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Mitotic Spindle (mitotic + spindle)
Selected AbstractsKinesin-5 is not essential for mitotic spindle elongation in DictyosteliumCYTOSKELETON, Issue 11 2008Irina Tikhonenko Abstract The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13, cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves in kif13, cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Shaggy/GSK-3, kinase localizes to the centrosome and to specialized cytoskeletal structures in DrosophilaCYTOSKELETON, Issue 6 2006Yves Bobinnec Abstract The assembly of a functional bipolar mitotic spindle requires an exquisite regulation of microtubule behavior in time and space. To characterize new elements of this machinery we carried out a GFP based "protein trap" screen and selected fusion proteins which localized to the spindle apparatus. By this method we identified Shaggy, the Drosophila homologue of glycogen synthase kinase-3, (GSK-3,), as a component of centrosomes. GSK-3, acting in the Wingless signaling pathway is involved in a vast range of developmental processes, from pattern formation to cell-fate specification, and is a key factor for cell proliferation in most animals. We exploited our Shaggy::GFP Drosophila line to analyze the subcellular localizations of GSK-3,/Shaggy and shed light on its multiple roles during embryogenesis. We found that Shaggy becomes enriched transiently in a variety of specialized cytoskeletal structures of the embryo, including centrosomes throughout mitosis, suggesting that this kinase is involved in the regulation of many aspects of the cytoskeleton function. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Glutamylated tubulin: Diversity of expression and distribution of isoformsCYTOSKELETON, Issue 1 2003Marie-Louise Kann Abstract Glutamylation of , and , tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility. Cell Motil. Cytoskeleton 55:14,25, 2003. © 2003 Wiley-Liss, Inc. [source] Asator, a tau-tubulin kinase homolog in Drosophila localizes to the mitotic spindleDEVELOPMENTAL DYNAMICS, Issue 12 2009Hongying Qi Abstract We have used a yeast two-hybrid interaction assay to identify Asator, a tau-tubulin kinase homolog in Drosophila that interacts directly with the spindle matrix protein Megator. Using immunocytochemical labeling by an Asator-specific mAb as well as by transgenic expression of a GFP-labeled Asator construct, we show that Asator is localized to the cytoplasm during interphase but redistributes to the spindle region during mitosis. Determination of transcript levels using qRT-PCR suggested that Asator is expressed throughout development but at relatively low levels. By P-element excision, we generated a null or strong hypomorphic Asatorexc allele that resulted in complete adult lethality when homozygous, indicating that Asator is an essential gene. That the observed lethality was caused by impaired Asator function was further supported by the partial restoration of viability by transgenic expression of Asator-GFP in the Asatorexc homozygous mutant background. The finding that Asator localizes to the spindle region during mitosis and directly can interact with Megator suggests that its kinase activity may be involved in regulating microtubule dynamics and microtubule spindle function. Developmental Dynamics 238:3248,3256, 2009. © 2009 Wiley-Liss, Inc. [source] The ,-tubulin complex protein Alp4 provides a link between the metaphase checkpoint and cytokinesis in fission yeastGENES TO CELLS, Issue 4 2002Leah Vardy Background:, The progression of cytokinesis requires cyclin B destruction by the anaphase promoting complex (APC/C) and, in fission yeast, activation of the septation initiation network (SIN) is also essential. The ,-tubulin complex (,-TuC) localizes to the centrosome throughout the cell cycle and is directly involved in the organization of the mitotic spindle. Results:, We have previously shown that the mutant defective in alp4+ (Spc97/GCP2) displays bipolar spindle defects due to a failure in the recruitment of the ,-TuC on to the spindle pole body (SPB, the centrosome equivalent). Here we show that in these mutants the Mad2 checkpoint is activated, yet septation proceeds due to the untimely activation of the SIN. The Sid1 kinase, the downstream effector of the SIN, is recruited prematurely to both, instead of only one, of the SPBs, which triggers septation despite the presence of monopolar spindles. Remarkably, cyclin B levels, which would normally have declined, remain high at the SPB in septated mutant cells. Conclusions:, We propose a novel role of the ,-TuC in inhibiting activation of the SIN until cyclin B is destroyed. Given the ubiquitous existence of the ,-TuC, this mechanism may be conserved throughout evolution and function to couple cytokinesis to mitotic exit. [source] Ultrastructural and antigenic properties of neural stem cells and their progeny in adult rat subventricular zoneGLIA, Issue 2 2009Alexandre I. Danilov Abstract Neural stem cells (NSCs) in the subventricular zone (SVZ) continuously generate olfactory bulb interneurons in the adult rodent brain. Based on their ultrastructural and antigenic properties, NSCs, transient amplifying precursor cells, and neuroblasts (B, C, and A cells, respectively) have been distinguished in mouse SVZ. Here, we aimed to identify these cell types in rat SVZ ultrastructurally and at the light microscopy level, and to determine the antigenic properties of each cell type using gold and fluorescence immunolabeling. We found astrocytes with single cilia (NSCs, correspond to B cells) and neuroblasts (A cells). We also observed mitotic cells, ependymal cells, displaced ependymal cells, and mature astrocytes. In contrast, transient amplifying precursor cells (C cells) were not detected. The NSCs and neuroblasts had epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor alpha (PDGFR,) expressed on the ciliary apparatus and were the only cell types incorporating the proliferation marker BrdU. Throughout mitosis, EGFR and PDGFR, were associated with the microtubule of the mitotic spindle. Ependymal and displaced ependymal cells also expressed EGFR and PDGFR, on their cilia but did not incorporate BrdU. Our findings indicate that the NSCs in adult rat SVZ give rise directly to neuroblasts. During mitosis, the NSCs disassemble the primary cilium and symmetrically distribute EGFR and PDGFR, among their progeny. © 2008 Wiley-Liss, Inc. [source] Cover Picture: J. Basic Microbiol.JOURNAL OF BASIC MICROBIOLOGY, Issue 1 20101/2010 This issue gives excellent examples for basic mycology, both from mushroom forming basidioymcetes and ascomycetes, including yeasts. The composite title photo shows two fungi, one basidiomycete and a yeast. A mitotic spindle of the mushroom forming basidiomycete Schizophyllum commune, stained for tubulin by immunofl uorescence and for DNA using DAPI (photo: Elke-Martina Jung, Jena) is shown, as well as a fruitbody forming in culture (insert on the left, photo: Nicole Knabe, Jena). Endocytosis of fl uorescein labelled, linear DNA is shown in a second insert (green stain) which is accompanied by a phase-contrast picture of the Saccharomyces cerevisiae cells used (see article by Lang et al. in this issue). (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Centrosome function in normal and tumor cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2006Satish Sankaran Abstract Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process. J. Cell. Biochem. 99: 1240,1250, 2006. © 2006 Wiley-Liss, Inc. [source] Intensity-based signal separation algorithm for accurate quantification of clustered centrosomes in tissue sectionsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 12 2006Markus C. Fleisch Abstract Centrosomes are small organelles that organize the mitotic spindle during cell division and are also involved in cell shape and polarity. Within epithelial tumors, such as breast cancer, and some hematological tumors, centrosome abnormalities (CAs) are common, occur early in disease etiology, and correlate with chromosomal instability and disease stage. In situ quantification of CA by optical microscopy is hampered by overlap and clustering of these organelles, which appear as focal structures. CA has been frequently associated with Tp53 status in premalignant lesions and tumors. Here the authors described an approach to accurately quantify centrosome frequencies in tissue sections and tumors, independently of background or noise levels. Applying simple optical rules in nondeconvolved conventional 3D images of stained tissue sections, the authors showed that they could evaluate more accurately and rapidly centrosome frequencies than with traditional investigator-based visual analysis or threshold-based techniques. The resulting population-based frequency of centrosomes per nucleus could then be used to approximate the proportion of cells with CA in that same population. This was done by taking into account baseline centrosome amplification and proliferation rates measured in the tissue. Using this technique, the authors showed that 20,30% of cells have amplified centrosomes in Tp53 null mammary tumors. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [source] Cytotoxic and antimitotic effects of N -containing Monascus metabolites studied using immortalized human kidney epithelial cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 4-5 2006Anja Knecht Abstract Recently the first Monascus metabolites with a pyridine ring were detected, the monascopyridines A and B. They are formally dehydrogenated derivatives of the red rice pigments rubropunctamine and monascorubramine. Because of their structural similarity, the toxicological effects of these secondary metabolites were studied using immortalized human kidney epithelial cells. The cytotoxicity was determined with the following different endpoint detection methods: metabolic activity, trypan blue exclusion, and electronic cell counting. The compounds led to EC50 values between 11 and 31 ,mol/L but the pigments caused a stronger reduction of the cell viability. Also, the apoptotic potential was examined by measuring caspase 3 activity and detecting apoptotic bodies, but none of the tested compounds induced apoptosis. All four substances caused a rise of the mitotic index to about 9% (100 ,mol/L monascopyridine A and B) and 20% (25 ,mol/L rubropunctamine and monascorubramine). The significant decrease of the ratio of cells in the ana- and telophase to cells in the prometa- and metaphase proved a stop of the mitosis at the meta- to anaphase control point. The compounds caused mitotic arrest and the formation of structural damages like c-mitosis through interaction with the mitotic spindle. These effects point to an aneuploidy inducing potential, which is linked to cancer formation. [source] Eomesodermin is expressed in mouse oocytes and pre-implantation embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005Josie McConnell Abstract T-box genes are a highly conserved family of genes encoding transcription factors, which share a conserved DNA binding domain (the T-box). Appropriate temporal and spatial expression of this gene family is critical for gastrulation and organogenesis in a number of species. The T-box containing gene Eomesodermin was first identified in Xenopus, where it plays a critical role in mesoderm formation. In situ analyses in mice have described the expression patterns of the mouse ortholog of this gene mEomesodermin (mEomes) at the time of implantation and during fetal development. Additional studies involving the disruption of the mEomes gene, have demonstrated an additional role for mEomes in trophoblast formation. However, these analyses did not address the possibility that maternally encoded or pre-blastocyst zygotic transcription of mEomes may also contribute to embryonic development. We show here that mEomes mRNA is present prior to blastocyst formation, and that the protein product of mEomes is associated with nuclear DNA during oocyte development and persistently localizes within all nuclei of the preimplantation embryo until the early blastocyst stage. mEomes protein is associated with the meiotic spindle in the unfertilized egg and with the mitotic spindle at each cell division. Our results are consistent with mEomesodermin having a role in early preimplantation development and inner cell mass formation in addition to its function in the trophoblast lineage. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Increased expression and nuclear localization of the centrosomal kinase Nek2 in human testicular seminomas,THE JOURNAL OF PATHOLOGY, Issue 3 2009Federica Barbagallo Abstract Protein kinases that regulate the centrosome cycle are often aberrantly controlled in neoplastic cells. Changes in their expression or activity can lead to perturbations in centrosome duplication, potentially leading to chromosome segregation errors and aneuploidy. Testicular germ cell tumours (TGCTs) are characterized by amplification of centrosomes through unknown mechanisms. Herein, we report that Nek2, a centrosomal kinase required for centrosome disjunction and formation of the mitotic spindle, is up-regulated in human testicular seminomas as compared to control testes or other types of testicular germ cell tumours. In addition, Nek2 activity is also increased in human seminomas, as demonstrated by immunokinase assays. Analysis by immunohistochemistry indicated that Nek2 is prevalently localized in the nucleus of neoplastic cells of primary human seminomas. Such nuclear localization and the up-regulation of Nek2 protein were also observed in the Tcam-2 seminoma cell line. We demonstrate that nuclear localization of Nek2 is a feature of the more undifferentiated germ cells of mouse testis and correlates with expression of the stemness markers OCT4 and PLZF. These studies suggest that up-regulation of Nek2 is a frequent event in human seminomas and that this may participate in the onset or progression of neoplastic transformation through deregulation of centrosome duplication and/or nuclear events in germ cells. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Shugoshin: a centromeric guardian senses tensionBIOESSAYS, Issue 6 2005Sarah E. Goulding To ensure accurate chromosome segregation during mitosis, the spindle checkpoint monitors chromosome alignment on the mitotic spindle. Indjeian and colleagues have investigated the precise role of the shugoshin 1 protein (Sgo1p) in this process in budding yeast.1 The Sgo proteins were originally identified as highly conserved proteins that protect cohesion at centromeres during the first meiotic division. Together with other recent findings,2 the study highlighted here has identified Sgo1 as a component that informs the mitotic spindle checkpoint when spindle tension is perturbed. This discovery has provided a molecular link between sister chromatid cohesion and tension-sensing at the kinetochore,microtubule interface. BioEssays 27:588,591, 2005. © 2005 Wiley Periodicals, Inc. [source] Aurora A selective inhibitor MLN8237 suppresses the growth and survival of HTLV-1-infected T-cells in vitroCANCER SCIENCE, Issue 5 2010Mariko Tomita Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle. We have shown previously that Aurora A expression is increased aberrantly in human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and primary adult T-cell leukemia cells, and a pan-Aurora kinase inhibitor, which inhibits both Aurora A and Aurora B kinases, reduces viability and induces apoptosis in these cells. However, the specific effects of Aurora A inhibition on HTLV-1-infected T-cells are poorly understood. In this study, we addressed this question by comparing the effects of MLN8237, a selective inhibitor of Aurora A, on cell viability, cell cycle progression, and induction of apoptosis in HTLV-1-infected and -uninfected T-cell lines. MLN8237 reduced the viability of HTLV-1-infected T-cell lines within 24 h, but its effects on that of HTLV-1-uninfected T-cell lines were moderate. MLN8237 induced early apoptosis of HTLV-1-infected T-cell lines without induction of polyploidy. It induced p53 and p21 expression in HTLV-1-infected but not in -uninfected T-cell lines, suggesting that MLN8237-treated HTLV-1-infected T-cell lines exit from mitosis and activate a p53-dependent postmitotic G1 checkpoint, leading to G1 arrest followed by the induction of apoptosis. Our results suggest that specific inhibition of Aurora A kinase is a potentially useful therapeutic strategy in the treatment of adult T-cell leukemia and that further in vivo exploration is warranted. (Cancer Sci 2010; 101: 1204,1211) [source] Live-cell analysis of mitotic spindle formation in taxol-treated cellsCYTOSKELETON, Issue 8 2008Jessica E. Hornick Abstract Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-, tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible,but before NEB is complete,results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Hepatitis B virus X protein affects S phase progression leading to chromosome segregation defects by binding to damaged DNA binding protein 1,HEPATOLOGY, Issue 5 2008Silvia Martin-Lluesma Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC), but its role in the transformation process remains unclear. HBV encodes a small protein, known as HBx, which is required for infection and has been implicated in hepatocarcinogenesis. Here we show that HBx induces lagging chromosomes during mitosis, which in turn leads to formation of aberrant mitotic spindles and multinucleated cells. These effects require the binding of HBx to UV-damaged DNA binding protein 1 (DDB1), a protein involved in DNA repair and cell cycle regulation, and are unexpectedly attributable to HBx interfering with S-phase progression and not directly with mitotic events. HBx also affects S-phase and induces lagging chromosomes when expressed from its natural viral context and, consequently, exhibits deleterious activities in dividing, but not quiescent, hepatoma cells. Conclusion: In addition to its reported role in promoting HBV replication, the binding of HBx to DDB1 may induce genetic instability in regenerating hepatocytes and thereby contribute to HCC development, thus making this HBV,host protein interaction an attractive target for new therapeutic intervention. (HEPATOLOGY 2008.) [source] Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cell-free extracts,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2003Takashi Tatsumoto Abstract The ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases. ECT2 contains motifs of cell cycle regulators at its N-terminal domain. We previously showed that ECT2 plays a critical role in cytokinesis. Here, we report a potential role of XECT2, the Xenopus homologue of the human ECT2, in spindle assembly in cell-free Xenopus egg extracts. Cloned XECT2 cDNA encodes a 100 kDa protein closely related to human ECT2. XECT2 is specifically phosphorylated in M phase extracts. Affinity-purified anti-XECT2 antibody strongly inhibited mitosis in Xenopus cell-free extracts. Instead of bipolar spindles, where chromosomes are aligned at the metaphase plane in control extracts, the addition of anti-XECT2 resulted in the appearance of abnormal spindles including monopolar and multipolar spindles as well as bipolar spindles with misaligned chromosomes. In these in vitro synthesized spindle structures, XECT2 was found to tightly associate with mitotic spindles. The N-terminal half of XECT2 lacking the catalytic domain also strongly inhibited spindle assembly in vitro, resulting in the formation of mitotic spindles with a low density. Among the representative Rho GTPases, a dominant-negative form of Cdc42 strongly inhibited spindle assembly in vitro. These results suggest that the Rho family GTPase Cdc42 and its exchange factor XECT2 are critical regulators of spindle assembly in Xenopus egg extracts. Published 2003 Wiley-Liss, Inc., [source] The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)PHYCOLOGICAL RESEARCH, Issue 1 2001Ilias Dimitriadis SUMMARY Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 ,mol L,1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome-associated MT almost disappeared, and a massive assembly of cortical/subcortical, well-organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome-associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles. [source] | |||||||||||||