HCS-MMA system combines different phenotypic information of cellular images obtained from three-channel acquisitions to distinguish and label individual cells at various phases of mitosis. The proposed system can also be used to extract and count the number of cells in each phase in cell-based assay experiments and archive the extracted data into a structured database for more sophisticated statistical and data analysis. To recognize different mitotic phases, binary patterns are set up based on a known biological mitotic spindle model to characterize cellular morphology of actin, microtubules, and DNA. To illustrate its utility, the HCS-MMA system has been applied to screen the quantitative response of 320 different drug compounds in suppressing Monastrol. The results are validated and evaluated by comparing the performance of HCS-MMA with visual analysis, as well as clustering of the drug compounds under evaluation.

[Consolidation of cytoskeleton during plant spindle formation. I. Abnormalities of spindle integrity in meiosis]. / Konsolidatsiia tsitoskeleta pri formirovanii veretena deleniia v rastitel'noi kletke.

The process of formation of multiple spindles in mononucleate pollen mother cells during meiosis in general wide hybrids F1 and haploids is described. It is assumed that the abnormality may be caused by aberration of a special process at late prometaphase providing for the spindle consolidation.

Meiotic mutants of Medicago sativa show altered levels of alpha- and beta-tubulin.

We have analysed the level of accumulation of alpha- and beta-tubulin polypeptides in flowers collected from different meiotic mutants of alfalfa (Medicago sativa L.). The H33 mutant previously identified as a producer of male and female gametes with the somatic chromosome number (2n gametes) as a result of defective spindle orientation or, more rarely, abnormal cytokinesis, showed a higher level of alpha- and beta-tubulin compared to control diploid plants and approximately the same level as control tetraploid plants. A higher level of tubulin was likewise observed in diploid plants displaying abnormalities in spindle orientation and cytokinesis, which had gone through 3-4 cycles of phenotypic recurrent selection to increase 2n gamete production. A similar analysis was performed on another class of Medicago meiotic mutants characterized by production of 4n pollen (jumbo pollen, due to the absence of cytokinesis at the end of meiosis) and 2n eggs. Again, the level of alpha- and beta-tubulin was found to be higher in the mutants than in diploid controls. We conclude that meiotic defects, such as abnormal spindle orientation or cytokinesis leading to the formation of 2n gametes, determine an increased level of tubulin, the main constituent of plant microtubules (MTs).

Light and electron microscopic immunocytochemical localization of two major proteins in garlic bulb.

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur-containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA-stimulated human short-term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti-40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.

Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles.

A human cDNA expression library was screened using anti-centrin antibodies to obtain a cDNA clone encoding human centrin. The cDNA clone contains an open reading frame of 516 base pairs and predicts a product of 172 amino acids with a calculated molecular mass of 19,528 and a pI of 4.61. Sequence analysis demonstrates that human centrin and centrins from higher plants, protozoa, algae, Xenopus and the yeast CDC31 gene product are closely related members of a subfamily of the EF-hand superfamily of calcium-binding proteins. The human centrin sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation and western blot studies from HeLa cells confirm that human centrin is a protein of approximately 20,000 M(r) as predicted from the cDNA clone. Indirect immunofluorescence analysis of HeLa cells demonstrates that centrin is localized at the centrosome of interphase cells and that it redistributes to the region of the spindle poles during mitosis. When taken together with earlier genetic studies, these results demonstrate that centrin is a ubiquitous component of centrosomes and mitotic spindle poles of diverse organisms and suggest that centrin plays a role in centrosome separation at the time of mitosis.

Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells.

MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.

Three-dimensional ultrastructure of generative cell mitosis in the pollen tube of Nicotiana tabacum.

Generative cell mitosis was examined in stylar-grown pollen tubes of Nicotiana tabacum using serial sectioning, transmission electron microscopy and computer-assisted reconstruction. Before mitosis, the generative cell has a cage-like organization of cytoplasmic microtubules. The mitotic spindle forms when the cytoplasmic microtubules reduce in frequency and kinetochore microtubules form in an area delimited by sheets of endoplasmic reticulum; no preprophase band of microtubules is observed. At metaphase, 21 pairs of kinetochores are distributed unevenly along the length and depth of the cell without the formation of a strictly planar metaphase plate. The metaphase spindle is highly oblique, with diffuse subpoles distributed along the sides of the cell, colocalized with endoplasmic reticulum lamellae. From these dispersed subpoles the kinetochore bundles emanate, closely associated with tubular endoplasmic reticulum. Anaphase consists of three principal processes: convergence of diffused mitotic poles, shortening of the kinetochore bundles, and the elongation of the spindle by an average of nearly 50%. At mid-anaphase, a phragmoplast begins to form, mainly by the assembly of new microtubules at the equatorial area, which form as a cluster of numerous short microtubules. Cytokinesis is essentially conventional, with centrifugal cell plate formation. Cytoplasmic microtubules are restored in the newly formed "brother" sperm cells in a distribution similar to that in the generative cell but fewer in number.