An astral simulacrum of the central spindle accounts for normal, spindle-less, and anucleate cytokinesis in echinoderm embryos

PubMed Central

Su, Kuan-Chung; Bement, William M.; Petronczki, Mark; von Dassow, George

2014-01-01

Cytokinesis in animal cells depends on spindle-derived spatial cues that culminate in Rho activation, and thereby actomyosin assembly, in a narrow equatorial band. Although the nature, origin, and variety of such cues have long been obscure, one component is certainly the Rho activator Ect2. Here we describe the behavior and function of Ect2 in echinoderm embryos, showing that Ect2 migrates from spindle midzone to astral microtubules in anaphase and that Ect2 shapes the pattern of Rho activation in incipient furrows. Our key finding is that Ect2 and its binding partner Cyk4 accumulate not only at normal furrows, but also at furrows that form in the absence of associated spindle, midzone, or chromosomes. In all these cases, the cell assembles essentially the same cytokinetic signaling ensemble—opposed astral microtubules decorated with Ect2 and Cyk4. We conclude that if multiple signals contribute to furrow induction in echinoderm embryos, they likely converge on the same signaling ensemble on an analogous cytoskeletal scaffold. PMID:25298401

Differential Diagnosis of Benign Spindle Cell Lesions.

PubMed

Magro, Gaetano

2018-03-01

Spindle cell lesions of the breast cover a wide spectrum of diseases ranging from reactive tumor-like lesions to high-grade malignant tumors. The recognition of the benign spindle cell tumor-like lesions (nodular fasciitis; reactive spindle cell nodule after biopsy, inflammatory pseudotumor/inflammatory myofibroblastic tumor; fascicular variant of pseudoangiomatous stromal hyperplasia) and tumors (myofibroblastoma, benign fibroblastic spindle cell tumor, leiomyoma, schwannoma, spindle cell lipoma, solitary fibrous tumor, myxoma) is crucial to avoid confusion with morphologically similar but more aggressive bland-appearing spindle cell tumors, such as desmoid-type fibromatosis, low-grade (fibromatosis-like) spindle cell carcinoma, low-grade fibrosarcoma/myofibroblastic sarcoma and dermatofibrosarcoma protuberans. Copyright © 2017 Elsevier Inc. All rights reserved.

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis.

PubMed

Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

2014-01-01

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).

NUCLEOPORINS NPP-1, NPP-3, NPP-4, NPP-11 and NPP-13 ARE REQUIRED FOR PROPER SPINDLE ORIENTATION IN C. ELEGANS

PubMed Central

Schetter, Aaron; Askjaer, Peter; Piano, Fabio; Mattaj, Iain; Kemphues, Kenneth

2006-01-01

Nucleoporins are components of the nuclear pore, which is required for nucleo-cytoplasmic transport. We report a role for a subclass of nucleoporins in orienting the mitotic spindle in C. elegans embryos. RNAi-mediated depletion of any of five putative nucleoporins npp-1, npp-3, npp-4, npp-11, and npp-13 leads to indistinguishable spindle orientation defects. Transgenic worms expressing NPP-1::GFP or NPP-11::GFP show GFP localization at the nuclear envelope, consistent with their predicted function. NPP-1 interacts with the other nucleoporins in yeast two-hybrid assays suggesting that the proteins affect spindle orientation by a common process. The failed orientation phenotype of npp-1(RNAi) is at least partially epistatic to the ectopic spindle rotation in the AB blastomere of par-3 mutant embryos. This suggests that NPP-1 contributes to the mechanics of spindle orientation. However, NPP-1 is also required for PAR-6 asymmetry at the two-cell stage, indicating that nucleoporins may be required to define cortical domains in the germ line blasotmere P1. Nuclear envelope structure is abnormal in npp-1(RNAi) embryos but the envelope maintains its integrity and most nuclear proteins we assayed accumulate normally. These findings raise the possibility that these nucleoporins may have direct roles in orienting the mitotic spindle and the maintenance of cell polarity. PMID:16325795

Is cohesin required for spindle-pole-body/centrosome cohesion?

PubMed Central

Jin, Hui; Avey, Martin

2012-01-01

Centrosomes are microtubule-organizing centers that nucleate spindle microtubules during cell division. In budding yeast, the centrosome, often referred to as the spindle pole body, shares structural components with the centriole, the central core of the animal centrosome. The parental centrosome is duplicated when DNA replication takes place. Like sister chromatids tethered together by cohesin, duplicated centrosomes are linked and then separate to form the bipolar spindle necessary for chromosome segregation. Recent studies have shown that cohesin is also localized to the animal centrosome and is perhaps directly involved in engaging paired centrioles. Here we discuss the potential role of cohesin in mediating spindle-pole-body cohesion in the context of yeast meiosis. We propose that the coordination of chromosome segregation with centrosome cohesion and duplication is mediated by the antagonistic interaction between the Aurora kinase and the Polo kinase and that the role of cohesin in centrosome regulation appears to be indirect in budding yeast. PMID:22482005

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick

Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugationmore » to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.« less

Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts

PubMed Central

Shimamoto, Yuta; Kapoor, Tarun M.

2014-01-01

SUMMARY To explain how micron-sized cellular structures generate and respond to forces we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based set-up to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multi-mode imaging system allows tracking of microtubules, chromosomes and needle tips. This set-up can be used to analyze the viscoelastic properties of the spindle on time-scales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 days. PMID:22538847

Anaphase B

PubMed Central

Scholey, Jonathan M.; Civelekoglu-Scholey, Gul; Brust-Mascher, Ingrid

2016-01-01

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical “modules” that cooperate to mediate pole–pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation. PMID:27941648

Mechanics of kinetochore microtubules and their interactions with chromosomes during cell division

NASA Astrophysics Data System (ADS)

Nazockdast, Ehssan; Fürthauer, Sebastian; Redemann, Stephanie; Baumgart, Johannes; Lindow, Norbert; Kratz, Andrea; Prohaska, Steffen; Müller-Reichert, Thomas; Shelley, Michael

2016-11-01

The accurate segregation of chromosomes, and subsequent cell division, in Eukaryotic cells is achieved by the interactions of an assembly of microtubules (MTs) and motor-proteins, known as the mitotic spindle. We use a combination of our computational platform for simulating cytoskeletal assemblies and our structural data from high-resolution electron tomography of the mitotic spindle, to study the kinetics and mechanics of MTs in the spindle, and their interactions with chromosomes during chromosome segregation in the first cell division in C.elegans embryo. We focus on kinetochore MTs, or KMTs, which have one end attached to a chromosome. KMTs are thought to be a key mechanical component in chromosome segregation. Using exploratory simulations of MT growth, bending, hydrodynamic interactions, and attachment to chromosomes, we propose a mechanical model for KMT-chromosome interactions that reproduces observed KMT length and shape distributions from electron tomography. We find that including detailed hydrodynamic interactions between KMTs is essential for agreement with the experimental observations.

Microtubule Dynamics Scale with Cell Size to Set Spindle Length and Assembly Timing.

PubMed

Lacroix, Benjamin; Letort, Gaëlle; Pitayu, Laras; Sallé, Jérémy; Stefanutti, Marine; Maton, Gilliane; Ladouceur, Anne-Marie; Canman, Julie C; Maddox, Paul S; Maddox, Amy S; Minc, Nicolas; Nédélec, François; Dumont, Julien

2018-05-21

Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume. Copyright © 2018 Elsevier Inc. All rights reserved.

[Pulmonary sarcomatoid carcinoma].

PubMed

Antoine, Martine; Vieira, Thibault; Fallet, Vincent; Hamard, Cécile; Duruisseaux, Michael; Cadranel, Jacques; Wislez, Marie

2016-01-01

Pulmonary sarcomatoid carcinomas are a rare group of tumors accounting for about one percent of non-small cell lung carcinoma (NSCLC). In 2015, the World Health Organization classification united under this name all the carcinomas with sarcomatous-like component with spindle cell or giant cell appearance, or associated with a sarcomatous component sometimes heterologous. There are five subtypes: pleomorphic carcinoma, spindle cell carcinoma, giant cell carcinoma, carcinosarcoma and pulmonary blastoma. Clinical characteristics are not specific from the other subtypes of NSCLC. Epithelial to mesenchymal transition pathway may play a key role. Patients, usually tobacco smokers, are frequently symptomatic. Tumors are voluminous more often peripherical than central, with strong fixation on FDG TEP CT. Distant metastases are frequent with atypical visceral locations. These tumors have poorer prognosis than the other NSCLC subtypes because of great aggressivity, and frequent chemoresistance. Here we present pathological description and a review of literature with molecular features in order to better describe these tumors and perhaps introduce new therapeutics. Copyright © 2016. Published by Elsevier Masson SAS.

Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.

PubMed

Tauchman, Eric C; Boehm, Frederick J; DeLuca, Jennifer G

2015-12-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore-microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore-microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.

Stable kinetochore–microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells

PubMed Central

Tauchman, Eric C.; Boehm, Frederick J.; DeLuca, Jennifer G.

2015-01-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. PMID:26620470

Mucinous breast carcinoma with myoepithelial-like spindle cells.

PubMed

Miyake, Yasuyuki; Hirokawa, Mitsuyoshi; Norimatsu, Yoshiaki; Kanahara, Takuo; Monobe, Yasumasa; Ohno, Setsuyo; Miyamoto, Tomoyuki; Yakushiji, Hiromasa; Sakaguchi, Takuya; Aratake, Yatsuki; Ohno, Eiji

2009-06-01

Appearance of spindle cells has been believed as a benign index of breast cytology. But, we have frequently observed the spindle cells in smears from mucinous carcinoma of the breast. Here, we characterized the biochemical nature of the spindle cells, so as to clarify their identity in cytology. Nineteen cases of breast mucinous carcinoma were used for cytological examination. The spindle cells were located at edges of tumor cell nests and in the backgrounds of cytological specimens. Immunohistological examination revealed that the spindle cells exhibited both immunoreactivity against carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA). Immunoreactivity against vimentin, cytokeratin, or alpha-smooth muscle actin was, however, not observed. The mode of distribution of biochemical markers suggests that the positive cells for anti-CEA antibody and anti-EMA antibody are tumor cells compressed by mucin, while the vimentin-positive cells are fibroblasts. We assert that the presence of spindle cells can be a characteristic feature of mucinous carcinoma of the breast. Discrimination of the spindle cells in mucinous carcinoma from myoepithelial cells and naked bipolar nuclei in benign lesions was established here. It should facilitate precise diagnosis of breast cancer. (c) 2009 Wiley-Liss, Inc.

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA.

PubMed

Liro, Małgorzata J; Rose, Lesilee S

2016-11-01

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. Copyright © 2016 by the Genetics Society of America.

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA

PubMed Central

Liro, Małgorzata J.; Rose, Lesilee S.

2016-01-01

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. PMID:27672093

Intercentrosomal angular separation during mitosis plays a crucial role for maintaining spindle stability

NASA Astrophysics Data System (ADS)

Sutradhar, S.; Basu, S.; Paul, R.

2015-10-01

Cell division through proper spindle formation is one of the key puzzles in cell biology. In most mammalian cells, chromosomes spontaneously arrange to achieve a stable bipolar spindle during metaphase which eventually ensures proper segregation of the DNA into the daughter cells. In this paper, we present a robust three-dimensional mechanistic model to investigate the formation and maintenance of a bipolar mitotic spindle in mammalian cells under different physiological constraints. Using realistic parameters, we test spindle viability by measuring the spindle length and studying the chromosomal configuration. The model strikingly predicts a feature of the spindle instability arising from the insufficient intercentrosomal angular separation and impaired sliding of the interpolar microtubules. In addition, our model successfully reproduces chromosomal patterns observed in mammalian cells, when activity of different motor proteins is perturbed.

A rare case of malignant pediatric ectomesenchymoma arising from the cerebrum.

PubMed

Kun, Yao; Duan, Zejun; Mei, Xi; Xu, Ying; Li, Jiuzhou; Li, Shouwei; Qi, Xueling

2015-01-01

Malignant ectomesenchymoma is a rare tumor that contains both ectodermal and mesenchymal elements. So far, only 7 patients with a manifestation in the cerebrum (with confirmed clinicopathological data) have been reported. A 4-year-old girl was present at our hospital with a 3-week history of intermittent sudden dizzy with no apparent cause. MRI showed an irregular enhanced lesion in the left frontal-parietal lobe and lateral ventricle with peripheral gadolinium-enhancement with a significant surrounding edema. Total removal of the tumor was performed. Histological examination of the resected tumor revealed a mixed astrocytoma and anaplastic ependymoma component with undifferentiated mesenchymal spindle cell component. Generally speaking, the main malignant part in most cases of malignant ectomesenchymoma (MEM) is the mesenchymal component. In the present case, the malignant component was both in the mesenchymal and ectodermal part. In particular, the mesenchymal part was mainly composed of spindle cells, and the ectodermal part primarily consisted of gliomatous component and anaplastic ependymoma component. The patient was then treated with chemotherapy and as regard to the prognosis, there was no evidence of tumor recurrence at the 5 months' follow-up. The long term follow-up is still in progress.

Expanding the histologic spectrum of mucinous tubular and spindle cell carcinoma of the kidney.

PubMed

Fine, Samson W; Argani, Pedram; DeMarzo, Angelo M; Delahunt, Brett; Sebo, Thomas J; Reuter, Victor E; Epstein, Jonathan I

2006-12-01

Mucinous tubular and spindle cell carcinomas (MTSCs) are polymorphic neoplasms characterized by small, elongated tubules lined by cuboidal cells and/or cords of spindled cells separated by pale mucinous stroma. Nonclassic morphologic variants and features of MTSC have not been well studied. We identified 17 previously unreported MTSCs from Surgical Pathology and consultative files of the authors and their respective institutions and studied their morphologic features. A total of 10/17 cases were considered "classic," as described above, with 5/10 showing at least focal (20% to 50%) tubular predominance without apparent mucinous matrix. Alcian blue staining revealed abundant (>50%) mucin in all classic cases. Seven of 17 MTSCs were classified as "mucin-poor," with little to no extracellular mucin appreciable by hematoxylin and eosin. Four of these cases showed equal tubular and spindled morphology, 2 cases showed spindle cell predominance (70%; 95%), and 1 case showed tubular predominance (90%). In 5/7 mucin-poor cases, staining for Alcian blue revealed scant (<10%) mucin in cellular areas with the other 2 cases having 30% mucin. Unusual histologic features identified in the 17 cases were: foamy macrophages (n=8), papillations/well formed papillae (n=6/n=1), focal clear cells in tubules (n=3), necrosis (n=3), oncocytic tubules (n=2; 40%, 5%), numerous small vacuoles (n=2), heterotopic bone (n=1), psammomatous calcification (n=1), and nodular growth with lymphocytic cuffing (n=1). An exceptional case contained a well-circumscribed, HMB45-positive angiomyolipoma within the MTSC. MTSCs may be "mucin-poor" and show a marked predominance of either of its principal morphologic components, which coupled with the presence of other unusual features such as clear cells, papillations, foamy macrophages, and necrosis, may mimic other forms of renal cell carcinoma. Pathologists must be aware of the spectrum of histologic findings within MTSCs to ensure their accurate diagnosis.

TFE3 Translocation Associated Perivascular Epithelioid Cell Neoplasm (PEComa) of the Gynecologic Tract: Morphology, Immunophenotype, Differential Diagnosis

PubMed Central

Schoolmeester, J. Kenneth; Dao, Linda N.; Sukov, William R.; Park, Kay J.; Murali, Rajmohan; Hameed, Meera R.; Soslow, Robert A.

2016-01-01

TFE3 translocation associated PEComa is a distinct form of perivascular epithelioid cell neoplasm, the features of which are poorly defined owing to their general infrequency and limited prior reports with confirmed rearrangement or fusion totaling nine cases. Recent investigation has found a lack of TSC gene mutation in these tumors compared to their nonrearranged counterparts which underscores the importance of recognizing the translocated variant due to hypothetical ineffectiveness of targeted mTOR inhibitor therapy. Six cases were identified and TFE3 rearrangement was confirmed by FISH. Patient age ranged 46 to 66 years (median 50) and none had a history of tuberous sclerosis complex. Three cases arose in the uterine corpus, one in the vagina, and one pelvic tumor and one pulmonary tumor were likely a recurrence/metastasis from a probable uterine primary. Five cases had purely clear cell epithelioid morphology that showed a spectrum of atypia while one case had a mixture of clear cell epithelioid and spindle cells. A mostly consistent immunophenotype was observed in the purely clear cell epithelioid cases: each demonstrated diffuse TFE3, HMB45, CathepsinK labeling, either focal or no melanA staining and variably weak reactivity to smooth muscle markers. The mixed clear cell epithelioid and spindle cell case had a similar pattern in its epithelioid component, but strong muscle marker positivity in its spindle cell component. Follow up ranged 1 to 57 months. Three cases demonstrated aggressive behavior and three cases had no evidence of recurrence. Both GYN-specific and traditional sets of criteria for malignancy were evaluated. The GYN model showed improved inclusion and specificity in comparison to the traditional model. PMID:25517951

TFE3 translocation-associated perivascular epithelioid cell neoplasm (PEComa) of the gynecologic tract: morphology, immunophenotype, differential diagnosis.

PubMed

Schoolmeester, J Kenneth; Dao, Linda N; Sukov, William R; Wang, Lu; Park, Kay J; Murali, Rajmohan; Hameed, Meera R; Soslow, Robert A

2015-03-01

TFE3 translocation-associated PEComa is a distinct form of perivascular epithelioid cell neoplasm, the features of which are poorly defined owing to their general infrequency and limited prior reports with confirmed rearrangement or fusion. Recent investigation has found a lack of TSC gene mutation in these tumors compared with their nonrearranged counterparts, which underscores the importance of recognizing the translocated variant because of hypothetical ineffectiveness of targeted mTOR inhibitor therapy. Six cases were identified, and TFE3 rearrangement was confirmed by fluorescence in situ hybridization. Patient age ranged from 46 to 66 years (median 50 y), and none had a history of a tuberous sclerosis complex. Three cases arose in the uterine corpus, 1 in the vagina, 1 pelvic tumor, and 1 pulmonary tumor that was likely a recurrence/metastasis from a probable uterine primary. Five cases had clear cell epithelioid morphology that showed a spectrum of atypia, while 1 case had a mixture of clear cell epithelioid and spindle cells. A mostly consistent immunophenotype was observed in the clear cell epithelioid cases: each demonstrated diffuse TFE3, HMB45, cathepsinK labeling, either focal or no melanA staining, and variably weak reactivity to smooth muscle markers. The mixed clear cell epithelioid and spindle cell case had a similar expression pattern in its epithelioid component but strong muscle marker positivity in its spindle cell component. Follow-up ranged from 1 to 57 months. Three cases demonstrated aggressive behavior, and 3 cases had no evidence of recurrence. Both GYN-specific and traditional sets of criteria for malignancy were evaluated. The GYN model showed improved inclusion and specificity in comparison to the traditional model.

Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors.

PubMed

Prodon, François; Chenevert, Janet; Hébras, Céline; Dumollard, Rémi; Faure, Emmanuel; Gonzalez-Garcia, Jose; Nishida, Hiroki; Sardet, Christian; McDougall, Alex

2010-06-01

Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

Primary pure spindle cell carcinoma (sarcomatoid carcinoma) of the ovary: A case report with immunohistochemical study.

PubMed

Giordano, Giovanna; Berretta, Roberto; Silini, Enrico

2016-08-05

In the ovary, sarcomatoid carcinoma has been reported only as mural nodules in epithelial malignant or borderline serous or mucinous cystic neoplasms, and in teratomas. In this paper we report a rare case of a solid sarcomatoid carcinoma of the ovary, without accompanying component of giant cells, pleomorphic cells, or glandular and other epithelial structures. This case report refers to a sarcomatoid carcinoma of the ovary in in a 57 year-old woman with abdominal pain. Macroscopically, the neoplasm was a 15x10x5 cm ovarian mass that featured gray white solid fleshy areas, interspersed with areas of necrosis, hemorrhage and cystic spaces filled with thick fluid. The epithelial differentiation of the tumor was demonstrated by strong and diffuse reactivity to CAM5.2 and focal immunoreactivity to EMA. A diagnosis of malignant mesenchymal tumor was excluded due to negativity for desmin, smooth muscle actin, caldesmon, CD34, CD10, and myoglobin. Neural, neuroendocrine neoplasm, melanoma and Perivascular Epithelioid Cell Tumor (PEComa) were excluded because of negativity for S100, chromogranin, synaptophysin and HMB45. Primary ovarian spindle cell carcinoma is a rare neoplasm, which must be considered in the differential diagnosis of solid ovarian mass with spindle cell appearance. This case adds to our knowledge of the biological behavior of these rare neoplasms. The distinction from true sarcomas and carcinosarcomas is important because of the more favorable prognosis of the spindle cell carcinomas. However their diagnosis necessitates a careful tissue sampling and immunohistochemical staining.

Akap350 Recruits Eb1 to The Spindle Poles, Ensuring Proper Spindle Orientation and Lumen Formation in 3d Epithelial Cell Cultures.

PubMed

Almada, Evangelina; Tonucci, Facundo M; Hidalgo, Florencia; Ferretti, Anabela; Ibarra, Solange; Pariani, Alejandro; Vena, Rodrigo; Favre, Cristián; Girardini, Javier; Kierbel, Arlinet; Larocca, M Cecilia

2017-11-02

The organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.

Dedifferentiated liposarcoma of the retro-peritoneum: histologically low-grade type.

PubMed

Parakh, Rugvedita Satyajeet; Zawar, Meera Prem; Gadgil, Pradeep Achyut; Kaujalagi, Neeraja Shashikant

2010-01-01

A dedifferentiated liposarcoma of the retro-peritoneum of a 45-year-old female is described in this case report. It showed dedifferentiation into a histological low-grade type and thus defies the traditional definition of dedifferentiated liposarcoma. The excised specimen was a huge, multi-nodular encapsulated mass surrounding the kidney and infiltrating into it. The mass showed a dedifferentiated focus different in color from the surrounding tumor and containing areas of necrosis and hemorrhage. The sections from the dedifferentiated part of the tumor appeared predominantly as benign spindle cell component on histology but the tumor was infiltrating into the kidney. Hence, a close and long-term follow-up is expected in such cases though they look benign. In the retro-peritoneum, a lipoma-like well-differentiated liposarcoma with spindle cell component, like the present tumor, which shows dedifferentiation, should not be overlooked.

Measuring mitotic spindle dynamics in budding yeast

NASA Astrophysics Data System (ADS)

Plumb, Kemp

In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

Sequential activities of Dynein, Mud and Asp in centrosome-spindle coupling maintain centrosome number upon mitosis.

PubMed

Bosveld, Floris; Ainslie, Anna; Bellaïche, Yohanns

2017-10-15

Centrosomes nucleate microtubules and are tightly coupled to the bipolar spindle to ensure genome integrity, cell division orientation and centrosome segregation. While the mechanisms of centrosome-dependent microtubule nucleation and bipolar spindle assembly have been the focus of numerous works, less is known about the mechanisms ensuring the centrosome-spindle coupling. The conserved NuMA protein (Mud in Drosophila ) is best known for its role in spindle orientation. Here, we analyzed the role of Mud and two of its interactors, Asp and Dynein, in the regulation of centrosome numbers in Drosophila epithelial cells. We found that Dynein and Mud mainly initiate centrosome-spindle coupling prior to nuclear envelope breakdown (NEB) by promoting correct centrosome positioning or separation, while Asp acts largely independently of Dynein and Mud to maintain centrosome-spindle coupling. Failure in the centrosome-spindle coupling leads to mis-segregation of the two centrosomes into one daughter cell, resulting in cells with supernumerary centrosomes during subsequent divisions. Altogether, we propose that Dynein, Mud and Asp operate sequentially during the cell cycle to ensure efficient centrosome-spindle coupling in mitosis, thereby preventing centrosome mis-segregation to maintain centrosome number. © 2017. Published by The Company of Biologists Ltd.

Regulating positioning and orientation of mitotic spindles via cell size and shape

NASA Astrophysics Data System (ADS)

Li, Jingchen; Jiang, Hongyuan

2018-01-01

Proper location of the mitotic spindle is critical for chromosome segregation and the selection of the cell division plane. However, how mitotic spindles sense cell size and shape to regulate their own position and orientation is still largely unclear. To investigate this question systematically, we used a general model by considering chromosomes, microtubule dynamics, and forces of various molecular motors. Our results show that in cells of various sizes and shapes, spindles can always be centered and oriented along the long axis robustly in the absence of other specified mechanisms. We found that the characteristic time of positioning and orientation processes increases with cell size. Spindles sense the cell size mainly by the cortical force in small cells and by the cytoplasmic force in large cells. In addition to the cell size, the cell shape mainly influences the orientation process. We found that more slender cells have a faster orientation process, and the final orientation is not necessarily along the longest axis but is determined by the radial profile and the symmetry of the cell shape. Finally, our model also reproduces the separation and repositioning of the spindle poles during the anaphase. Therefore, our work provides a general tool for studying the mitotic spindle across the whole mitotic phase.

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring.

PubMed

Gregory, Stephen L; Ebrahimi, Saman; Milverton, Joanne; Jones, Whitney M; Bejsovec, Amy; Saint, Robert

2008-01-08

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

PubMed Central

Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-01-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

Glandular differentiation in dedifferentiated chondrosarcoma: molecular evidence of a rare phenomenon.

PubMed

Jour, George; Liu, Yajuan; Ricciotti, Robert; Pritchard, Colin; Hoch, Benjamin L

2015-09-01

Epithelial glandular differentiation in dedifferentiated chondrosarcoma has not been described. Our patient was a 64-year-old man with a history of prostate cancer status post-radiation and hormonal therapy. On screening bone scan, he was found to have increased uptake in his right femoral shaft. Biopsy revealed intermediate-grade conventional chondrosarcoma. Subsequent femoral resection was remarkable for an intermediate-grade chondrosarcomatous component juxtaposed to an area composed of anastomosing nests and cords of malignant epithelial cells showing nuclear atypia and increased mitotic activity. A fibroblastic-appearing spindle cell population was intimately associated with the epithelial cells. The epithelial cells labeled with 34bE12, AE1/AE3, EMA, and Vimentin (both spindled and epithelial components) while being negative for prostate-specific antigen, prostate specific acid phosphatase, cytokeratin 20, thyroid transcription factor-1, and CDX2. The patient developed local recurrence 9 months after the initial resection but has had no metastatic disease and consistently undetectable prostate-specific antigen levels. Deep parallel sequencing of the dedifferentiated component showed a nonsynonymous mutation at exon 4 of IDH1 gene at codon R132 leading to a substitution of arginine, with serine confirming glandular differentiation in dedifferentiated chondrosarcoma. Copyright © 2015 Elsevier Inc. All rights reserved.

NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

PubMed Central

Seldin, Lindsey; Muroyama, Andrew; Lechler, Terry

2016-01-01

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures. DOI: http://dx.doi.org/10.7554/eLife.12504.001 PMID:26765568

Mechanisms of plant spindle formation.

PubMed

Zhang, Han; Dawe, R Kelly

2011-04-01

In eukaryotes, the formation of a bipolar spindle is necessary for the equal segregation of chromosomes to daughter cells. Chromosomes, microtubules and kinetochores all contribute to spindle morphogenesis and have important roles during mitosis. A unique property of flowering plant cells is that they entirely lack centrosomes, which in animals have a major role in spindle formation. The absence of these important structures suggests that plants have evolved novel mechanisms to assure chromosome segregation. In this review, we highlight some of the recent studies on plant mitosis and argue that plants utilize a variation of "spindle self-organization" that takes advantage of the early polarity of plant cells and accentuates the role of kinetochores in stabilizing the spindle midzone in prometaphase.

Flattop regulates basal body docking and positioning in mono- and multiciliated cells

PubMed Central

Gegg, Moritz; Böttcher, Anika; Burtscher, Ingo; Hasenoeder, Stefan; Van Campenhout, Claude; Aichler, Michaela; Walch, Axel; Grant, Seth G N; Lickert, Heiko

2014-01-01

Planar cell polarity (PCP) regulates basal body (BB) docking and positioning during cilia formation, but the underlying mechanisms remain elusive. In this study, we investigate the uncharacterized gene Flattop (Fltp) that is transcriptionally activated during PCP acquisition in ciliated tissues. Fltp knock-out mice show BB docking and ciliogenesis defects in multiciliated lung cells. Furthermore, Fltp is necessary for kinocilium positioning in monociliated inner ear hair cells. In these cells, the core PCP molecule Dishevelled 2, the BB/spindle positioning protein Dlg3, and Fltp localize directly adjacent to the apical plasma membrane, physically interact and surround the BB at the interface of the microtubule and actin cytoskeleton. Dlg3 and Fltp knock-outs suggest that both cooperatively translate PCP cues for BB positioning in the inner ear. Taken together, the identification of novel BB/spindle positioning components as potential mediators of PCP signaling might have broader implications for other cell types, ciliary disease, and asymmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.03842.001 PMID:25296022

Inflammatory myofibroblastic tumors in two dogs.

PubMed

Knight, C; Fan, E; Riis, R; McDonough, S

2009-03-01

Two soft tissue masses from different locations in 2 dogs were submitted for histopathologic examination. Each was well demarcated and consisted of interweaving streams of bland spindle cells among which numerous plasma cells and lymphocytes were scattered. All the spindle cells reacted strongly to antibodies against vimentin and calponin, whereas a subset of the spindle cells expressed smooth muscle actin and desmin. Immunohistochemistry results were consistent with a myofibroblastic derivation for the spindle-cell population and the diagnosis of inflammatory myofibroblastic tumor (IMT) was made. This is the second report of IMT in the veterinary literature.

Mutations in CENPE define a novel kinetochore-centromeric mechanism for Microcephalic Primordial Dwarfism

PubMed Central

Mirzaa, Ghayda M.; Vitre, Benjamin; Carpenter, Gillian; Abramowicz, Iga; Gleeson, Joseph G.; Paciorkowski, Alex R.; Cleveland, Don W.; Dobyns, William B.; O’Driscoll, Mark

2015-01-01

Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of Primary Microcephaly (PM) and Microcephalic Primordial Dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organisation, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated Microcephalic Osteodysplastic Primordial Dwarfism type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans. PMID:24748105

Mutations in CENPE define a novel kinetochore-centromeric mechanism for microcephalic primordial dwarfism.

PubMed

Mirzaa, Ghayda M; Vitre, Benjamin; Carpenter, Gillian; Abramowicz, Iga; Gleeson, Joseph G; Paciorkowski, Alex R; Cleveland, Don W; Dobyns, William B; O'Driscoll, Mark

2014-08-01

Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of primary microcephaly (PM) and microcephalic primordial dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organization, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated microcephalic osteodysplastic primordial dwarfism-type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.

Automated High-Throughput Quantification of Mitotic Spindle Positioning from DIC Movies of Caenorhabditis Embryos

PubMed Central

Cluet, David; Spichty, Martin; Delattre, Marie

2014-01-01

The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye. PMID:24763198

Mechanical Forces Program the Orientation of Cell Division during Airway Tube Morphogenesis.

PubMed

Tang, Zan; Hu, Yucheng; Wang, Zheng; Jiang, Kewu; Zhan, Cheng; Marshall, Wallace F; Tang, Nan

2018-02-05

Oriented cell division plays a key role in controlling organogenesis. The mechanisms for regulating division orientation at the whole-organ level are only starting to become understood. By combining 3D time-lapse imaging, mouse genetics, and mathematical modeling, we find that global orientation of cell division is the result of a combination of two types of spindles with distinct spindle dynamic behaviors in the developing airway epithelium. Fixed spindles follow the classic long-axis rule and establish their division orientation before metaphase. In contrast, rotating spindles do not strictly follow the long-axis rule and determine their division orientation during metaphase. By using both a cell-based mechanical model and stretching-lung-explant experiments, we showed that mechanical force can function as a regulatory signal in maintaining the stable ratio between fixed spindles and rotating spindles. Our findings demonstrate that mechanical forces, cell geometry, and oriented cell division function together in a highly coordinated manner to ensure normal airway tube morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Automated mitotic spindle tracking suggests a link between spindle dynamics, spindle orientation, and anaphase onset in epithelial cells

PubMed Central

Larson, Matthew E.; Bement, William M.

2017-01-01

Proper spindle positioning at anaphase onset is essential for normal tissue organization and function. Here we develop automated spindle-tracking software and apply it to characterize mitotic spindle dynamics in the Xenopus laevis embryonic epithelium. We find that metaphase spindles first undergo a sustained rotation that brings them on-axis with their final orientation. This sustained rotation is followed by a set of striking stereotyped rotational oscillations that bring the spindle into near contact with the cortex and then move it rapidly away from the cortex. These oscillations begin to subside soon before anaphase onset. Metrics extracted from the automatically tracked spindles indicate that final spindle position is determined largely by cell morphology and that spindles consistently center themselves in the XY-plane before anaphase onset. Finally, analysis of the relationship between spindle oscillations and spindle position relative to the cortex reveals an association between cortical contact and anaphase onset. We conclude that metaphase spindles in epithelia engage in a stereotyped “dance,” that this dance culminates in proper spindle positioning and orientation, and that completion of the dance is linked to anaphase onset. PMID:28100633

The Centrosome and Its Duplication Cycle

PubMed Central

Fu, Jingyan; Hagan, Iain M.; Glover, David M.

2015-01-01

The centrosome was discovered in the late 19th century when mitosis was first described. Long recognized as a key organelle of the spindle pole, its core component, the centriole, was realized more than 50 or so years later also to comprise the basal body of the cilium. Here, we chart the more recent acquisition of a molecular understanding of centrosome structure and function. The strategies for gaining such knowledge were quickly developed in the yeasts to decipher the structure and function of their distinctive spindle pole bodies. Only within the past decade have studies with model eukaryotes and cultured cells brought a similar degree of sophistication to our understanding of the centrosome duplication cycle and the multiple roles of this organelle and its component parts in cell division and signaling. Now as we begin to understand these functions in the context of development, the way is being opened up for studies of the roles of centrosomes in human disease. PMID:25646378

Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation

PubMed Central

Markus, Steven M.; Lee, Wei-Lih

2011-01-01

During animal development, microtubules (MTs) play a major role in directing cellular and subcellular patterning, impacting cell polarization and subcellular organization, thereby affecting cell fate determination and tissue architecture. In particular, when progenitor cells divide asymmetrically along an anterior-posterior or apical-basal axis, MTs must coordinate the position of the mitotic spindle with the site of cell division to ensure normal distribution of cell fate determinants and equal sequestration of genetic material into the two daughter cells. Emerging data from diverse model systems have led to the prevailing view that, during mitotic spindle positioning, polarity cues at the cell cortex signal for the recruitment of NuMA and the minus-end directed MT motor cytoplasmic dynein.1 The NuMA/dynein complex is believed to connect, in turn, to the mitotic spindle via astral MTs, thus aligning and tethering the spindle, but how this connection is achieved faithfully is unclear. Do astral MTs need to search for and then capture cortical NuMA/dynein? How does dynein capture the astral MTs emanating from the correct spindle pole? Recently, using the classical model of asymmetric cell division—budding yeast S. cerevisiae—we successfully demonstrated that astral MTs assume an active role in cortical dynein targeting, in that astral MTs utilize their distal plus ends to deliver dynein to the daughter cell cortex, the site where dynein activity is needed to perform its spindle alignment function. This observation introduced the novel idea that, during mitotic spindle orientation processes, polarity cues at the cell cortex may actually signal to prime the cortical receptors for MT-dependent dynein delivery. This model is consistent with the observation that dynein/dynactin accumulate prominently at the astral MT plus ends during metaphase in a wide range of cultured mammalian cells. PMID:22754610

Unusual recurrent tongue spindle cell carcinoma with marked anaplasia occurring at the site of glossectomy for a well-differentiated squamous cell carcinoma: A case report.

PubMed

Okuyama, Kohei; Fujita, Shuichi; Yanamoto, Souichi; Naruse, Tomofumi; Sakamoto, Yuki; Kawakita, Akiko; Omori, Keisuke; Tsuchihashi, Hiroki; Umeda, Masahiro

2017-09-01

Spindle cell carcinoma (SpCC), which predominantly arises in the oral, pharyngeal and laryngeal mucosal tissues, is composed of a mixture of squamous and sarcomatoid components. The present study describes the case of a 62-year-old woman with SpCC recurrence 4 years after an initial surgery to remove a well-differentiated primary squamous cell carcinoma (SCC) of the tongue. The recurrent tumor was spherical and located deep within the tongue tissue, which differs from the typical manifestation of ulcerated masses of the mucosa. The majority of cases of recurrence involving SpCC are associated with radiotherapeutic treatment of the primary malignancy; however, the patient in the present study had not received postoperative radiotherapy for SCC. Furthermore, the recurrent tumor in the present case exhibited marked anaplasia and sarcomatoid features, and the absence of SCC elements upon biopsy rendered histological diagnosis difficult. In summary, the present findings suggest that immunohistochemical examination and identification of SCC components are essential for ensuring the accuracy of the histological diagnosis of recurrent SpCC following a primary epithelial malignancy.

Unusual recurrent tongue spindle cell carcinoma with marked anaplasia occurring at the site of glossectomy for a well-differentiated squamous cell carcinoma: A case report

PubMed Central

Okuyama, Kohei; Fujita, Shuichi; Yanamoto, Souichi; Naruse, Tomofumi; Sakamoto, Yuki; Kawakita, Akiko; Omori, Keisuke; Tsuchihashi, Hiroki; Umeda, Masahiro

2017-01-01

Spindle cell carcinoma (SpCC), which predominantly arises in the oral, pharyngeal and laryngeal mucosal tissues, is composed of a mixture of squamous and sarcomatoid components. The present study describes the case of a 62-year-old woman with SpCC recurrence 4 years after an initial surgery to remove a well-differentiated primary squamous cell carcinoma (SCC) of the tongue. The recurrent tumor was spherical and located deep within the tongue tissue, which differs from the typical manifestation of ulcerated masses of the mucosa. The majority of cases of recurrence involving SpCC are associated with radiotherapeutic treatment of the primary malignancy; however, the patient in the present study had not received postoperative radiotherapy for SCC. Furthermore, the recurrent tumor in the present case exhibited marked anaplasia and sarcomatoid features, and the absence of SCC elements upon biopsy rendered histological diagnosis difficult. In summary, the present findings suggest that immunohistochemical examination and identification of SCC components are essential for ensuring the accuracy of the histological diagnosis of recurrent SpCC following a primary epithelial malignancy. PMID:28781811

A Functional Relationship between NuMA and Kid Is Involved in Both Spindle Organization and Chromosome Alignment in Vertebrate CellsV⃞

PubMed Central

Levesque, Aime A.; Howard, Louisa; Gordon, Michael B.; Compton, Duane A.

2003-01-01

We examined spindle morphology and chromosome alignment in vertebrate cells after simultaneous perturbation of the chromokinesin Kid and either NuMA, CENP-E, or HSET. Spindle morphology and chromosome alignment after simultaneous perturbation of Kid and either HSET or CENP-E were no different from when either HSET or CENP-E was perturbed alone. However, short bipolar spindles with organized poles formed after perturbation of both Kid and NuMA in stark contrast to splayed spindle poles observed after perturbation of NuMA alone. Spindles were disorganized if Kid, NuMA, and HSET were perturbed, indicating that HSET is sufficient for spindle organization in the absence of Kid and NuMA function. In addition, chromosomes failed to align efficiently at the spindle equator after simultaneous perturbation of Kid and NuMA despite appropriate kinetochore-microtubule interactions that generated chromosome movement at normal velocities. These data indicate that a functional relationship between the chromokinesin Kid and the spindle pole organizing protein NuMA influences spindle morphology, and we propose that this occurs because NuMA forms functional linkages between kinetochore and nonkinetochore microtubules at spindle poles. In addition, these data show that both Kid and NuMA contribute to chromosome alignment in mammalian cells. PMID:12972545

A yeast gene essential for regulation of spindle pole duplication.

PubMed Central

Baum, P; Yip, C; Goetsch, L; Byers, B

1988-01-01

In eucaryotic cells, duplication of spindle poles must be coordinated with other cell cycle functions. We report here the identification in Saccharomyces cerevisiae of a temperature-sensitive lethal mutation, esp1, that deregulates spindle pole duplication. Mutant cells transferred to the nonpermissive temperature became unable to continue DNA synthesis and cell division but displayed repeated duplication of their spindle pole bodies. Although entry into this state after transient challenge by the nonpermissive temperature was largely lethal, rare survivors were recovered and found to have become increased in ploidy. If the mutant cells were held in G0 or G1 during exposure to the elevated temperature, they remained viable and maintained normal numbers of spindle poles. These results suggest dual regulation of spindle pole duplication, including a mechanism that promotes duplication as cells enter the division cycle and a negative regulatory mechanism, controlled by ESP1, that limits duplication to a single occurrence in each cell division cycle. Tetrad analysis has revealed that ESP1 resides at a previously undescribed locus on the right arm of chromosome VII. Images PMID:3072479

Intramedullary spindle cell hemangioma: case report.

PubMed

Nasser, Rani; Ashayeri, Kimberly; Legatt, Alan D; Houten, John K

2016-09-01

The authors describe the case of a 48-year-old man found to have the first reported intramedullary spinal cord spindle cell hemangioma. Previous research indicates that spindle cell hemangiomas are rarely found in the spine. Only 3 previous cases exist, all in the intradural, extramedullary space. In the present case, gross-total resection of the tumor was possible with no loss of function from baseline. This report presents the successful resection of the first reported intramedullary spindle cell hemangioma and reports 4-month follow-up, demonstrating the biological behavior of this rare tumor.

Human Nek7-interactor RGS2 is required for mitotic spindle organization.

PubMed

de Souza, Edmarcia Elisa; Hehnly, Heidi; Perez, Arina Marina; Meirelles, Gabriela Vaz; Smetana, Juliana Helena Costa; Doxsey, Stephen; Kobarg, Jörg

2015-01-01

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.

Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

PubMed

Tuder, R M; Karasek, M A; Bensch, K G

1990-02-01

The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces: Mechanics of mitotic spindle positioning.

PubMed

Howard, Jonathon; Garzon-Coral, Carlos

2017-11-01

Tissues are shaped and patterned by mechanical and chemical processes. A key mechanical process is the positioning of the mitotic spindle, which determines the size and location of the daughter cells within the tissue. Recent force and position-fluctuation measurements indicate that pushing forces, mediated by the polymerization of astral microtubules against- the cell cortex, maintain the mitotic spindle at the cell center in Caenorhabditis elegans embryos. The magnitude of the centering forces suggests that the physical limit on the accuracy and precision of this centering mechanism is determined by the number of pushing microtubules rather than by thermally driven fluctuations. In cells that divide asymmetrically, anti-centering, pulling forces generated by cortically located dyneins, in conjunction with microtubule depolymerization, oppose the pushing forces to drive spindle displacements away from the center. Thus, a balance of centering pushing forces and anti-centering pulling forces localize the mitotic spindles within dividing C. elegans cells. © 2017 The Authors. BioEssays published by Wiley Periodicals, Inc.

Warts phosphorylates Mud to promote Pins-mediated mitotic spindle orientation in Drosophila independent of Yorkie

PubMed Central

Dewey, Evan B.; Sanchez, Desiree; Johnston, Christopher A.

2015-01-01

SUMMARY Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily-conserved cell proliferation pathway. PMID:26592339

Warts phosphorylates mud to promote pins-mediated mitotic spindle orientation in Drosophila, independent of Yorkie.

PubMed

Dewey, Evan B; Sanchez, Desiree; Johnston, Christopher A

2015-11-02

Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here, we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily conserved cell proliferation pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

CENP-W Plays a Role in Maintaining Bipolar Spindle Structure

PubMed Central

Kaczmarczyk, Agnieszka; Sullivan, Kevin F.

2014-01-01

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. ‘Spindle free’ nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability. PMID:25329824

Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila male accessory gland cells.

PubMed

Taniguchi, Kiichiro; Kokuryo, Akihiko; Imano, Takao; Minami, Ryunosuke; Nakagoshi, Hideki; Adachi-Yamada, Takashi

2014-12-20

In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however, such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as the central spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them by characterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells are binucleate. The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middle of the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered the M-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape, spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of NuMA, regulated the processes responsible for these three features, the classical isoform Mud(PBD) and the two newly characterized isoforms Mud(L) and Mud(S) regulated them differently: Mud(L) repressed cell rounding, Mud(PBD) and Mud(S) oriented the spindle along the apico-basal axis, and Mud(S) and Mud(L) repressed central spindle assembly. Importantly, overexpression of Mud(S) induced binucleation even in standard proliferating cells such as those in imaginal discs. We characterized the binucleation in the Drosophila male accessory gland and examined mechanisms that regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by three structurally different isoforms of Mud regulate cell rounding, spindle orientation and central spindle assembly in binucleation. We also propose that Mud(S) is a key regulator triggering cytokinesis skipping in binucleation processes.

Inscuteable Regulates the Pins-Mud Spindle Orientation Pathway

PubMed Central

Mauser, Jonathon F.; Prehoda, Kenneth E.

2012-01-01

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud). PMID:22253744

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

PubMed Central

Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

2015-01-01

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.09384.001 PMID:26406118

Human Nek7-interactor RGS2 is required for mitotic spindle organization

PubMed Central

de Souza, Edmarcia Elisa; Hehnly, Heidi; Perez, Arina Marina; Meirelles, Gabriela Vaz; Smetana, Juliana Helena Costa; Doxsey, Stephen; Kobarg, Jörg

2015-01-01

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization. PMID:25664600

Arsenite inhibits mitotic division and perturbs spindle dynamics in HeLa S3 cells.

PubMed

Huang, S C; Lee, T C

1998-05-01

Arsenical compounds, known to be human carcinogens, were shown to disturb cell cycle progression and induce cytogenetic alterations in a variety of cell systems. We report here that a 24 h treatment of arsenite induced mitotic accumulation in human cell lines. HeLa S3 and KB cells were most susceptible: 35% of the total cell population was arrested at the mitotic stage after treatment with 5 microM sodium arsenite in HeLa S3 cells and after 10 microM in KB cells. Under a microscope, we observed abnormal mitotic figures in arsenite-arrested mitotic cells, including deranged chromosome congression, elongated polar distance of mitotic spindle, and enhanced microtubule immunofluorescence. The spindle microtubules of arsenite-arrested mitotic cells were more resistant to nocodazole-induced dissolution than those of control mitotic cells. According to turbidity assay, arsenite at concentrations below 100 microM significantly enhanced polymerization of tubulins. Since spindle dynamics play a crucial role in mitotic progression, our results suggest that arsenite-induced mitotic arrest may be due to arsenite's effects on attenuation of spindle dynamics.

Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes

PubMed Central

Hornick, Jessica E.; Mader, Christopher C.; Tribble, Emily K.; Bagne, Cydney C.; Vaughan, Kevin T.; Shaw, Sidney L.; Hinchcliffe, Edward H.

2011-01-01

Summary The role of centrosomes/centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1, 2, 3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays – termed an amphiaster (“a star on both sides” [4]) – that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC re-assembled, and prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split/separate before NEB, and these entered mitosis with persistent monastral spindles. The chromatin-mediated RAN-GTP pathway could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but in its absence, the fidelity of bipolar spindle assembly is highly compromised. PMID:21439826

The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

PubMed Central

Levesque, Aime A.; Compton, Duane A.

2001-01-01

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression. PMID:11564754

27 T ultra-high static magnetic field changes orientation and morphology of mitotic spindles in human cells

PubMed Central

Zhang, Lei; Hou, Yubin; Li, Zhiyuan; Ji, Xinmiao; Wang, Ze; Wang, Huizhen; Tian, Xiaofei; Yu, Fazhi; Yang, Zhenye; Pi, Li; Mitchison, Timothy J; Lu, Qingyou; Zhang, Xin

2017-01-01

Purified microtubules have been shown to align along the static magnetic field (SMF) in vitro because of their diamagnetic anisotropy. However, whether mitotic spindle in mammalian cells can be aligned by magnetic field has not been experimentally proved. In particular, the biological effects of SMF of above 20 T (Tesla) on mammalian cells have never been reported. Here we found that in both CNE-2Z and RPE1 human cells spindle orients in 27 T SMF. The direction of spindle alignment depended on the extent to which chromosomes were aligned to form a planar metaphase plate. Our results show that the magnetic torque acts on both microtubules and chromosomes, and the preferred direction of spindle alignment relative to the field depends more on chromosome alignment than microtubules. In addition, spindle morphology was also perturbed by 27 T SMF. This is the first reported study that investigated the mammalian cellular responses to ultra-high magnetic field of above 20 T. Our study not only found that ultra-high magnetic field can change the orientation and morphology of mitotic spindles, but also provided a tool to probe the role of spindle orientation and perturbation in developmental and cancer biology. DOI: http://dx.doi.org/10.7554/eLife.22911.001 PMID:28244368

SLK-dependent activation of ERMs controls LGN–NuMA localization and spindle orientation

PubMed Central

Machicoane, Mickael; de Frutos, Cristina A.; Fink, Jenny; Rocancourt, Murielle; Lombardi, Yannis; Garel, Sonia; Piel, Matthieu

2014-01-01

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane–actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN–NuMA force generator complexes and hence for proper spindle orientation. PMID:24958772

O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization*

PubMed Central

Tan, Ee Phie; Caro, Sarah; Potnis, Anish; Lanza, Christopher; Slawson, Chad

2013-01-01

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events. PMID:23946484

Parvalbumin-positive interneurons mediate neocortical-hippocampal interactions that are necessary for memory consolidation

PubMed Central

Xia, Frances; Richards, Blake A; Tran, Matthew M; Josselyn, Sheena A

2017-01-01

Following learning, increased coupling between spindle oscillations in the medial prefrontal cortex (mPFC) and ripple oscillations in the hippocampus is thought to underlie memory consolidation. However, whether learning-induced increases in ripple-spindle coupling are necessary for successful memory consolidation has not been tested directly. In order to decouple ripple-spindle oscillations, here we chemogenetically inhibited parvalbumin-positive (PV+) interneurons, since their activity is important for regulating the timing of spiking activity during oscillations. We found that contextual fear conditioning increased ripple-spindle coupling in mice. However, inhibition of PV+ cells in either CA1 or mPFC eliminated this learning-induced increase in ripple-spindle coupling without affecting ripple or spindle incidence. Consistent with the hypothesized importance of ripple-spindle coupling in memory consolidation, post-training inhibition of PV+ cells disrupted contextual fear memory consolidation. These results indicate that successful memory consolidation requires coherent hippocampal-neocortical communication mediated by PV+ cells. PMID:28960176

Sleep spindle and K-complex detection using tunable Q-factor wavelet transform and morphological component analysis

PubMed Central

Lajnef, Tarek; Chaibi, Sahbi; Eichenlaub, Jean-Baptiste; Ruby, Perrine M.; Aguera, Pierre-Emmanuel; Samet, Mounir; Kachouri, Abdennaceur; Jerbi, Karim

2015-01-01

A novel framework for joint detection of sleep spindles and K-complex events, two hallmarks of sleep stage S2, is proposed. Sleep electroencephalography (EEG) signals are split into oscillatory (spindles) and transient (K-complex) components. This decomposition is conveniently achieved by applying morphological component analysis (MCA) to a sparse representation of EEG segments obtained by the recently introduced discrete tunable Q-factor wavelet transform (TQWT). Tuning the Q-factor provides a convenient and elegant tool to naturally decompose the signal into an oscillatory and a transient component. The actual detection step relies on thresholding (i) the transient component to reveal K-complexes and (ii) the time-frequency representation of the oscillatory component to identify sleep spindles. Optimal thresholds are derived from ROC-like curves (sensitivity vs. FDR) on training sets and the performance of the method is assessed on test data sets. We assessed the performance of our method using full-night sleep EEG data we collected from 14 participants. In comparison to visual scoring (Expert 1), the proposed method detected spindles with a sensitivity of 83.18% and false discovery rate (FDR) of 39%, while K-complexes were detected with a sensitivity of 81.57% and an FDR of 29.54%. Similar performances were obtained when using a second expert as benchmark. In addition, when the TQWT and MCA steps were excluded from the pipeline the detection sensitivities dropped down to 70% for spindles and to 76.97% for K-complexes, while the FDR rose up to 43.62 and 49.09%, respectively. Finally, we also evaluated the performance of the proposed method on a set of publicly available sleep EEG recordings. Overall, the results we obtained suggest that the TQWT-MCA method may be a valuable alternative to existing spindle and K-complex detection methods. Paths for improvements and further validations with large-scale standard open-access benchmarking data sets are discussed. PMID:26283943

Mps1/TTK: a novel target and biomarker for cancer.

PubMed

Xie, Yuan; Wang, Anqiang; Lin, Jianzhen; Wu, Liangcai; Zhang, Haohai; Yang, Xiaobo; Wan, Xueshuai; Miao, Ruoyu; Sang, Xinting; Zhao, Haitao

2017-02-01

Monopolar spindle1 (Mps1, also known as TTK) is the core component of the spindle assembly checkpoint, which functions to ensure proper distribution of chromosomes to daughter cells. Mps1 is hardly detectable in normal organs except the testis and placenta. However, high levels of Mps1 are found in many types of human malignancies, including glioblastoma, thyroid carcinoma, breast cancer, and other cancers. Several Mps1 inhibitors can inhibit the proliferation of cancer cells and exhibit demonstrable survival benefits. Mps1 can be utilized as a new immunogenic epitope, which is able to induce potent cytotoxic T lymphocyte activity against cancer cells while sparing normal cells. Some clinical trials have validated its safety, immunogenicity and clinical response. Thus, Mps1 may be a novel target for cancer therapy. Mps1 is differentially expressed between normal and malignant tissues, indicating its potential as a molecular biomarker for diagnosis. Meanwhile, the discovery that it clearly correlates with recurrence and survival time suggests it may serve as an independent prognostic biomarker as well.

Dedifferentiated liposarcoma involving the spleen and splenic hilum: a report of a case with a rare growth pattern.

PubMed

Nishikawa, Gen; Minamiguchi, Sachiko; Hata, Hiroaki; Ogiso, Satoshi; Yamaguchi, Takashi; Otani, Tethushi; Ikai, Iwao

2015-01-01

We present a rare case of dedifferentiated liposarcoma confined to the spleen and splenic hilum. An 81-year-old man was referred to our hospital with a large asymptomatic splenic tumor. The patient underwent splenectomy, and the adipose tissue surrounding the splenic hilum was also resected. Microscopically, the tumor mainly consisted of high-grade spindle cells similar to those seen in undifferentiated pleomorphic liposarcoma. In the splenic hilum, scattered atypical cells were detected in the sclerosing component and adipose tissue. Immunohistochemically, both the spindle cells in the spleen and the atypical cells in the splenic hilum were positive for MDM2 and CDK4. The histopathologic diagnosis was dedifferentiated liposarcoma derived from an atypical lipomatous tumor/well-differentiated liposarcoma of the adipose tissue in the splenic hilum with extension into the spleen. Dedifferentiated liposarcoma in the spleen and splenic hilum should be considered as a differential diagnosis of splenic tumors.

Spinal spindle cell haemangioma: an atypical location.

PubMed

Talan-Hranilović, J; Vucić, M; Sajko, T; Bedek, D; Tomić, K; Lupret, V

2007-03-01

We present a case of the 31-year-old male patient who complained of weakness in both legs and progressed slowly. Neuroimagine of the thoracic spine showed an intraspinal, extradural mass lesion, measuring 5.3 x 1.2 cm at the Th1-Th3 level. Histologically the lesion was a spindle cell haemangioma composed of dilated vascular spaces and a proliferation of bland appearing interspersed spindle cells. Immunohistochemical analysis was diffusely positive for VIM, SMA and focally for CD34. This lesion is uncommon and shows a predilection for distal extremities. Spindle cell haemangioma within the spine has not been previously reported in the literature.

Microtubule-dependent regulation of mitotic protein degradation

PubMed Central

Song, Ling; Craney, Allison; Rape, Michael

2014-01-01

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex. Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C-activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C-substrates provides a mechanism for the selective disposal of cell cycle regulators that have fulfilled their mitotic roles. PMID:24462202

The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

PubMed Central

Mountain, Vicki; Simerly, Calvin; Howard, Louisa; Ando, Asako; Schatten, Gerald; Compton, Duane A.

1999-01-01

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes. PMID:10525540

Analysis of metaplastic breast carcinoma: FNAC; histopathology and immunohistochemistry are complementary for diagnosis.

PubMed

Abd El Hafez, Amal; Shawky, Abd El-Aty

2013-01-01

Metaplastic breast carcinoma (MBC) is a rare malignancy comprised of ductal, squamous and/or mesenchymal elements with problematic diagnosis. This study analyses MBC identifying its cytologic and histologic features and emphasizing the combined role of FNAC, histopathology and immunohistochemistry (IHC) in its diagnosis. Cytology and histopathology files search yielded 21 cases identified as MBC from January 2005 to December 2010. FNAC and the histopathology slides were re-examined for the presence and frequency of various elements. Cytological and histopathological diagnoses were made and the cases subtyped according to WHO classification. Cytokeratin and vimenten IHC were used to confirm diagnosis when required. On FNAC, 52.4% were diagnosed as malignant, 9.5% as suspicious for malignancy and 38.1% as benign lesions. Most frequent cytologic findings were squamous and spindle cell elements (52.4% each). Histopathology revealed 76.2% pure epithelial tumors and 23.8% mixed epithelial-mesenchymal tumors. Squamous cell carcinoma was the most frequent histological subtype (33.3%). Carcinosarcomas were dimorphic on IHC& spindle cell carcinomas were positive for both cytokeratin and vimentin. Presence of dual components, squamous, spindle elements, mesenchymal fragments and necrosis in moderate to high cellularity breast FNAC provides clues for the diagnosis of MBC. FNAC; histopathology and IHC complement for diagnosis.

AIRE is a critical spindle-associated protein in embryonic stem cells

PubMed Central

Gu, Bin; Lambert, Jean-Philippe; Cockburn, Katie; Gingras, Anne-Claude; Rossant, Janet

2017-01-01

Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator (Aire), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that Aire was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE’s mitotic function. Combined maternal and zygotic knockout further revealed Aire’s critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for Aire and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying Aire mutations. DOI: http://dx.doi.org/10.7554/eLife.28131.001 PMID:28742026

Cellular angiofibroma with atypia or sarcomatous transformation: clinicopathologic analysis of 13 cases.

PubMed

Chen, Eleanor; Fletcher, Christopher D M

2010-05-01

Cellular angiofibroma is a mesenchymal neoplasm that is characterized by a bland spindle cell component, morphologically reminiscent of spindle cell lipoma, and thick-walled vessels. The tumor occurs equally in men and women and usually arises in the inguino-scrotal or vulvovaginal regions. An earlier study of 51 cases from our group showed that the tumor follows a benign course without any tendency for recurrence. In 1 case, an intralesional microscopic nodule of pleomorphic liposarcoma was observed. The biologic significance of atypia or sarcomatous transformation in cellular angiofibroma remains uncertain. In this study, we characterized clinicopathologic features in 13 cases of cellular angiofibroma with morphologic atypia or sarcomatous transformation. Thirteen cases with atypia or sarcomatous transformation among 154 usual cellular angiofibromas identified between 1993 and 2009 were retrieved from consultation files. There were 12 females and 1 male ranging in age from 39 to 71 years (median age, 46 y). Tumor size ranged from 1.2 to 7.5 cm. In 11 cases, the tumors occurred in the vulva. One case each occurred in the paratesticular and hip regions. Most tumors were located in subcutaneous tissue. There were 4 cases of cellular angiofibroma with atypia. Three showed severely atypical cells as scattered foci within the cellular angiofibroma. One case showed a discrete nodule of atypical cells. There were 9 cases of cellular angiofibroma with morphologic features of sarcomatous transformation. In each case, abrupt transition to a discrete sarcomatous component was seen. Of these 9 cases, the sarcomatous component in 2 cases showed features of pleomorphic liposarcoma with multivacuolated lipoblasts readily identified. Three of these 9 cases showed discrete nodule(s) closely resembling atypical lipomatous tumor within usual cellular angiofibroma. In the remaining 4 cases, the sarcomatous component was composed of pleomorphic spindle cells arranged in various patterns. By immunohistochemistry, atypical cells and sarcomatous areas showed either multifocal or more diffuse p16 expression compared with either scattered or negative expression in the conventional cellular angiofibroma. The 3 cases with atypical lipomatous tumor-like areas were negative for MDM-2 and CDK4. Follow-up information was available for 7 patients (range from 2 to 75 mo; median: 14 mo). Six patients did not develop recurrence or metastasis. One patient died of metastatic carcinoma of unknown primary site 27 months after the diagnosis of cellular angiofibroma with sarcomatous transformation. Cellular angiofibroma with atypia or morphologic sarcomatous transformation occurs predominantly in the subcutaneous tissue of the vulva and, as yet, shows no evident tendency to recur based on limited clinical follow-up available for 7 cases. The sarcomatous component can show variable features including atypical lipomatous tumor, pleomorphic liposarcoma, and pleomorphic sarcoma NOS. Overexpression of p16 in the atypical cells and sarcomatous component suggests a possible underlying molecular mechanism.

Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bala, Shashi; Kumar, Ajay; Soni, Shivani

2006-04-21

Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched withmore » the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.« less

Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

PubMed

Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit

2006-04-21

Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

Self-Organization and Forces in the Mitotic Spindle.

PubMed

Pavin, Nenad; Tolić, Iva M

2016-07-05

At the onset of division, the cell forms a spindle, a precise self-constructed micromachine composed of microtubules and the associated proteins, which divides the chromosomes between the two nascent daughter cells. The spindle arises from self-organization of microtubules and chromosomes, whose different types of motion help them explore the space and eventually approach and interact with each other. Once the interactions between the chromosomes and the microtubules have been established, the chromosomes are moved to the equatorial plane of the spindle and ultimately toward the opposite spindle poles. These transport processes rely on directed forces that are precisely regulated in space and time. In this review, we discuss how microtubule dynamics and their rotational movement drive spindle self-organization, as well as how the forces acting in the spindle are generated, balanced, and regulated.

Microtubule dynamics in cell division: exploring living cells with polarized light microscopy.

PubMed

Inoué, Shinya

2008-01-01

This Perspective is an account of my early experience while I studied the dynamic organization and behavior of the mitotic spindle and its submicroscopic filaments using polarized light microscopy. The birefringence of spindle filaments in normally dividing plant and animal cells, and those treated by various agents, revealed (a) the reality of spindle fibers and fibrils in healthy living cells; (b) the labile, dynamic nature of the molecular filaments making up the spindle fibers; (c) the mode of fibrogenesis and action of orienting centers; and (d) force-generating properties based on the disassembly and assembly of the fibrils. These studies, which were carried out directly on living cells using improved polarizing microscopes, in fact predicted the reversible assembly properties of microtubules.

Overexpression of Mps1 in colon cancer cells attenuates the spindle assembly checkpoint and increases aneuploidy.

PubMed

Ling, Youguo; Zhang, Xiaojuan; Bai, Yuanyuan; Li, Ping; Wei, Congwen; Song, Ting; Zheng, Zirui; Guan, Kai; Zhang, Yanhong; Zhang, Buchang; Liu, Xuedong; Ma, Runlin Z; Cao, Cheng; Zhong, Hui; Xu, Quanbin

2014-08-08

The spindle assembly checkpoint kinase Mps1 is highly expressed in several types of cancers, but its cellular involvement in tumorigenesis is less defined. Herein, we confirm that Mps1 is overexpressed in colon cancer tissues. Further, we find that forced expression of Mps1 in the colon cancer cell line SW480 enables cells to become resistant to both Mps1 inhibition-induced checkpoint depletion and cell death. Overexpression of Mps1 also increases genome instability in tumor cells owing to a weakened spindle assembly checkpoint. Collectively, our findings suggest that high levels of Mps1 contribute to tumorigenesis by attenuating the spindle assembly checkpoint. Copyright © 2014 Elsevier Inc. All rights reserved.

Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152

PubMed Central

Katta, Santharam S.; Chen, Jingjing; Gardner, Jennifer M.; Friederichs, Jennifer M.; Smith, Sarah E.; Gogol, Madelaine; Unruh, Jay R.; Slaughter, Brian D.; Jaspersen, Sue L.

2015-01-01

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication. PMID:26510791

F-actin mechanics control spindle centring in the mouse zygote

NASA Astrophysics Data System (ADS)

Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

2016-01-01

Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

PubMed Central

Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W.; Wainman, Alan

2015-01-01

Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs. PMID:26020779

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses

PubMed Central

2014-01-01

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels. PMID:24479647

A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

PubMed

Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi

2016-01-01

Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

Mathematical modeling and numerical simulation of the mitotic spindle orientation system.

PubMed

Ibrahim, Bashar

2018-05-21

The mitotic spindle orientation and position is crucial for the fidelity of chromosome segregation during asymmetric cell division to generate daughter cells with different sizes or fates. This mechanism is best understood in the budding yeast Saccharomyces cerevisiae, named the spindle position checkpoint (SPOC). The SPOC inhibits cells from exiting mitosis until the mitotic spindle is properly oriented along the mother-daughter polarity axis. Despite many experimental studies, the mechanisms underlying SPOC regulation remains elusive and unexplored theoretically. Here, a minimal mathematical is developed to describe SPOC activation and silencing having autocatalytic feedback-loop. Numerical simulations of the nonlinear ordinary differential equations (ODEs) model accurately reproduce the phenotype of SPOC mechanism. Bifurcation analysis of the nonlinear ODEs reveals the orientation dependency on spindle pole bodies, and how this dependence is altered by parameter values. These results provide for systems understanding on the molecular organization of spindle orientation system via mathematical modeling. The presented mathematical model is easy to understand and, within the above mentioned context, can be used as a base for further development of quantitative models in asymmetric cell-division. Copyright © 2018. Published by Elsevier Inc.

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and CytokinesisD⃞V⃞

PubMed Central

Chen, Qian; Li, Hui; De Lozanne, Arturo

2006-01-01

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring. PMID:16339076

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

PubMed

Kim, Jiyoung; Kim, Yeon-Soo

2018-06-06

Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

Spindle Cell Rhabdomyosarcoma of Bone with FUS-TFCP2 Fusion: Confirmation of a Very Recently Described Rhabdomyosarcoma Subtype.

PubMed

Dashti, Nooshin K; Wehrs, Rebecca N; Thomas, Brittany C; Nair, Asha; Davila, Jaime; Buckner, Jan C; Martinez, Anthony P; Sukov, William R; Halling, Kevin C; Howe, Benjamin M; Folpe, Andrew L

2018-05-14

Rhabdomyosarcomas of bone are extremely rare, with fewer than 10 reported cases. A very rare subtype of spindle cell/sclerosing rhabdomyosarcoma harboring a FUS-TFCP2 fusion and involving both soft tissue and bone locations has very recently been reported. We report only the fourth case of this unusual, clinically aggressive rhabdomyosarcoma. A previously-well 72-year old male presented with a destructive lesion of the mandible. Morphological and immunohistochemical study of a needle biopsy and the subsequent resection showed a spindle cell rhabdomyosarcoma. RNA-seq, RT-PCR and FISH confirmed the presence of the FUS-TFCP2 fusion. Spindle cell rhabdomyosarcomas carrying the FUS-TFCP2 fusion are very rare rhabdomyosarcoma variants with osseous predilection. The classification and differential diagnosis of this unusual molecular variant of spindle cell/ sclerosing rhabdomyosarcoma are discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Pericentromere tension is self-regulated by spindle structure in metaphase

PubMed Central

Chacón, Jeremy M.; Mukherjee, Soumya; Schuster, Breanna M.; Clarke, Duncan J.

2014-01-01

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4–6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted. PMID:24821839

Pericentromere tension is self-regulated by spindle structure in metaphase.

PubMed

Chacón, Jeremy M; Mukherjee, Soumya; Schuster, Breanna M; Clarke, Duncan J; Gardner, Melissa K

2014-05-12

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4-6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted.

CENTROSOMES AND MICROTUBULES DURING MEIOSIS IN THE MUSHROOM BOLETUS RUBINELLUS

PubMed Central

McLaughlin, David J.

1971-01-01

The double centrosome in the basidium of Boletus rubinellus has been observed in three planes with the electron microscope at interphase preceding nuclear fusion, at prophase I, and at interphase I. It is composed of two components connected by a band-shaped middle part. At anaphase I a single, enlarged centrosome is found at the spindle pole, which is attached to the cell membrane. Microtubules mainly oriented parallel to the longitudinal axis of the basidium are present at prefusion, prophase I and interphase I. Cytoplasmic microtubules are absent when the spindle is present. The relationship of the centrosome in B. rubinellus to that in other organisms and the role of the cytoplasmic microtubules are discussed. PMID:4329156

The spindle protein CHICA mediates localization of the chromokinesin Kid to the mitotic spindle.

PubMed

Santamaria, Anna; Nagel, Susanna; Sillje, Herman H W; Nigg, Erich A

2008-05-20

Microtubule-based motor proteins provide essential forces for bipolar organization of spindle microtubules and chromosome movement, prerequisites of chromosome segregation during the cell cycle. Here, we describe the functional characterization of a novel spindle protein, termed "CHICA," that was originally identified in a proteomic survey of the human spindle apparatus [1]. We show that CHICA localizes to the mitotic spindle and is both upregulated and phosphorylated during mitosis. CHICA-depleted cells form shorter spindles and fail to organize a proper metaphase plate, highly reminiscent of the phenotype observed upon depletion of the chromokinesin Kid, a key mediator of polar ejection forces [2-6]. We further show that CHICA coimmunoprecipitates with Kid and is required for the spindle localization of Kid without affecting its chromosome association. Moreover, upon depletion of either CHICA or Kid (or both proteins simultaneously), chromosomes collapse onto the poles of monastrol-induced monopolar spindles. We conclude that CHICA represents a novel interaction partner of the chromokinesin Kid that is required for the generation of polar ejection forces and chromosome congression.

Intra-spindle Microtubule Assembly Regulates Clustering of Microtubule-Organizing Centers during Early Mouse Development.

PubMed

Watanabe, Sadanori; Shioi, Go; Furuta, Yasuhide; Goshima, Gohta

2016-04-05

Errors during cell division in oocytes and early embryos are linked to birth defects in mammals. Bipolar spindle assembly in early mouse embryos is unique in that three or more acentriolar microtubule-organizing centers (MTOCs) are initially formed and are then clustered into two spindle poles. Using a knockout mouse and live imaging of spindles in embryos, we demonstrate that MTOC clustering during the blastocyst stage requires augmin, a critical complex for MT-dependent MT nucleation within the spindle. Functional analyses in cultured cells with artificially increased numbers of centrosomes indicate that the lack of intra-spindle MT nucleation, but not loss of augmin per se or overall reduction of spindle MTs, is the cause of clustering failure. These data suggest that onset of mitosis with three or more MTOCs is turned into a typical bipolar division through augmin-dependent intra-spindle MT assembly. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Multipolar Spindle Pole Coalescence Is a Major Source of Kinetochore Mis-Attachment and Chromosome Mis-Segregation in Cancer Cells

PubMed Central

Silkworth, William T.; Nardi, Isaac K.; Scholl, Lindsey M.; Cimini, Daniela

2009-01-01

Many cancer cells display a CIN (Chromosome Instability) phenotype, by which they exhibit high rates of chromosome loss or gain at each cell cycle. Over the years, a number of different mechanisms, including mitotic spindle multipolarity, cytokinesis failure, and merotelic kinetochore orientation, have been proposed as causes of CIN. However, a comprehensive theory of how CIN is perpetuated is still lacking. We used CIN colorectal cancer cells as a model system to investigate the possible cellular mechanism(s) underlying CIN. We found that CIN cells frequently assembled multipolar spindles in early mitosis. However, multipolar anaphase cells were very rare, and live-cell experiments showed that almost all CIN cells divided in a bipolar fashion. Moreover, fixed-cell analysis showed high frequencies of merotelically attached lagging chromosomes in bipolar anaphase CIN cells, and higher frequencies of merotelic attachments in multipolar vs. bipolar prometaphases. Finally, we found that multipolar CIN prometaphases typically possessed γ-tubulin at all spindle poles, and that a significant fraction of bipolar metaphase/early anaphase CIN cells possessed more than one centrosome at a single spindle pole. Taken together, our data suggest a model by which merotelic kinetochore attachments can easily be established in multipolar prometaphases. Most of these multipolar prometaphase cells would then bi-polarize before anaphase onset, and the residual merotelic attachments would produce chromosome mis-segregation due to anaphase lagging chromosomes. We propose this spindle pole coalescence mechanism as a major contributor to chromosome instability in cancer cells. PMID:19668340

Response to apatinib in chemotherapy-failed advanced spindle cell breast carcinoma.

PubMed

Zhou, Na; Liu, Congmin; Hou, Helei; Zhang, Chuantao; Liu, Dong; Wang, Guanqun; Liu, Kewei; Zhu, Jingjuan; Lv, Hongying; Li, Tianjun; Zhang, Xiaochun

2016-11-01

Spindle cell carcinoma of the breast is a rare subtype of metaplastic carcinoma, and no effective chemotherapy special for metaplastic carcinoma exists until now. As spindle cell carcinomas of the breast are typically "Triple Negative", endocrine therapy and molecular therapy targeted to Her2 might not be favorable, resulting in poor prognosis. Apatinib is currently being tested in patients with breast or lung cancers. Here we report a successful case using Apatinib to treat spindle cell carcinoma of breast.A 52- year- old woman presented with a gradually enlarged lump in left breast, which was revealed to be a triple-negative spindle cell carcinoma, underwent a modified radical mastectomy. After the first line chemotherapy with Cyclophosphamide and Epirubicin, multiple metastases in bilateral lung and left anterior thoracic wall appeared. After disease progressed with therapy of Bevacizumab combined with Albumin-bound Paclitaxel and Cisplatin, we treated the patient with Apatinib according to her VEGFR expression, which showed nearly complete response and controllable and tolerated side effects. Next-generation sequencing analysis of the tumor specimen and real time ctDNA was performed to observe the mutated gene numbers matched with therapeutic effect. The present case can help to provide a new and effective therapy strategy to treat advanced spindle cell carcinoma.

Response to apatinib in chemotherapy-failed advanced spindle cell breast carcinoma

PubMed Central

Zhou, Na; Liu, Congmin; Hou, Helei; Zhang, Chuantao; Liu, Dong; Wang, Guanqun; Liu, Kewei; Zhu, Jingjuan; Lv, Hongying; Li, Tianjun; Zhang, Xiaochun

2016-01-01

Spindle cell carcinoma of the breast is a rare subtype of metaplastic carcinoma, and no effective chemotherapy special for metaplastic carcinoma exists until now. As spindle cell carcinomas of the breast are typically “Triple Negative”, endocrine therapy and molecular therapy targeted to Her2 might not be favorable, resulting in poor prognosis. Apatinib is currently being tested in patients with breast or lung cancers. Here we report a successful case using Apatinib to treat spindle cell carcinoma of breast. A 52- year- old woman presented with a gradually enlarged lump in left breast, which was revealed to be a triple-negative spindle cell carcinoma, underwent a modified radical mastectomy. After the first line chemotherapy with Cyclophosphamide and Epirubicin, multiple metastases in bilateral lung and left anterior thoracic wall appeared. After disease progressed with therapy of Bevacizumab combined with Albumin-bound Paclitaxel and Cisplatin, we treated the patient with Apatinib according to her VEGFR expression, which showed nearly complete response and controllable and tolerated side effects. Next-generation sequencing analysis of the tumor specimen and real time ctDNA was performed to observe the mutated gene numbers matched with therapeutic effect. The present case can help to provide a new and effective therapy strategy to treat advanced spindle cell carcinoma. PMID:27738308

Spatial signals link exit from mitosis to spindle position.

PubMed

Falk, Jill Elaine; Tsuchiya, Dai; Verdaasdonk, Jolien; Lacefield, Soni; Bloom, Kerry; Amon, Angelika

2016-05-11

In budding yeast, if the spindle becomes mispositioned, cells prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN is a signaling cascade that localizes to spindle pole bodies (SPBs) and activates the phosphatase Cdc14. There are two competing models that explain MEN regulation by spindle position. In the 'zone model', exit from mitosis occurs when a MEN-bearing SPB enters the bud. The 'cMT-bud neck model' posits that cytoplasmic microtubule (cMT)-bud neck interactions prevent MEN activity. Here we find that 1) eliminating cMT- bud neck interactions does not trigger exit from mitosis and 2) loss of these interactions does not precede Cdc14 activation. Furthermore, using binucleate cells, we show that exit from mitosis occurs when one SPB enters the bud despite the presence of a mispositioned spindle. We conclude that exit from mitosis is triggered by a correctly positioned spindle rather than inhibited by improper spindle position.

MAD2 expression in oral squamous cell carcinoma and its relationship to tumor grade and proliferation.

PubMed

Rizzardi, Clara; Torelli, Lucio; Schneider, Manuela; Giudici, Fabiola; Zandona, Lorenzo; Biasotto, Matteo; Di Lenarda, Roberto; Melato, Mauro

2014-12-01

Defects in the cell-cycle surveillance mechanism, called the spindle checkpoint, might contribute to the chromosomal instability observed in human cancers, including oral squamous cell carcinoma. MAD2 and BUBR1 are key components of the spindle checkpoint, whose role in oral carcinogenesis and clinical relevance still need to be elucidated. We analyzed the expression of MAD2 in 49 cases of oral squamous cell carcinoma by immunohistochemistry and compared the findings with clinicopathological parameters, proliferative activity, BUBR1 expression and DNA ploidy. MAD2 was over-expressed in 18 (36.7%) cases. Tumors with over-expression of MAD2 were associated with the progression of histological grade from well to poor differentiation (p<0.001), the extent of lymph nodes involvement (PN) (p=0.0339) and Ki-67 labeling index (p<0.001). MAD2 may be involved in oral carcinogenesis and may represent an important prognostic factor associated with a more malignant phenotype of oral squamous cell carcinoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Squamous cell carcinoma with sarcomatous stroma in the nasal cavity of a dog.

PubMed

Bosward, K L; Kessell, A E; Lucy, R J

2004-09-01

This is a report of an unusual squamous cell carcinoma in the nasal cavity of a dog. A 13-year-old Golden Retriever was presented with a unilateral nasal and ocular discharge. Although a nasal tumour was suspected, initial diagnostic investigations were unrewarding, and, with worsening clinical signs, the dog was euthanatized. Necropsy examination confirmed the presence of a nasal tumour that was composed histologically of both a well-differentiated squamous cell carcinoma component blending with a predominant spindle cell component. Immunohistochemical staining with anti-human keratin/cytokeratin (AE1/AE3, CAM 5.2 and broad spectrum cytokeratin), Vimentin, Desmin, smooth muscle actin and S-100 protein supported a diagnosis of a squamous cell carcinoma with (pseudo) sarcomatous stroma.

A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞

PubMed Central

Paluh, Janet L.; Nogales, Eva; Oakley, Berl R.; McDonald, Kent; Pidoux, Alison L.; Cande, W. Z.

2000-01-01

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component. PMID:10749926

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo

PubMed Central

1986-01-01

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self- reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes. PMID:3941154

The MAPK Signaling Cascade is a Central Hub in the Regulation of Cell Cycle, Apoptosis and Cytoskeleton Remodeling by Tripeptidyl-Peptidase II

PubMed Central

Sompallae, Ramakrishna; Stavropoulou, Vaia; Houde, Mathieu; Masucci, Maria G.

2008-01-01

Tripeptidyl-peptidase II (TPPII) is a serine peptidase highly expressed in malignant Burkitt’s lymphoma cells (BL). We have previously shown that overexpression of TPPII correlates with chromosomal instability, centrosomal and mitotic spindle abnormalities and resistance to apoptosis induced by spindle poisons. Furthermore, TPPII knockdown by RNAi was associated with endoreplication and the accumulation of polynucleated cells that failed to complete cell division, indicating a role of TPPII in the cell cycle. Here we have applied a global approach of gene expression analysis to gain insights on the mechanism by which TPPII regulates this phenotype. mRNA profiling of control and TPPII knockdown BL cells identified one hundred and eighty five differentially expressed genes. Functional categorization of these genes highlighted major physiological functions such as apoptosis, cell cycle progression, cytoskeleton remodeling, proteolysis, and signal transduction. Pathways and protein interactome analysis revealed a significant enrichment in components of MAP kinases signaling. These findings suggest that TPPII influences a wide network of signaling pathways that are regulated by MAPKs and exerts thereby a pleiotropic effect on biological processes associated with cell survival, proliferation and genomic instability. PMID:19787088

Dedifferentiated liposarcoma of thigh with chondrosarcomatous dedifferentiated component.

PubMed

Yoon, Richard S; Benevenia, Joseph; Beebe, Kathleen S; Hameed, Meera

2010-11-01

Liposarcomas are common soft-issue sarcomas arising predominantly in deep soft tissue and the retroperitoneum with varied mortality and recurrence rates, largely dependent on histologic type. Thought to arise de novo, liposarcomas typically are classified into 5 types based on strict morphologic characteristics: well-differentiated, dedifferentiated, myxoid, round cell, and pleomorphic. More specifically, dedifferentiated liposarcoma, a common type most prevalent in the retroperitoneum, often has 2 distinct components, a well-differentiated lipomatous component and a dedifferentiated nonlipomatous component composed of sarcomas, such as myxofibrosarcomas or other spindle-cell sarcomas. Morphology typically ranges from low- to high-grade components, most commonly exhibiting myxofibrosarcoma and malignant fibrous histiocytoma components. However, the case reported in this article is unique-the dedifferentiated component exhibited only chondrosarcomatous differentiation-and it is, to our knowledge, the first such case to be described.

Exclusive destruction of mitotic spindles in human cancer cells.

PubMed

Visochek, Leonid; Castiel, Asher; Mittelman, Leonid; Elkin, Michael; Atias, Dikla; Golan, Talia; Izraeli, Shai; Peretz, Tamar; Cohen-Armon, Malka

2017-03-28

We identified target proteins modified by phenanthrenes that cause exclusive eradication of human cancer cells. The cytotoxic activity of the phenanthrenes in a variety of human cancer cells is attributed by these findings to post translational modifications of NuMA and kinesins HSET/kifC1 and kif18A. Their activity prevented the binding of NuMA to α-tubulin and kinesins in human cancer cells, and caused aberrant spindles. The most efficient cytotoxic activity of the phenanthridine PJ34, caused significantly smaller aberrant spindles with disrupted spindle poles and scattered extra-centrosomes and chromosomes. Concomitantly, PJ34 induced tumor growth arrest of human malignant tumors developed in athymic nude mice, indicating the relevance of its activity for cancer therapy.

Metastatic breast carcinomas display genomic and transcriptomic heterogeneity

PubMed Central

Weigelt, Britta; Ng, Charlotte KY; Shen, Ronglai; Popova, Tatiana; Schizas, Michail; Natrajan, Rachael; Mariani, Odette; Stern, Marc-Henri; Norton, Larry; Vincent-Salomon, Anne; Reis-Filho, Jorge S

2015-01-01

Metaplastic breast carcinoma is a rare and aggressive histologic type of breast cancer, preferentially displaying a triple-negative phenotype. We sought to define the transcriptomic heterogeneity of metaplastic breast cancers on the basis of current gene expression microarray-based classifiers, and to determine whether these tumors display gene copy number profiles consistent with those of BRCA1-associated breast cancers. Twenty-eight consecutive triple-negative metaplastic breast carcinomas were reviewed, and the metaplastic component present in each frozen specimen was defined (ie, spindle cell, squamous, chondroid metaplasia). RNA and DNA extracted from frozen sections with tumor cell content >60% were subjected to gene expression (Illumina HumanHT-12 v4) and copy number profiling (Affymetrix SNP 6.0), respectively. Using the best practice PAM50/claudin-low microarray-based classifier, all metaplastic breast carcinomas with spindle cell metaplasia were of claudin-low subtype, whereas those with squamous or chondroid metaplasia were preferentially of basal-like subtype. Triple-negative breast cancer subtyping using a dedicated website (http://cbc.mc.vanderbilt.edu/tnbc/) revealed that all metaplastic breast carcinomas with chondroid metaplasia were of mesenchymal-like subtype, spindle cell carcinomas preferentially of unstable or mesenchymal stem-like subtype, and those with squamous metaplasia were of multiple subtypes. None of the cases was classified as immunomodulatory or luminal androgen receptor subtype. Integrative clustering, combining gene expression and gene copy number data, revealed that metaplastic breast carcinomas with spindle cell and chondroid metaplasia were preferentially classified as of integrative clusters 4 and 9, respectively, whereas those with squamous metaplasia were classified into six different clusters. Eight of the 26 metaplastic breast cancers subjected to SNP6 analysis were classified as BRCA1-like. The diversity of histologic features of metaplastic breast carcinomas is reflected at the transcriptomic level, and an association between molecular subtypes and histology was observed. BRCA1-like genomic profiles were found only in a subset (31%) of metaplastic breast cancers, and were not associated with a specific molecular or histologic subtype. PMID:25412848

Very High Load Capacity Air Bearing Spindle for Large Diamond Turning Machines

DTIC Science & Technology

2010-06-08

testing and a surplus air bearing rotary table has been located. A prototype spindle has been designed to work with the table. 15. SUBJECT TERMS...MSFC) • PROTOTYPE SPINDLE DESIGN June 8, 2010Mirror Technology Workshop 3 Introduction • DT is a proven method of manufacturing aspheric off-axis... designed to hold in a strain-free condition. This spindle development is aimed at producing 3 meter diameter components. This requirement results in the

Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells.

PubMed

von Schubert, Conrad; Cubizolles, Fabien; Bracher, Jasmine M; Sliedrecht, Tale; Kops, Geert J P L; Nigg, Erich A

2015-07-07

Equal mitotic chromosome segregation is critical for genome integrity and is monitored by the spindle assembly checkpoint (SAC). We have previously shown that the consensus phosphorylation motif of the essential SAC kinase Monopolar spindle 1 (Mps1) is very similar to that of Polo-like kinase 1 (Plk1). This prompted us to ask whether human Plk1 cooperates with Mps1 in SAC signaling. Here, we demonstrate that Plk1 promotes checkpoint signaling at kinetochores through the phosphorylation of at least two Mps1 substrates, including KNL-1 and Mps1 itself. As a result, Plk1 activity enhances Mps1 catalytic activity as well as the recruitment of the SAC components Mad1:C-Mad2 and Bub3:BubR1 to kinetochores. We conclude that Plk1 strengthens the robustness of SAC establishment at the onset of mitosis and supports SAC maintenance during prolonged mitotic arrest. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Spindle neurons of the human anterior cingulate cortex

NASA Technical Reports Server (NTRS)

Nimchinsky, E. A.; Vogt, B. A.; Morrison, J. H.; Hof, P. R.; Bloom, F. E. (Principal Investigator)

1995-01-01

The human anterior cingulate cortex is distinguished by the presence of an unusual cell type, a large spindle neuron in layer Vb. This cell has been noted numerous times in the historical literature but has not been studied with modern neuroanatomic techniques. For instance, details regarding the neuronal class to which these cells belong and regarding their precise distribution along both ventrodorsal and anteroposterior axes of the cingulate gyrus are still lacking. In the present study, morphological features and the anatomic distribution of this cell type were studied using computer-assisted mapping and immunocytochemical techniques. Spindle neurons are restricted to the subfields of the anterior cingulate cortex (Brodmann's area 24), exhibiting a greater density in anterior portions of this area than in posterior portions, and tapering off in the transition zone between anterior and posterior cingulate cortex. Furthermore, a majority of the spindle cells at any level is located in subarea 24b on the gyral surface. Immunocytochemical analysis revealed that the neurofilament protein triple was present in a large percentage of these neurons and that they did not contain calcium-binding proteins. Injections of the carbocyanine dye DiI into the cingulum bundle revealed that these cells are projection neurons. Finally, spindle cells were consistently affected in Alzheimer's disease cases, with an overall loss of about 60%. Taken together, these observations indicate that the spindle cells of the human cingulate cortex represent a morphological subpopulation of pyramidal neurons whose restricted distribution may be associated with functionally distinct areas.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

PubMed Central

Sawin, K E; Mitchison, T J

1995-01-01

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7753799

The rhizoplast of chrysomonads, a basal body-nucleus connector that polarises the dividing spindle.

PubMed

Brugerolle, G; Mignot, J-P

2003-09-01

An ultrastructure study of the rhizoplast in Synura petersenii, Mallomonas fastigiata, and M. insignis shows that it consists of 15-20 striated rootlets that form a claw or an incomplete cone over the nucleus. These rootlets course along one face of the nucleus between the nuclear membrane and the cis-face of the Golgi stack of cisternae. They converge and merge above the nucleus, forming a stub attached to the proximal section of the two basal bodies. These cross-striated rootlets are composed of closely packed longitudinal microfibrils. By immunofluorescence, the basal bodies and the rootlets forming the claw were decorated by the anti-centrin monoclonal antibody ICL19 raised against the Paramecium tetraurelia acidic centrin protein and by two antibodies raised against the striated parabasal and costal striated fibres of trichomonads. Only the anti-centrin monoclonal antibody 20H5 raised against Chlamydomonas reinhardtii centrin strongly labelled the 20-22 kDa protein bands from the extracted cytoskeleton of S. petersenii by immunoblotting. Electron micrographs of mitosis in S. petersenii cells revealed that the segregated pairs of basal bodies are linked by the striated rootlets of the rhizoplast to the poles of the mitotic spindle. The spindle microtubules arise perpendicularly from the striated rootlets of the basal body-nucleus connector forming the centrosome. In conclusion, in these cells there is a basal body-nucleus connector similar to that of C. reinhardtii and other chlorophytes. It contains centrin proteins, it is involved in the linkage of the basal bodies to the nucleus and is a component of the spindle pole body or centrosome in the dividing cell.

Spindle cell oncocytoma of the pituitary gland with follicle-like component: organotypic differentiation to support its origin from folliculo-stellate cells.

PubMed

Vajtai, Istvan; Beck, Jürgen; Kappeler, Andreas; Hewer, Ekkehard

2011-08-01

Spindle cell oncocytoma (SCO) is a rare, non-adenomatous tumor originating from the anterior pituitary gland. Composed of fusiform, mitochondrion-rich cells sharing several immunophenotypic and ultrastructural properties with folliculo-stellate cells (FSC), SCO has been proposed to represent a neoplastic counterpart of the latter. To date, however, SCO has failed to meet one criterion commonly used in histological-based taxonomy and diagnostics; that of recapitulating any of FSCs' morphologically defined developmental or physiological states. We describe a unique example of SCO wherein a conventional fascicular texture was seen coexisting with and organically merging into follicle-like arrangements. The sellar tumor of 2.7 × 2.6 × 2.5 cm was transphenoidally resected from a 55-year old female. Preoperative magnetic resonance imaging indicated an isointense, contrast enhancing mass with suprasellar extension. Histology showed multiple rudimentary to well-formed, follicle-like cavities on a classical spindle cell background; while all the participating cells exhibited an SCO immunophenotype, including positivity for S100 protein, vimentin, EMA, Bcl-2, and TTF-1, as well as staining with the antimitochondrial antibody 113-1. Conversely no expression of GFAP, follicular-epithelial cytokeratin, carcinoembryonic antigen, or anterior pituitary hormones was detected. Ultrastructurally, tumor cells facing follicular lumina displayed organelles of epithelial specialization, in particular surface microvilli and apical tight junctions. This constellation is felt to be reminiscent of FSCs' metaplastic transition to follicular epithelium, as observed during embryonic development and physiological renewal of the hormone-secreting parenchyma. Such finding is apt to being read as a supporting argument for SCO's descent from the FSC lineage.

Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

PubMed

Schrader, Thorsten; Münter, Klaus; Kleine-Ostmann, Thomas; Schmid, Ernst

2008-12-01

The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.

Development of ipsilateral chest wall spindle cell carcinoma in a patient with invasive ductal breast carcinoma during postoperative adjuvant therapy: A case report.

PubMed

Li, Kaifu; Kang, Hua; Wang, Yajun; Hai, Tao; Wang, Bixiao

2018-05-01

Metaplastic breast carcinoma (MBC) is rare subtype of breast carcinoma and is regarded as ductal carcinoma that undergoes metaplasia into a glandular growth pattern. Spindle cell carcinoma (SPC) is a subtype of MBC with a predominant spindle cell component. The patient was a 52-year-old female with invasive ductal breast carcinoma who underwent a modified radical mastectomy and an axillary node dissection. A new lump was observed underneath the surgical site between the pectoralis major and pectoralis minor muscles 45 days after the patient underwent sequential postoperative chemotherapy and radiotherapy. It was speculated that the new lesion had developed during postoperative adjuvant therapy. And the new lesion was regarded as a recurrence. We performed a wide dissection of the tumor with negative margins. The pathology of the tumor indicated SPC. Then, the patient received chemotherapy and demonstrated a poor response. Local recurrence and pulmonary metastasis developed shortly afterwards, and the patient succumbed to the disease within 5 months. Local recurrence with metaplastic SPC transformed from invasive ductal breast carcinoma during postoperative chemotherapy and radiotherapy is rare. The failure of subsequent chemotherapy and the progression of disease indicate the aggressive nature of SPC and its decreased sensitivity to chemotherapy and radiotherapy. Further studies must be performed to improve the prognosis of these patients.

Mutational analysis using Sanger and next generation sequencing in sporadic spindle cell hemangiomas: A study of 19 cases.

PubMed

Ten Broek, Roel W; Bekers, Elise M; de Leng, Wendy W J; Strengman, Eric; Tops, Bastiaan B J; Kutzner, Heinz; Leeuwis, Jan Willem; van Gorp, Joost M; Creytens, David H; Mentzel, Thomas; van Diest, Paul J; Eijkelenboom, Astrid; Flucke, Uta

2017-12-01

Spindle cell hemangioma (SCH) is a distinct vascular soft-tissue lesion characterized by cavernous blood vessels and a spindle cell component mainly occurring in the distal extremities of young adults. The majority of cases harbor heterozygous mutations in IDH1/2 sporadically or rarely in association with Maffucci syndrome. However, based on mosaicism and accordingly a low percentage of lesional cells harboring a mutant allele, detection can be challenging. We tested 19 sporadic SCHs by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), conventional next generation sequencing (NGS), and NGS using a single molecule molecular inversion probes (smMIP)-based library preparation to compare their diagnostic value. Out of 10 cases tested by Sanger sequencing and 2 analyzed using MLPA, 4 and 1, respectively, revealed a mutation in IDH1 (p.R132C). The 7 remaining negative cases and additional 6 cases were investigated using smMIP/NGS, showing hot spot mutations in IDH1 (p.R132C) (8 cases) and IDH2 (3 cases; twice p.R172S and once p.R172G, respectively). One case was negative. Owing to insufficient DNA quality and insufficient coverage, 2 cases were excluded. In total, in 16 out of 17 cases successfully tested, an IDH1/2 mutation was found. Given that IDH1/2 mutations were absent in 161 other vascular lesions tested by smMIP/NGS, the mutation can be considered as highly specific for SCH. © 2017 Wiley Periodicals, Inc.

The Physics of the Metaphase Spindle.

PubMed

Oriola, David; Needleman, Daniel J; Brugués, Jan

2018-05-20

The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.

Spatial signals link exit from mitosis to spindle position

PubMed Central

Falk, Jill Elaine; Tsuchiya, Dai; Verdaasdonk, Jolien; Lacefield, Soni; Bloom, Kerry; Amon, Angelika

2016-01-01

In budding yeast, if the spindle becomes mispositioned, cells prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN is a signaling cascade that localizes to spindle pole bodies (SPBs) and activates the phosphatase Cdc14. There are two competing models that explain MEN regulation by spindle position. In the 'zone model', exit from mitosis occurs when a MEN-bearing SPB enters the bud. The 'cMT-bud neck model' posits that cytoplasmic microtubule (cMT)-bud neck interactions prevent MEN activity. Here we find that 1) eliminating cMT– bud neck interactions does not trigger exit from mitosis and 2) loss of these interactions does not precede Cdc14 activation. Furthermore, using binucleate cells, we show that exit from mitosis occurs when one SPB enters the bud despite the presence of a mispositioned spindle. We conclude that exit from mitosis is triggered by a correctly positioned spindle rather than inhibited by improper spindle position. DOI: http://dx.doi.org/10.7554/eLife.14036.001 PMID:27166637

Assessing the Contributions of Motor Enzymes and Microtubule Dynamics to Mitotic Chromosome Motions.

PubMed

McIntosh, J Richard

2017-10-06

During my graduate work with Keith Porter, I became fascinated by the mitotic spindle, an interest that has motivated much of my scientific work ever since. I began spindle studies by using electron microscopes, instruments that have made significant contributions to our understanding of spindle organization. Such instruments have helped to elucidate the distributions of spindle microtubules, the interactions among them, their molecular polarity, and their associations with both kinetochores and spindle poles. Our lab has also investigated some processes of spindle physiology: microtubule dynamics, the actions of microtubule-associated proteins (including motor enzymes), the character of forces generated by specific spindle components, and factors that control mitotic progression. Here, I give a personal perspective on some of this intellectual history and on what recent discoveries imply about the mechanisms of chromosome motion.

BCOR-CCNB3-positive soft tissue sarcoma with round-cell and spindle-cell histology: a series of four cases highlighting the pitfall of mimicking poorly differentiated synovial sarcoma.

PubMed

Li, Wan-Shan; Liao, I-Chuang; Wen, Mei-Chin; Lan, Howard Haw-Chang; Yu, Shih-Chen; Huang, Hsuan-Ying

2016-11-01

BCOR-CCNB3 sarcoma is a genetically defined undifferentiated malignancy with Ewing sarcoma (ES)-like round cells, and preferentially affects the bones of male adolescents. Sarcomas harbouring BCOR-CCNB3 rarely arise from soft tissues; therefore, we aimed to report four cases to expand the clinicopathological spectrum. By reverse transcription polymerase chain reaction and confirmatory sequencing, we detected a BCOR-CCNB3 transcript in primary undifferentiated sarcomas of the deep musculature of four male patients, comprising two teenagers (aged 14 and 17 years) and two adults (aged 34 and 44 years). The tumours originated in the back (n = 2), pelvis (n = 1), and thigh (n = 1), and were 70-140 mm in size (mean, 107 mm). All tumours showed sheets of primitive round or ovoid cells with vesicular nuclei, active mitosis (28-41/10 high-power fields), variably prominent nucleoli, and geographical necrosis. This major component transformed into fascicles of elongated spindle cells with staghorn vessels and a myxoid reticular stroma, accounting for 10-50% of areas. All cases were positive for CD99, three were positive for TLE1, and one was positive for EMA, indicating poorly differentiated synovial sarcomas (PDSSs). Nuclear cyclin B3 reactivity was present in all cases, but not in molecularly confirmed atypical ESs and PDSSs. At the last follow-up (median, 13.5 months), one patient had died of lung metastasis, two were alive with tumours, and one was tumour-free. BCOR-CCNB3-positive sarcomas may primarily occur in soft tissues of adults and show PDSS-mimicking round-cell and spindle-cell histology with aggressive behaviour. Cyclin B3 is useful for selecting candidates for BCOR-CCNB3 molecular testing. © 2016 John Wiley & Sons Ltd.

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

PubMed

Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

2016-08-02

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

Expression of Progesterone Receptor Membrane Component-2 Within the Immature Rat Ovary and Its Role in Regulating Mitosis and Apoptosis of Spontaneously Immortalized Granulosa Cells1

PubMed Central

Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K.; Peluso, John J.

2014-01-01

ABSTRACT Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular 3H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development. PMID:24990806

Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

NASA Astrophysics Data System (ADS)

Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

2014-03-01

Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

Physical limits on kinesin-5–mediated chromosome congression in the smallest mitotic spindles

PubMed Central

McCoy, Kelsey M.; Tubman, Emily S.; Claas, Allison; Tank, Damien; Clancy, Shelly Applen; O’Toole, Eileen T.; Berman, Judith; Odde, David J.

2015-01-01

A characteristic feature of mitotic spindles is the congression of chromosomes near the spindle equator, a process mediated by dynamic kinetochore microtubules. A major challenge is to understand how precise, submicrometer-scale control of kinetochore micro­tubule dynamics is achieved in the smallest mitotic spindles, where the noisiness of microtubule assembly/disassembly will potentially act to overwhelm the spatial information that controls microtubule plus end–tip positioning to mediate congression. To better understand this fundamental limit, we conducted an integrated live fluorescence, electron microscopy, and modeling analysis of the polymorphic fungal pathogen Candida albicans, which contains one of the smallest known mitotic spindles (<1 μm). Previously, ScCin8p (kinesin-5 in Saccharomyces cerevisiae) was shown to mediate chromosome congression by promoting catastrophe of long kinetochore microtubules (kMTs). Using C. albicans yeast and hyphal kinesin-5 (Kip1p) heterozygotes (KIP1/kip1∆), we found that mutant spindles have longer kMTs than wild-type spindles, consistent with a less-organized spindle. By contrast, kinesin-8 heterozygous mutant (KIP3/kip3∆) spindles exhibited the same spindle organization as wild type. Of interest, spindle organization in the yeast and hyphal states was indistinguishable, even though yeast and hyphal cell lengths differ by two- to fivefold, demonstrating that spindle length regulation and chromosome congression are intrinsic to the spindle and largely independent of cell size. Together these results are consistent with a kinesin-5–mediated, length-dependent depolymerase activity that organizes chromosomes at the spindle equator in C. albicans to overcome fundamental noisiness in microtubule self-assembly. More generally, we define a dimensionless number that sets a fundamental physical limit for maintaining congression in small spindles in the face of assembly noise and find that C. albicans operates very close to this limit, which may explain why it has the smallest known mitotic spindle that still manifests the classic congression architecture. PMID:26354423

Male gametogenesis without centrioles.

PubMed

Riparbelli, Maria Giovanna; Callaini, Giuliano

2011-01-15

The orientation of the mitotic spindle plays a central role in specifying stem cell-renewal by enabling interaction of the daughter cells with external cues: the daughter cell closest to the hub region is instructed to self-renew, whereas the distal one starts to differentiate. Here, we have analyzed male gametogenesis in DSas-4 Drosophila mutants and we have reported that spindle alignment and asymmetric divisions are properly executed in male germline stem cells that lack centrioles. Spermatogonial divisions also correctly proceed in the absence of centrioles, giving rise to cysts of 16 primary spermatocytes. By contrast, abnormal meiotic spindles assemble in primary spermatocytes. These results point to different requirements for centrioles during male gametogenesis of Drosophila. Spindle formation during germ cell mitosis may be successfully supported by an acentrosomal pathway that is inadequate to warrant the proper execution of meiosis. Copyright © 2010 Elsevier Inc. All rights reserved.

HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

PubMed Central

Jiang, Jihong; Kuan, Chia-Wei; Fotovati, Abbas; Chu, Tony LH; He, Zhengcheng; Lengyell, Tess C; Li, Huaibiao; Kroll, Torsten; Li, Amanda M; Goldowitz, Daniel; Frappart, Lucien; Ploubidou, Aspasia; Patel, Millan S; Pilarski, Linda M; Simpson, Elizabeth M; Lange, Philipp F; Allan, Douglas W

2017-01-01

Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of Hmmr-knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development. PMID:28994651

Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases.

PubMed

Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C

2014-05-01

Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.

The chromokinesin Kid is required for maintenance of proper metaphase spindle size.

PubMed

Tokai-Nishizumi, Noriko; Ohsugi, Miho; Suzuki, Emiko; Yamamoto, Tadashi

2005-11-01

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle

PubMed Central

1979-01-01

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement. PMID:479316

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast.

PubMed

Yukawa, Masashi; Kawakami, Tomoki; Okazaki, Masaki; Kume, Kazunori; Tang, Ngang Heok; Toda, Takashi

2017-12-01

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end-directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end-directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly. © 2017 Yukawa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soeda, Shuhei; Nakayama, Yuji, E-mail: nakayama@mb.kyoto-phu.ac.jp; Department of Biochemistry and Molecular Biology, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414

Src-family tyrosine kinases are aberrantly activated in cancers, and this activation is associated with malignant tumor progression. v-Src, encoded by the v-src transforming gene of the Rous sarcoma virus, is a mutant variant of the cellular proto-oncogene c-Src. Although investigations with temperature sensitive mutants of v-Src have shown that v-Src induces many oncogenic processes, the effects on cell division are unknown. Here, we show that v-Src inhibits cellular proliferation of HCT116, HeLa S3 and NIH3T3 cells. Flow cytometry analysis indicated that inducible expression of v-Src results in an accumulation of 4N cells. Time-lapse analysis revealed that binucleation is induced throughmore » the inhibition of cytokinesis, a final step of cell division. The localization of Mklp1, which is essential for cytokinesis, to the spindle midzone is inhibited in v-Src-expressing cells. Intriguingly, Aurora B, which regulates Mklp1 localization at the midzone, is delocalized from the spindle midzone and the midbody but not from the metaphase chromosomes upon v-Src expression. Mklp2, which is responsible for the relocation of Aurora B from the metaphase chromosomes to the spindle midzone, is also lost from the spindle midzone. These results suggest that v-Src inhibits cytokinesis through the delocalization of Mklp1 and Aurora B from the spindle midzone, resulting in binucleation. -- Highlights: • v-Src inhibits cell proliferation of HCT116, HeLa S3 and NIH3T3 cells. • v-Src induces binucleation together with cytokinesis failure. • v-Src causes delocalization of Mklp1, Aurora B and INCENP from the spindle midzone.« less

TFG-MET fusion in an infantile spindle cell sarcoma with neural features.

PubMed

Flucke, Uta; van Noesel, Max M; Wijnen, Marc; Zhang, Lei; Chen, Chun-Liang; Sung, Yun-Shao; Antonescu, Cristina R

2017-09-01

An increasing number of congenital and infantile sarcomas displaying a primitive, monomorphic spindle cell phenotype have been characterized to harbor recurrent gene fusions, including infantile fibrosarcoma and congenital spindle cell rhabdomyosarcoma. Here, we report an unusual spindle cell sarcoma presenting as a large and infiltrative pelvic soft tissue mass in a 4-month-old girl, which revealed a novel TFG-MET gene fusion by whole transcriptome RNA sequencing. The tumor resembled the morphology of an infantile fibrosarcoma with both fascicular and patternless growth, however, it expressed strong S100 protein immunoreactivity, while lacking SOX10 staining and retaining H3K27me3 expression. Although this immunoprofile suggested partial neural/neuroectodermal differentiation, overall features were unusual and did not fit into any known tumor types (cellular schwannoma, MPNST), raising the possibility of a novel pathologic entity. The TFG-MET gene fusion expands the genetic spectrum implicated in the pathogenesis of congenital spindle cell sarcomas, with yet another example of kinase oncogenic activation through chromosomal translocation. The discovery of this new fusion is significant since the resulting MET activation can potentially be inhibited by targeted therapy, as MET inhibitors are presently available in clinical trials. © 2017 Wiley Periodicals, Inc.

Biophysical Aspects of Spindle Evolution

NASA Astrophysics Data System (ADS)

Farhadifar, Reza; Baer, Charlie; Needleman, Daniel

2011-03-01

The continual propagation of genetic material from one generation to the next is one of the most basic characteristics of all organisms. In eukaryotes, DNA is segregated into the two daughter cells by a highly dynamic, self-organizing structure called the mitotic spindle. Mitotic spindles can show remarkable variability between tissues and organisms, but there is currently little understanding of the biophysical and evolutionary basis of this diversity. We are studying how spontaneous mutations modify cell division during nematode development. By comparing the mutational variation - the raw material of evolution - with the variation present in nature, we are investigating how the mitotic spindle is shaped over the course of evolution. This combination of quantitative genetics and cellular biophysics gives insight into how the structure and dynamics of the spindle is formed through selection, drift, and biophysical constraints.

CDKL5 localizes at the centrosome and midbody and is required for faithful cell division.

PubMed

Barbiero, Isabella; Valente, Davide; Chandola, Chetan; Magi, Fiorenza; Bergo, Anna; Monteonofrio, Laura; Tramarin, Marco; Fazzari, Maria; Soddu, Silvia; Landsberger, Nicoletta; Rinaldo, Cinzia; Kilstrup-Nielsen, Charlotte

2017-07-24

The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.

Chronic exposure to particulate chromate induces spindle assembly checkpoint bypass in human lung cells.

PubMed

Wise, Sandra S; Holmes, Amie L; Xie, Hong; Thompson, W Douglas; Wise, John Pierce

2006-11-01

One of the hallmarks of lung cancer is chromosome instability (CIN), particularly a tetraploid phenotype, which is normally prevented by the spindle assembly checkpoint. Hexavalent chromium Cr(VI) is an established human lung carcinogen, and Cr(VI) induces tumors at lung bifurcation sites where Cr(VI) particles impact and persist. However, the effects of Cr(VI) on the spindle assembly checkpoint are unknown and little is known about prolonged exposure to particulate Cr(VI). Accordingly, we investigated particulate Cr(VI)-induced bypass of the spindle assembly checkpoint after several days of exposure in WHTBF-6 cells. We found that lead chromate indeed induces spindle assembly checkpoint bypass in human lung cells, as 72, 96, and 120 h treatments with 0.5 or 1 microg/cm2 lead chromate induced significant increases in the percentage of cells with aberrant mitotic figures. For example, treatment with 1 microg/cm2 lead chromate for 96 h induced 11, 12.3, and 14% of cells with premature anaphase, centromere spreading and premature centromere division, respectively. In addition, we found a disruption of mitosis with more cells accumulating in anaphase; cells treated for 96 h increased from 18% in controls to 31% in cells treated with lead chromate. To confirm involvement of the spindle assembly checkpoint, Mad2 expression was used as a marker. Mad2 expression was decreased in cells exposed to chronic treatments of lead chromate, consistent with disruption of the checkpoint. We also found concentration- and time-dependent increases in tetraploid cells, which continued to grow and form colonies. When cells were treated with chronic lead alone there was no increase in aberrant mitotic cells or polyploidy; however, chronic exposure to a soluble Cr(VI) showed an increase in aberrant mitotic cells and polyploidy. These data suggest that lead chromate does induce CIN and may be one mechanism in the development of Cr(VI)-induced lung cancer.

Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

PubMed Central

Gergely, Zachary R.; Crapo, Ammon; Hough, Loren E.; McIntosh, J. Richard; Betterton, Meredith D.

2016-01-01

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen. PMID:27146110

Spindle cell sarcoma of the vulva with myofibroblastic differentiation.

PubMed

Adeleye, Amanda J; Palmeri, Nicholas; Wang, Shih-Hsiu J; Liu-Jarin, Xiaolin; Wright, Jason D

2015-04-01

Primary vulvar sarcomas are rare lesions of the lower genital tract. We report the case of a patient with a spindle cell sarcoma of the vulva. A 44-year-old woman presented with a painless vulvar mass. Vulvar biopsy demonstrated a spindle cell sarcoma with myofibroblastic differentiation. Pretreatment evaluation revealed no evidence of metastatic disease, and magnetic resonance imaging found no local masses. The patient underwent right radical vulvectomy with negative margins and tolerated the procedure well. Women undergoing gynecologic care should have routine evaluation of the vulva to detect these rare neoplasms.

Identified Cellular Correlates of Neocortical Ripple and High-Gamma Oscillations during Spindles of Natural Sleep.

PubMed

Averkin, Robert G; Szemenyei, Viktor; Bordé, Sándor; Tamás, Gábor

2016-11-23

Ultra-high-frequency network events in the hippocampus are instrumental in a dialogue with the neocortex during memory formation, but the existence of transient ∼200 Hz network events in the neocortex is not clear. Our recordings from neocortical layer II/III of freely behaving rats revealed field potential events at ripple and high-gamma frequencies repeatedly occurring at troughs of spindle oscillations during sleep. Juxtacellular recordings identified subpopulations of fast-spiking, parvalbumin-containing basket cells with epochs of firing at ripple (∼200 Hz) and high-gamma (∼120 Hz) frequencies detected during spindles and centered with millisecond precision at the trough of spindle waves in phase with field potential events but phase shifted relative to pyramidal cell firing. The results suggest that basket cell subpopulations are involved in spindle-nested, high-frequency network events that hypothetically provide repeatedly occurring neocortical temporal reference states potentially involved in mnemonic processes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Staining for factor VIII related antigen and Ulex europaeus agglutinin I (UEA-I) in 230 tumours. An assessment of their specificity for angiosarcoma and Kaposi's sarcoma.

PubMed

Leader, M; Collins, M; Patel, J; Henry, K

1986-11-01

In this study we examined the staining reactivity of commercially available antisera to factor VIII related antigen (F VIII RAg) and Ulex europaeus agglutinin I (UEA-I) on sections from 230 formalin fixed paraffin embedded tumours. These included 196 sarcomas, 20 carcinomas and 14 angiomas. All angiomas showed positive staining for F VIII RAg; all carcinomas showed negative staining; the vasoformative areas of all angiosarcomas stained positively but only four of six angiosarcomas showed positive staining of their solid areas; of seven Kaposi's sarcomas, all showed positive staining of vessels and six showed positive staining of the spindle cell component. In the remaining 181 non-vascular sarcomas there was a false positive result in four tumours (2.2%), three of which had a history of irradiation. Pre-radiotherapy biopsies of these three tumours stained negatively with anti-F VIII RAg. UEA-I was demonstrated in all the angiomas studied, in all angiosarcomas (including the solid components) and in well-formed vessels of all Kaposi's sarcomas, but only in the spindle cell component of 3/6. However, there was an unacceptably high rate of false positive staining amongst the carcinomas and non-vascular sarcomas. In conclusion, F VIII RAg is a specific but not a sensitive marker of angiosarcomas; UEA-I is a sensitive but not a specific marker of angiosarcomas.

Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

PubMed Central

Yumoto, Takahito; Nakadate, Kazuhiko; Nakamura, Yuki; Sugitani, Yoshinobu; Sugitani-Yoshida, Reiko; Ueda, Shuichi; Sakakibara, Shin-ichi

2013-01-01

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs. PMID:24260314

EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

PubMed

Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

2016-08-17

EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

v-Src-induced nuclear localization of YAP is involved in multipolar spindle formation in tetraploid cells.

PubMed

Kakae, Keiko; Ikeuchi, Masayoshi; Kuga, Takahisa; Saito, Youhei; Yamaguchi, Naoto; Nakayama, Yuji

2017-01-01

The protein-tyrosine kinase, c-Src, is involved in a variety of signaling events, including cell division. We have reported that v-Src, which is a mutant variant of the cellular proto-oncogene, c-Src, causes delocalization of Aurora B kinase, resulting in a furrow regression in cytokinesis and the generation of multinucleated cells. However, the effect of v-Src on mitotic spindle formation is unknown. Here we show that v-Src-expressing HCT116 and NIH3T3 cells undergo abnormal cell division, in which cells separate into more than two cells. Upon v-Src expression, the proportion of multinucleated cells is increased in a time-dependent manner. Flow cytometry analysis revealed that v-Src increases the number of cells having a ≥4N DNA content. Microscopic analysis showed that v-Src induces the formation of multipolar spindles with excess centrosomes. These results suggest that v-Src induces multipolar spindle formation by generating multinucleated cells. Tetraploidy activates the tetraploidy checkpoint, leading to a cell cycle arrest of tetraploid cells at the G1 phase, in which the nuclear exclusion of the transcription co-activator YAP plays a critical role. In multinucleated cells that are induced by cytochalasin B and the Plk1 inhibitor, YAP is excluded from the nucleus. However, v-Src prevents this nuclear exclusion of YAP through a decrease in the phosphorylation of YAP at Ser127 in multinucleated cells. Furthermore, v-Src decreases the expression level of p53, which also plays a critical role in the cell cycle arrest of tetraploid cells. These results suggest that v-Src promotes abnormal spindle formation in at least two ways: generation of multinucleated cells and a weakening of the tetraploidy checkpoint. Copyright © 2016 Elsevier Inc. All rights reserved.

Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

PubMed Central

Onishi, Masayuki; Yeong, Foong May

2016-01-01

Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

An adult spindle cell rhabdomyosarcoma in the head and neck region with long-term survival: a case report

PubMed Central

2014-01-01

Introduction Spindle cell rhabdomyosarcoma of the head and neck is a very rare tumor in adults. We report on one case with long-term survival. Case presentation A 41-year-old nonsmoking Caucasian man presented in June 2007 with a painless swelling under his tongue. A diagnosis of a soft tissue sarcoma, and a myofibrosarcoma in particular, was made via biopsy. After multimodal treatment, including local and systemic therapy, our patient remained disease-free until September 2010. The local recurrence was treated unsuccessfully with various chemotherapy regimens. In September 2011, our patient underwent surgical resection again, and a spindle cell rhabdomyosarcoma was diagnosed. To analyze the mismatch between the original diagnosis of a myofibrosarcoma and the second diagnosis, the two specimens were reassessed, and a final diagnosis of a spindle cell rhabdomyosarcoma was made. In 2012 and 2013, our patient suffered further recurrences that were surgically treated, and he is still alive with disease six years and 10 months after the initial diagnosis in June 2007. Conclusions In adults, the spindle cell rhabdomyosarcoma tumor is very rare in the head and neck region. In contrast to childhood tumors, spindle cell rhabdomyosarcoma in adulthood is often associated with a poor prognosis. In the present case, the radical surgical treatment might have helped to prolong the patient’s overall survival, which has lasted more than six years. To our knowledge, this is the longest overall survival reported so far for this tumor entity in the head and neck region. PMID:24946859

A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling.

PubMed

Ji, Zhejian; Gao, Haishan; Jia, Luying; Li, Bing; Yu, Hongtao

2017-01-10

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1-Bub3 and BubR1-Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1-Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/C Cdc20 ) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1-Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

Downregulation of Protein 4.1R impairs centrosome function,bipolar spindle organization and anaphase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spence, Jeffrey R.; Go, Minjoung M.; Bahmanyar, S.

2006-03-17

Centrosomes nucleate and organize interphase MTs and areinstrumental in the assembly of the mitotic bipolar spindle. Here wereport that two members of the multifunctional protein 4.1 family havedistinct distributions at centrosomes. Protein 4.1R localizes to maturecentrioles whereas 4.1G is a component of the pericentriolar matrixsurrounding centrioles. To selectively probe 4.1R function, we used RNAinterference-mediated depletion of 4.1R without decreasing 4.1Gexpression. 4.1R downregulation reduces MT anchoring and organization atinterphase and impairs centrosome separation during prometaphase.Metaphase chromosomes fail to properly condense/align and spindleorganization is aberrant. Notably 4.1R depletion causes mislocalizationof its binding partner NuMA (Nuclear Mitotic Apparatus Protein),essential for spindle pole focusing,more » and disrupts ninein. Duringanaphase/telophase, 4.1R-depleted cells have lagging chromosomes andaberrant MT bridges. Our data provide functional evidence that 4.1R makescrucial contributions to centrosome integrity and to mitotic spindlestructure enabling mitosis and anaphase to proceed with the coordinatedprecision required to avoid pathological events.« less

Axin localizes to mitotic spindles and centrosomes in mitotic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Shi-Mun; Choi, Eun-Jin; Song, Ki-Joon

2009-04-01

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3{beta}) without producing any notable changes inmore » cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3{beta} in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.« less

From proto-mitosis to mitosis — An alternative hypothesis on the origin and evolution of the mitotic spindle

NASA Astrophysics Data System (ADS)

Roos, U.-P.

1984-03-01

Based on the assumption that the ancestral proto-eukaryote evolved from an ameboid prokarybte I propose the hypothesis that nuclear division of the proto-eukaryote was effected by the same system of contractile filaments it used for ameboid movement and cytosis. When the nuclear membranes evolved from the cell membrane, contractile filaments remained associated with them. The attachment site of the genome in the nuclear envelope was linked to the cell membrane by specialized contractile filaments. During protomitosis, i.e., nuclear and cell division of the proto-eukaryote, these filaments performed segregation of the chromosomes, whereas others constricted and cleaved the nucleus and the mother cell. When microtubules (MTs) had evolved in the cytoplasm, they also became engaged in nuclear division. Initially, an extranuolear bundle of MTs assisted chromosome segregation by establishing a defined axis. The evolutionary tendency then was towards an increasingly important role for MTs. Spindle pole bodies (SPBs) developed from the chromosomal attachment sites in the nuclear envelope and organized an extranuclear central spindle. The chromosomes remained attached to the SPBs during nuclear division. In a subsequent step the spindle became permanently lodged inside the nucleus. Chromosomes detached from the SPBs and acquired kinetochores and kinetochore-MTs. At first, this spindle segregated chromosomes by elongation, the kinetochore-MTs playing the role of static anchors. Later, spindle elongation was supplemented by poleward movement of the chromosomes. When dissolution of the nuclear envelope at the beginning of mitosis became a permanent feature, the open spindle of higher eukaryotes was born.

Mammalian neurogenesis requires Treacle-Plk1 for precise control of spindle orientation, mitotic progression, and maintenance of neural progenitor cells.

PubMed

Sakai, Daisuke; Dixon, Jill; Dixon, Michael J; Trainor, Paul A

2012-01-01

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1(+/-) mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly.

Mammalian Neurogenesis Requires Treacle-Plk1 for Precise Control of Spindle Orientation, Mitotic Progression, and Maintenance of Neural Progenitor Cells

PubMed Central

Sakai, Daisuke; Dixon, Jill; Dixon, Michael J.; Trainor, Paul A.

2012-01-01

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1 +/− mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly. PMID:22479190

An integrated overview of spatiotemporal organization and regulation in mitosis in terms of the proteins in the functional supercomplexes.

PubMed

Zheng, Yueyuan; Guo, Junjie; Li, Xu; Xie, Yubin; Hou, Mingming; Fu, Xuyang; Dai, Shengkun; Diao, Rucheng; Miao, Yanyan; Ren, Jian

2014-01-01

Eukaryotic cells may divide via the critical cellular process of cell division/mitosis, resulting in two daughter cells with the same genetic information. A large number of dedicated proteins are involved in this process and spatiotemporally assembled into three distinct super-complex structures/organelles, including the centrosome/spindle pole body, kinetochore/centromere and cleavage furrow/midbody/bud neck, so as to precisely modulate the cell division/mitosis events of chromosome alignment, chromosome segregation and cytokinesis in an orderly fashion. In recent years, many efforts have been made to identify the protein components and architecture of these subcellular organelles, aiming to uncover the organelle assembly pathways, determine the molecular mechanisms underlying the organelle functions, and thereby provide new therapeutic strategies for a variety of diseases. However, the organelles are highly dynamic structures, making it difficult to identify the entire components. Here, we review the current knowledge of the identified protein components governing the organization and functioning of organelles, especially in human and yeast cells, and discuss the multi-localized protein components mediating the communication between organelles during cell division.

The Clathrin-dependent Spindle Proteome*

PubMed Central

Rao, Sushma R.; Flores-Rodriguez, Neftali; Page, Scott L.; Wong, Chin; Robinson, Phillip J.; Chircop, Megan

2016-01-01

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a “moonlighting” role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. PMID:27174698

The Clathrin-dependent Spindle Proteome.

PubMed

Rao, Sushma R; Flores-Rodriguez, Neftali; Page, Scott L; Wong, Chin; Robinson, Phillip J; Chircop, Megan

2016-08-01

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stability of kinetochore-microtubule attachment and the role of different KMN network components in Drosophila.

PubMed

Feijão, Tália; Afonso, Olga; Maia, André F; Sunkel, Claudio E

2013-10-01

Kinetochores bind spindle microtubules and also act as signaling centers that monitor this interaction. Defects in kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for kinetochore-microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral kinetochore-microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral kinetochore-microtubule interactions are still functional allowing poleward kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient microtubule attachment, whereas Spc105 plays a predominant role in the kinetochore-microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability. Copyright © 2013 Wiley Periodicals, Inc.

Spindle cell oncocytomas and granular cell tumors of the pituitary are variants of pituicytoma.

PubMed

Mete, Ozgur; Lopes, Maria Beatriz; Asa, Sylvia L

2013-11-01

Pituicytomas are neoplasms that arise from pituicytes, which are specialized glia of the posterior pituitary. Pituicytes have 5 ultrastructural variants: light, dark, granular, ependymal, and oncocytic. Granular cell tumors of the pituitary gland are thought to arise from granular pituicytes. Spindle cell oncocytomas are considered to arise from folliculostellate cells, which are sustentacular cells of the adenohypophysis. Recent data suggest that, whereas pituicytes and all 3 tumor types are positive for TTF-1, folliculostellate cells are negative for TTF-1. We investigated 7 spindle cell oncocytomas, 4 pituicytomas, and 3 granular cell tumors for their genetic (BRAF(V600E) mutation and BRAF-KIAA fusion), immunohistochemical (GFAP, vimentin, S100 protein, olig2, IDH1-R132H, NF, galectin-3, chromogranin-A, CD56, EMA, CAM5.2, CD68, TTF-1, and bcl-2), and ultrastructural features to refine their classification. All tumors had nuclear positivity for TTF-1 and were negative for CAM5.2, chromogranin-A, and NF. GFAP, vimentin, S100, galectin-3, EMA, and CD68 were variably positive in the majority of the 3 tumor groups. Olig2 was only positive in 1 pituicytoma. Whereas granular cell tumors were negative for bcl-2 and CD56, pituicytomas and spindle cell oncocytomas showed variable positivity. All tumors were negative with the IDH1-R132H mutation-specific antibody, and none had evidence of BRAF alterations (BRAF(V600E) mutation and BRAF-KIAA fusion). Diffuse TTF-1 expression in nontumorous pituicytes, pituicytomas, spindle cell oncocytomas, and granular cell tumors indicates a common pituicyte lineage. The ultrastructural variants of pituicytes are reflected in these 3 morphologic variants of tumors arising from these cells. We propose the terminology "oncocytic pituicytomas" and "granular cell pituicytomas" to refine the classification of these lesions.

Myofibroblastoma Breast with Unusual Morphological Features. Cytohistopathogical Diagnostic Pitfalls and Role of Immunohistochemistry-Review of Literature

PubMed Central

Shivali, B.; S., Kataria; Chandramouleeswari, K.; Anita, S.

2013-01-01

Myofibroblastoma (MFB) is a rare mesenchymal tumour, derived from mammary stromal fibro-myofibroblasts, with diverse biological and morphological behaviour. Large and cellular myofibroblastomas, especially those with epitheliod like cells, can mimic various spindle cell lesions and metaplastic carcinomas, thus posing diagnostic challenge. A 50–year woman presented with slow growing, painless lump in the left breast. Fine Needle Aspiration (FNA) smears showed predominant atypical spindle cell population, pleomorphic epithelial like cells and giant cells. Cytodiagnosis of atypical spindle cell lesion with the possibility of metaplastic carcinoma was suggested. Histopathological examination showed fascicles of spindle cell population admixed with epithelial like cells, atypical cells and tumour giant cells, thus raising differential diagnosis of metaplastic carcinoma, low grade spindle cell sarcoma and myofibroblastic tumour. Lymph nodes were negative for metastatic deposits. Immunohistochemistry revealed variable coexpression of markers for vimentin, fibronectin, CD34, SMA (smooth muscle actin), but negative expression for , S-100, CD99, CK7 (cytokeratin 7), HMWK (high molecular weight keratin), ER (oestrogen receptor) and PR(progesterone receptors). Diagnosis of cellular myofibroblastoma with mixed unusual morphological features was defined, based on both histological and immunohistochemical features. MFB may cause a potential diagnostic pitfall while interpreting FNA and histopathological sections due to its wide differential diagnosis. The distinction of MFB from its cytohistological mimics of malignancy is crucial to avoid unnecessary extensive procedures. The case report emphasizes the role of immunohistochemistry as gold standard in diagnosis of MFB. The case is also being presented because of its large size and rare mixed unusual morphological features. PMID:24298520

Centromeric chromatin and its dynamics in plants.

PubMed

Lermontova, Inna; Sandmann, Michael; Mascher, Martin; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2015-07-01

Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast

PubMed Central

McDonald, Heather B.; Byers, Breck

1997-01-01

Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/ CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810– 32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1–green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication. PMID:9151663

Photodynamic effects induced by meso-tris(pentafluorophenyl)corrole and its cyclodextrin conjugates on cytoskeletal components of HeLa cells.

PubMed

Barata, Joana F B; Zamarrón, Alicia; Neves, M Graça P M S; Faustino, M Amparo F; Tomé, Augusto C; Cavaleiro, José A S; Röder, Beate; Juarranz, Ángeles; Sanz-Rodríguez, Francisco

2015-03-06

The aim of this work was to synthesize new corrole β-cyclodextrin conjugates βCD1 (with one β-cyclodextrin moiety) and βCD2 (with two β-cyclodextrin moieties) from 5,10,15-tris(pentafluorophenyl)corrole (TPFC) and to test in vitro the efficacy of these compounds towards tumoral HeLa cells. No dark cytotoxicity was observed for TPFC and βCD1 at the concentration used for PDT cell treatment, even during long incubation periods (24 h). Fluorescence microscopy showed that TPFC and βCD1 accumulate in HeLa cells at lysosomes and in the Golgi apparatus, respectively. The cell survival after the PDT treatment with visible light was dependent on light exposure level and compound concentration. βCD1 was able to penetrate efficiently in the cytoplasm of the HeLa cells. In particular, we have analyzed the photodynamic effect of the corrole derivatives on the microtubules of HeLa cells and the morphological alterations on the mitotic spindle. TPFC and βCD1 caused photocytotoxicity in tumoral HeLa cells and induced a rapid metaphase blockage of cells that also showed clearly altered configurations of the mitotic spindle. The results showed that TPFC has the highest photosensitizing efficiency on tumoral cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint.

PubMed

Hu, Xiangjing; Shen, Bin; Liao, Shangying; Ning, Yan; Ma, Longfei; Chen, Jian; Lin, Xiwen; Zhang, Daoqin; Li, Zhen; Zheng, Chunwei; Feng, Yanmin; Huang, Xingxu; Han, Chunsheng

2017-06-29

ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.

The Chromokinesin Kid Is Required for Maintenance of Proper Metaphase Spindle SizeD⃞

PubMed Central

Tokai-Nishizumi, Noriko; Ohsugi, Miho; Suzuki, Emiko; Yamamoto, Tadashi

2005-01-01

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes—imprecise chromosome alignment at metaphase and shortened spindles— exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization. PMID:16176979

Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes.

PubMed

Yi, Zi-Yun; Ma, Xue-Shan; Liang, Qiu-Xia; Zhang, Teng; Xu, Zhao-Yang; Meng, Tie-Gang; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan; Quan, Song

2016-12-19

Kif2a is a member of the Kinesin-13 microtubule depolymerases. Here, we report the expression, subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation. Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages, and then decreased slightly at the M II stage. Confocal microscopy identified that Kif2a localized to the meiotic spindle, especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase. Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes, reduced microtubule depolymerization, which led to significant pro-M I/M Iarrest and failure of first polar body (PB1) extrusion. Kif2a-depleted oocytes were also defective in spindle pole localization of γ-tubulin and showed spindle assembly checkpoint (SAC) protein Bub3 at the kinetochores even after 10 hr extended culture. These results demonstrate that Kif2a may act as a microtubule depolymerase, regulating microtubule dynamics, spindle assembly and chromosome congression, and thus cell cycle progression during mouse oocyte meiotic maturation.

LIS1 controls mitosis and mitotic spindle organization via the LIS1–NDEL1–dynein complex

PubMed Central

Moon, Hyang Mi; Youn, Yong Ha; Pemble, Hayley; Yingling, Jessica; Wittmann, Torsten; Wynshaw-Boris, Anthony

2014-01-01

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore–microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1–dynein complex. Overexpression of NDEL1–dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1–NDEL1–dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division. PMID:24030547

A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein

PubMed Central

Muhua, Li; Adames, Neil R.; Murphy, Michael D.; Shields, Colleen R.; Cooper, John A.

2008-01-01

Checkpoint controls ensure that events of the cell-division cycle are completed with fidelity and in the correct order. In budding yeast with a mutation in the motor protein dynein, the mitotic spindle is often misaligned and therefore slow to enter the neck between mother cell and budding daughter cell. When this occurs, cytokinesis (division of the cytoplasm into two) is delayed until the spindle is properly positioned1. Here we describe mutations that abolish this delay, indicating the existence of a new checkpoint mechanism. One mutation lies in the gene encoding the yeast homologue of EB1, a human protein that binds the adenomatous polyposis coli (APC) protein, a tumour suppressor. EB1 is located on microtubules of the mitotic spindle and is important in spindle assembly. EB1 may therefore, by associating with microtubules, contribute to the sensor mechanism that activates the checkpoint. Another mutation affects Stt4, a phosphatidylinositol-4-OH kinase. Cold temperature is an environmental stimulus that causes misalignment of the mitotic spindle in yeast and appears to activate this checkpoint mechanism. PMID:9624007

Identification and diversity of functional centromere satellites in the wild rice species Oryza brachyantha.

PubMed

Yi, Chuandeng; Zhang, Wenli; Dai, Xibin; Li, Xing; Gong, Zhiyun; Zhou, Yong; Liang, Guohua; Gu, Minghong

2013-12-01

The centromere is a key chromosomal component for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, allowing each sister chromatid to faithfully segregate to each daughter cell during cell division. It is not clear what types of sequences act as functional centromeres and how centromere sequences are organized in Oryza brachyantha, an FF genome species. In this study, we found that the three classes of centromere-specific CentO-F satellites (CentO-F1, CentO-F2, and CentOF3) in O. brachyantha share no homology with the CentO satellites in Oryza sativa. The three classes of CentO-F satellites are all located within the chromosomal regions to which the spindle fibers attach and are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons, CRR-F, in the O. brachyantha centromeres. Although these CentO-F satellites are quantitatively variable among 12 O. brachyantha centromeres, immunostaining with an antibody specific to CENH3 indicates that they are colocated with CENH3 in functional centromere regions. Our results demonstrate that the three classes of CentO-F satellites may be the major components of functional centromeres in O. brachyantha.

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis

PubMed Central

Fuchs, Margit; Lambert, Herman; Jetté, Alexandra; Elowe, Sabine; Landry, Jacques; Lavoie, Josée N.

2015-01-01

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation. PMID:26496431

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.

PubMed

Fuchs, Margit; Luthold, Carole; Guilbert, Solenn M; Varlet, Alice Anaïs; Lambert, Herman; Jetté, Alexandra; Elowe, Sabine; Landry, Jacques; Lavoie, Josée N

2015-10-01

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

A novel role of KIF3b in the seminoma cell cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Hao-Qing; Xiao, Yu-Xi; She, Zhen-Yu

KIF3b is a protein of the kinesin-2 family which plays an important role in intraflagellar transport. Testis cancer is a common cancer among young men. Its diagnostic rate is increasing and over half of the cases are seminomas. Many aspects of the mechanism and gene expression background of this cancer remain unclear. Using western-blotting and semi-quantitative PCR we found high protein levels of KIF3b enrichment in seminoma tissue despite the mRNA levels remaining equivalent to that of normal testicular tissues. The distribution of KIF3b was mainly in cells with division potential. Wound-healing assays and cell counting kit assays showed thatmore » the knockdown of KIF3b significantly suppressed cell migration ability, viability and number in HeLa cells. Immunofluorescence images during the cell cycle revealed that KIF3b tended to gather at the spindles and was enriched at the central spindle. This indicated that KIF3b may also have direct impacts upon spindle formation and cytokinesis. By counting the numbers of nuclei, spindles and cells, we found that the rates of multipolar division and multi-nucleation were raised in KIF3b-knockdown cells. In this way we demonstrate that KIF3b functions importantly in mitosis and may be essential to seminoma cell division and proliferation as well as being necessary for normal cell division. - Highlights: • A significant upregulation of KIF3b is detected in seminoma. • Knockdown of KIF3b impacts on cell proliferation and migration. • KIF3b may have direct impacts upon spindle formation and cytokinesis.« less

Desmoplastic melanoma morphology on Thinprep: a report of two cases

PubMed Central

Van Ells, Becky L; Madory, James E; Hoda, Rana S

2007-01-01

Background Desmoplastic melanoma is a variant of malignant melanoma that can range in appearance from sarcomatoid to scar-like. Cytomorphology of desmoplastic melanoma has been previously described on conventional smears; however, to our knowledge, detailed cytomorphology on ThinPrep has so far not been described. Herein, we describe the cytomorphology of two cases of desmoplastic melanoma on fine needle aspiration processed as ThinPrep slides and compare it to that seen on conventional smears. Pertinent immunocytochemical stains, performed on ThinPrep slides are also discussed. Case presentation The first case is a woman with a history of desmoplastic melanoma of the scalp with previous local recurrences and lymph node metastasis with a new submandibular mass. The second case is a man with a previously resected desmoplastic melanoma with his first local recurrence. Conventional smears, including air-dried Diff-Quik-stained and alcohol-fixed Papanicolaou-stained smears, demonstrated aggregates of pleomorphic spindle cells admixed with fibrous stroma and single spindle cells. In both cases, nuclei were elongated and plump with irregular nuclear contours, deep grooves, and folds. Chromatin was dark and coarse with either inconspicuous or multiple prominent nucleoli. Cytoplasm was located at the nuclear poles and was fine, wispy, and delicate. The background was clean with no evidence of necrosis or melanin pigment. Papanicolaou-stained ThinPrep slides were prepared from needle rinses and demonstrated excellent correlation of nuclear and cytoplasmic detail of single spindle cells to that seen on conventional smears with the exception of only slight decrease in nuclear size; however, nuclear and cytoplasmic detail of spindle cells embedded in stroma was markedly attenuated. Confirmatory immunostain for S-100 protein in both cases was performed on ThinPrep slides demonstrating crisp cytoplasmic staining in the spindle cells. Conclusion The cytomorphology of desmoplastic melanoma shows excellent correlation between cytomorphology of single spindle cells on conventional smears and on ThinPrep slides. The major difference noted on ThinPrep slides was attenuated nuclear and cytoplasmic detail of spindle cells embedded in fibrous stoma. PMID:17880690

EFHC1, a protein mutated in juvenile myoclonic epilepsy, associates with the mitotic spindle through its N-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijs, Laurence de; Lakaye, Bernard; Coumans, Bernard

2006-09-10

A novel gene, EFHC1, mutated in juvenile myoclonic epilepsy (JME) encodes a protein with three DM10 domains of unknown function and one putative EF-hand motif. To study the properties of EFHC1, we expressed EGFP-tagged protein in various cell lines. In interphase cells, the fusion protein was present in the cytoplasm and in the nucleus with specific accumulation at the centrosome. During mitosis EGFP-EFHC1 colocalized with the mitotic spindle, especially at spindle poles and with the midbody during cytokinesis. Using a specific antibody, we demonstrated the same distribution of the endogenous protein. Deletion analyses revealed that the N-terminal region of EFHC1more » is crucial for the association with the mitotic spindle and the midbody. Our results suggest that EFHC1 could play an important role during cell division.« less

Mycobacterium genavense-induced spindle cell pseudotumor in a pediatric hematopoietic stem cell transplant recipient: Case report and review of the literature.

PubMed

Coelho, Ritika; Hanna, Rabi; Flagg, Aron; Stempak, Lisa M; Ondrejka, Sarah; Procop, Gary W; Harrington, Susan; Zembillas, Anthony; Kusick, Karissa; Gonzalez, Blanca E

2017-04-01

We describe the first reported pediatric patient to our knowledge with a spindle cell pseudotumor caused by Mycobacterium genavense in a hematopoietic stem cell transplant recipient, and review the literature of such an entity in the transplant population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis

PubMed Central

Marston, Adele L.; Wassmann, Katja

2017-01-01

Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC) controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3), and Aurora B and C (Ipl1) will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid. PMID:29322045

Anterior uveal spindle cell tumor in a cat.

PubMed

Evans, Paige M; Lynch, Gwendolyn L; Dubielzig, Richard R

2010-11-01

To describe a case of anterior uveal spindle cell tumor in a cat with features similar to spindle cell tumor of blue eyed dogs. A 10-year-old female spayed domestic short-haired cat was referred for an iris mass OS. The mass was solitary, nodular, nonpigmented, located medially, and causing dyscoria. A diagnosis of a benign epithelial tumor was suggested by a FNA of the mass. The cat was lost to follow-up for 2 years, after which time she re-presented with glaucoma, blindness and grossly evident iridal mass enlargement OS. Transconjunctival enucleation was performed and the globe submitted for histopathology. Histopathology of the enucleated globe revealed the superior iris to be infiltrated and effaced by a large population of neoplastic spindle cells. The cells were arranged in streams and bundles and exhibited Antoni-A and Antoni-B tissue patterns, which are characteristic of Schwann cell tumors. Mitotic figures were rare and cellular pleomorphism moderate. Immunohistochemical staining was positive for S-100 protein and glial fibrillary acidic protein (GFAP), and negative for Melan-A. Interestingly, there was no histological evidence of glaucoma. Based on its histopathologic characteristics, this iris tumor was diagnosed as a Schwann cell variant of a peripheral nerve sheath tumor (PNST) closely resembling the spindle cell tumor of blue-eyed dogs. Anterior uveal PNST has not been previously reported in cats to the authors' knowledge. The presence of Antoni type A and type B tissue patterns along with immunohistochemical staining may facilitate a diagnosis of PNST and rule out malignant melanoma. © 2010 American College of Veterinary Ophthalmologists.

Myxoid atypical fibroxanthoma: a previously undescribed variant.

PubMed

Patton, A; Page, R; Googe, P B; King, R

2009-11-01

Atypical fibroxanthomas (AFX) are dermal-based cutaneous tumors typically found in sun-damaged skin of the elderly. Histologic variants include spindle cell, clear cell, osteoid, osteoclastic, chondroid, pigmented, and granular cell. To date, myxoid change in AFX, has not been described. Four cases were retrieved from the consultation and surgical pathology files of Knoxville Dermatopathology Laboratory, Knoxville, Tennessee during a 4-year period. The clinical and histologic findings were reviewed and Alcian blue/periodic acid-Schiff (PAS) stain and panel of immunohistochemical stains was obtained. All 4 lesions occurred as solitary lesions in elderly males on the head and neck (2 cases) and upper extremity (2 cases). Histologically all tumors demonstrated a well-circumscribed, cellular lesion centered in the dermis and composed of a mix of atypical pleomorphic and spindle cells in a prominent myxomatous background. A junctional component was absent and the tumors did not arise from the epidermis or adnexal structures. Subcutaneous involvement was absent in all cases. Tumor cells were negative for melanocytic and epithelial markers. Positive staining was noted with CD10 (3/4 cases) and vimentin (4/4 cases). Myxoid change in AFX is rare and previously undescribed in the English literature. Myxoid change may be a prominent finding in benign and malignant cutaneous tumors and awareness of this variant of AFX will avoid misdiagnosis.

Inversin modulates the cortical actin network during mitosis

PubMed Central

Werner, Michael E.; Ward, Heather H.; Phillips, Carrie L.; Miller, Caroline; Gattone, Vincent H.

2013-01-01

Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity defect, we investigated whether inversin plays a role in cell division. In developing nephrons from inv−/− mouse embryos we observed heterogeneity of nuclear size, increased cell membrane perimeters, cells with double cilia, and increased frequency of binuclear cells. Depletion of inversin by siRNA in cultured mammalian cells leads to an increase in bi- or multinucleated cells. While spindle assembly, contractile ring formation, or furrow ingression appears normal in the absence of inversin, mitotic cell rounding and the underlying rearrangement of the cortical actin cytoskeleton are perturbed. We find that inversin loss causes extensive filopodia formation in both interphase and mitotic cells. These cells also fail to round up in metaphase. The resultant spindle positioning defects lead to asymmetric division plane formation and cell division. In a cell motility assay, fibroblasts isolated from inv−/− mouse embryos migrate at half the speed of wild-type fibroblasts. Together these data suggest that inversin is a regulator of cortical actin required for cell rounding and spindle positioning during mitosis. Furthermore, cell division defects resulting from improper spindle position and perturbed actin organization contribute to altered nephron morphogenesis in the absence of inversin. PMID:23515530

Integrity and Function of the Saccharomyces cerevisiae Spindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

PubMed Central

Casey, Amanda K.; Dawson, T. Renee; Chen, Jingjing; Friederichs, Jennifer M.; Jaspersen, Sue L.; Wente, Susan R.

2012-01-01

The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother–daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs. PMID:22798490

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

PubMed

Matsuhara, Hirotada; Yamamoto, Ayumu

2016-01-01

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

PubMed

Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K; Peluso, John J

2014-08-01

Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development. © 2014 by the Society for the Study of Reproduction, Inc.

Cell Motility by Labile Association of Molecules

PubMed Central

Inoué, Shinya; Sato, Hidemi

1967-01-01

This article summarizes our current views on the dynamic structure of the mitotic spindle and its relation to mitotic chromosome movements. The following statements are based on measurements of birefringence of spindle fibers in living cells, normally developing or experimentally modified by various physical and chemical agents, including high and low temperatures, antimitotic drugs, heavy water, and ultraviolet microbeam irradiation. Data were also obtained concomitantly with electron microscopy employing a new fixative and through measurements of isolated spindle protein. Spindle fibers in living cells are labile dynamic structures whose constituent filaments (microtubules) undergo cyclic breakdown and reformation. The dynamic state is maintained by an equilibrium between a pool of protein molecules and their linearly aggregated polymers, which constitute the microtubules or filaments. In living cells under physiological conditions, the association of the molecules into polymers is very weak (absolute value of ΔF 25°C < 1 kcal), and the equilibrium is readily shifted to dissociation by low temperature or by high hydrostatic pressure. The equilibrium is shifted toward formation of polymer by increase in temperature (with a large increase in entropy: ΔS 25°C ≃ 100 eu) or by the addition of heavy water. The spindle proteins tend to polymerize with orienting centers as their geometrical foci. The centrioles, kinetochores, and cell plate act as orienting centers successively during mitosis. Filaments are more concentrated adjacent to an orienting center and yield higher birefringence. Astral rays, continuous fibers, chromosomal fibers, and phragmoplast fibers are thus formed by successive reorganization of the same protein molecules. During late prophase and metaphase, polymerization takes place predominantly at the kinetochores; in metaphase and anaphase, depolymerization is prevalent near the spindle poles. When the concentration of spindle protein is high, fusiform bundles of polymer are precipitated out even in the absence of obvious orienting centers. The shift of equilibrium from free protein molecules to polymer increases the length and number of the spindle microtubules or filaments. Slow depolymerization of the polymers, which can be brought about by low concentrations of colchicine or by gradual cooling, allows the filaments to shorten and perform work. The dynamic equilibrium controlled by orienting centers and other factors provides a plasusible mechanism by which chromosomes and other organelles, as well as the cell surface, are deformed or moved by temporarily organized arrays of microtubules or filaments. PMID:6058222

Self-organization mechanisms in the assembly and maintenance of bipolar spindles

NASA Astrophysics Data System (ADS)

Burbank, Kendra Stewart

Anastral, meiotic spindles are thought to be organized differently from astral, mitotic spindles, but the field has lacked basic structural information required to describe and model them, including the location of microtubule nucleating sites and minus ends. How the various components of spindles act together to establish and maintain the dynamic bipolar structure of spindles is not understood. We measure the distributions of oriented microtubules (MTs) in metaphase anastral spindles in Xenopus extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We find that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. This dads to the surprising conclusion that spindles contained many short MTs, not connected to the spindle poles. Based on these data, we propose a slide-and-cluster model based on four known molecular activities: MT nucleation near chromosomes, the sliding of MTs by a plus-enddirected motor, the clustering of their minus ends by a minus-end-directed motor, and the loss of MTs by dynamic instability. This work demonstrates how the interplay between two types of motors together with continual nucleation of MTs by chromosomes could organize the MTs into spindles. Our model applies to overlapping, nonkinetochore MTs in anastral spindles, and perhaps also to interpolar MTs in astral spindles. We show mathematically that the slide-and-cluster mechanism robustly forms bipolar spindles a stable steady-state length, sometimes with sharp poles. This model accounts for several experimental observations that were difficult to explain with existing models, and is the first self contained model for anastral spindle assembly, MT sliding (known as poleward flux), and spindle bistability. Our experimental results support the slide-and-cluster scenario; most significantly, we find that MT sliding slows near spindle poles, confirming the models primary prediction.

Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication

DTIC Science & Technology

2006-03-01

AD Award Number: W81XWH-05-1-0310 TITLE: Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication PRINCIPAL...Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication Sb. GRANT...Degradation, Cell Cycle, Spindle Pole Body 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON OF ABSTRACT OF

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis.

PubMed

He, Zhimin; Wu, Junyu; Su, Xiaonan; Zhang, Ye; Pan, Lixia; Wei, Huimin; Fang, Qiang; Li, Haitao; Wang, Da-Liang; Sun, Fang-Lin

2016-02-26

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Potato spindle tuber viroid detection in phloem exudates and guttation fluid of tomato plants (Solanum lycopersicum)

USDA-ARS?s Scientific Manuscript database

Potato spindle tuber viroid (PSTVd) is a single-stranded, non protein-encoding, covalently-closed circular RNA molecule (359nt) that infects many horticultural and agricultural crops. PSTVd is mechanically transmitted, replicates in the nucleus, and moves cell-to-cell through plasmodesmata. Though i...

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer.

PubMed

Mason, Jacqueline M; Wei, Xin; Fletcher, Graham C; Kiarash, Reza; Brokx, Richard; Hodgson, Richard; Beletskaya, Irina; Bray, Mark R; Mak, Tak W

2017-03-21

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 K i = 0.09 ± 0.02 nM; cellular Mps1 EC 50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.

Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer

PubMed Central

Mason, Jacqueline M.; Wei, Xin; Fletcher, Graham C.; Kiarash, Reza; Brokx, Richard; Hodgson, Richard; Beletskaya, Irina; Bray, Mark R.; Mak, Tak W.

2017-01-01

Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors. PMID:28270606

Time-sequential observation of spindle and phragmoplast orientation in BY-2 cells with altered cortical actin microfilament patterning.

PubMed

Kojo, Kei H; Yasuhara, Hiroki; Hasezawa, Seiichiro

2014-01-01

Precise division plane determination is essential for plant development. At metaphase, a dense actin microfilament meshwork appears on both sides of the cell center, forming a characteristic cortical actin microfilament twin peak pattern in BY-2 cells. We previously reported a strong correlation between altered cortical actin microfilament patterning and an oblique mitotic spindle orientation, implying that these actin microfilament twin peaks play a role in the regulation of mitotic spindle orientation. In the present study, time-sequential observation was used to reveal the progression from oblique phragmoplast to oblique cell plate orientation in cells with altered cortical actin microfilament patterning. In contrast to cells with normal actin microfilament twin peaks, oblique phragmoplast reorientation was rarely observed in cells with altered cortical actin microfilament patterning. These results support the important roles of cortical actin microfilament patterning in division plane orientation.

Time-sequential observation of spindle and phragmoplast orientation in BY-2 cells with altered cortical actin microfilament patterning.

PubMed

Kojo, Kei H; Yasuhara, Hiroki; Hasezawa, Seiichiro

2014-06-18

Precise division plane determination is essential for plant development. At metaphase, a dense actin microfilament meshwork appears on both sides of the cell center, forming a characteristic cortical actin microfilament twin peak pattern in BY-2 cells. We previously reported a strong correlation between altered cortical actin microfilament patterning and an oblique mitotic spindle orientation, implying that these actin microfilament twin peaks play a role in the regulation of mitotic spindle orientation. In the present study, time-sequential observation was used to reveal the progression from oblique phragmoplast to oblique cell plate orientation in cells with altered cortical actin microfilament patterning. In contrast to cells with normal actin microfilament twin peaks, oblique phragmoplast reorientation was rarely observed in cells with altered cortical actin microfilament patterning. These results support the important roles of cortical actin microfilament patterning in division plane orientation.

Dedifferentiated Chondrosarcoma of the Larynx.

PubMed

Fidai, Shiraz S; Ginat, Daniel T; Langerman, Alexander J; Cipriani, Nicole A

2016-09-01

Primary dedifferentiated chondrosarcoma occurring in the larynx is a rare head and neck malignancy. The cases reported in the literature suggest male gender predilection and variable clinical outcomes ranging from disease-free survival to disease-related death. Although a calcified matrix is suggestive of chondrosarcoma, the dedifferentiated component is not readily appreciated on conventional imaging modalities and thorough tissue sampling is necessary for confirming the diagnosis. Histologically, there is an abrupt transition from a well-differentiated chondrosarcoma to a high-grade spindle cell component, which can show focal heterologous differentiation. These features are exemplified in this sine qua non radiology-pathology correlation article.

Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans.

PubMed

Dejima, Katsufumi; Kang, Sukryool; Mitani, Shohei; Cosman, Pamela C; Chisholm, Andrew D

2014-11-01

Wnt signals orient mitotic spindles in development, but it remains unclear how Wnt signaling is spatially controlled to achieve precise spindle orientation. Here, we show that C. elegans syndecan (SDN-1) is required for precise orientation of a mitotic spindle in response to a Wnt cue. We find that SDN-1 is the predominant heparan sulfate (HS) proteoglycan in the early C. elegans embryo, and that loss of HS biosynthesis or of the SDN-1 core protein results in misorientation of the spindle of the ABar blastomere. The ABar and EMS spindles both reorient in response to Wnt signals, but only ABar spindle reorientation is dependent on a new cell contact and on HS and SDN-1. SDN-1 transiently accumulates on the ABar surface as it contacts C, and is required for local concentration of Dishevelled (MIG-5) in the ABar cortex adjacent to C. These findings establish a new role for syndecan in Wnt-dependent spindle orientation. © 2014. Published by The Company of Biologists Ltd.

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

PubMed Central

Colombié, Nathalie; Głuszek, A. Agata; Meireles, Ana M.; Ohkura, Hiroyuki

2013-01-01

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle. PMID:23785300

Malignant perivascular epithelioid cell neoplasm (PEComa) of the urinary bladder with TFE3 gene rearrangement: clinicopathologic, immunohistochemical, and molecular features.

PubMed

Williamson, Sean R; Bunde, Paula J; Montironi, Rodolfo; Lopez-Beltran, Antonio; Zhang, Shaobo; Wang, Mingsheng; Maclennan, Gregory T; Cheng, Liang

2013-10-01

Recently, a small subgroup of PEComas has been recognized to harbor rearrangements involving TFE3, a gene also involved in rearrangements in translocation-associated renal cell carcinomas and alveolar soft part sarcomas. The few TFE3 rearrangement-associated PEComas reported have exhibited distinctive pathologic characteristics contrasting to PEComas in general, including predominantly epithelioid nested or alveolar morphology and underexpression of muscle markers by immunohistochemistry. In this study, we report the clinicopathologic, immunohistochemical, and molecular features of a primary urinary bladder PEComa diagnosed by transurethral resection in a 55-year-old woman that clinically mimicked urothelial carcinoma. Light microscopy demonstrated mixed spindle cell and epithelioid morphology with the epithelioid component preferentially associated with blood vessels. Immunohistochemistry revealed positive staining for HMB45, tyrosinase, MiTF, cathepsin K, smooth muscle actin, and TFE3 protein. Fluorescence in situ hybridization for the TFE3 gene revealed a split signal pattern, indicating TFE3 rearrangement. X chromosome inactivation analysis demonstrated a clonal pattern despite the heterogenous appearance of the tumor. Unfortunately, despite surgical resection and sarcoma-directed therapy, the patient died of metastatic disease 12 months after diagnosis. This report adds to the known data regarding urinary bladder PEComas and PEComas with TFE3 rearrangement, indicating that both can pursue an aggressive course. Although the few reported TFE3-rearranged PEComas have predominantly lacked a spindle cell component and expression of smooth muscle actin and MiTF by immunohistochemistry, the findings in this study indicate that these features are sometimes present in TFE3-rearranged PEComas.

Spindle pole body component 25 homolog expressed by ECM stiffening is required for lung cancer cell proliferation.

PubMed

Jeong, Jangho; Keum, Seula; Kim, Daehwan; You, Eunae; Ko, Panseon; Lee, Jieun; Kim, Jaegu; Kim, Jung-Woong; Rhee, Sangmyung

2018-06-12

Accumulating evidence has shown that matrix stiffening in cancer tissue by the deposition of extracellular matrix (ECM) is closely related with severe tumor progression. However, much less is known about the genes affected by matrix stiffness and its signaling for cancer progression. In the current research, we investigated the differential gene expression of a non-small lung adenocarcinoma cell line, H1299, cultured under the conditions of soft (∼0.5 kPa) and stiff (∼40 kPa) matrices, mimicking the mechanical environments of normal and cancerous tissues, respectively. For integrated transcriptome analysis, the genes identified by ECM stiffening were compared with 8248 genes retrieved from The Cancer Genome Atlas Lung Adenocarcinoma (TCGA). In stiff matrix, 29 genes were significantly upregulated, while 75 genes were downregulated. The screening of hazard ratios for these genes using the Kaplan-Meier Plotter identified 8 genes most closely associated with cancer progression under the condition of matrix stiffening. Among these genes, spindle pole body component 25 homolog (SPC25) was one of the most up-regulated genes in stiff matrix and tumor tissue. Knockdown of SPC25 in H1299 cells using shRNA significantly inhibited cell proliferation with downregulation of the expression of checkpoint protein, Cyclin B1, under the condition of stiff matrix whereas the proliferation rate in soft matrix was not affected by SPC25 silencing. Thus, our findings provide novel key molecules for studying the relationship of extracellular matrix stiffening and cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

PubMed Central

Ji, Zhejian; Gao, Haishan; Jia, Luying; Li, Bing; Yu, Hongtao

2017-01-01

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment. DOI: http://dx.doi.org/10.7554/eLife.22513.001 PMID:28072388

[Morphological pathology of classic Kaposi's sarcoma. Ultrastructural studies and reflections on histogenesis].

PubMed

Holzhausen, H J; Stiller, D; Sachs, M

1988-01-01

Histological and electron-microscopic studies were conducted into biopsy material from cases of what is called the classical type of idiopathic Kaposi's sarcoma without acquired immunodeficiency syndrome. Ultrastructural analysis was conducted, with the view to characterizing a possible progenitor cell from which the angioblastic and fibroblastic elements were likely to originate. The biopsy material had been obtained from two males, aged 86 or 83 years, who had been afflicted with the disease for 18 or 8 years. The nodular lesions were typical of Kaposi's sarcoma and were, histologically, made up of variable mixtures of vascular and spindle cell elements. The angiomatous structures were a capillary meshwork or sinusoidal patterns lined by atypical endothelial cells. The spindle cell areas contained large numbers of slit-like spaces which were without endothelial lining but were stuffed with erythrocytes. Flattened endothelioid cells were recordable from semi-thin-sections of some clefts. Haemosiderin was, typically, deposited in places. Electron microscopically, the endothelial cells of vascular channels exhibited varying amounts of characteristic organelles, such as Weibel-Palade bodies, microfilaments and pinocytotic vesicles as well as basal membranes. Cells with typical endothelial markers, too, were detectable in solid sprouts or in capillary-like differentiations with narrow or small lumina. The spindle cell tumour areas consisted of fibroblastic cells with plenty of rough endoplasmic reticulum and surrounded by material of basal membrane nature. Also visible were solid, sprout-type multilayer cell complexes surrounded by basal membranes which exhibited undifferentiated or primitive cellular forms, endothelioid and pericytic. Transitional forms from these complexes to the above vascular tumours or the spindle-cell formations were detectable. These ultrastructural findings might be interpreted to the effect that an angioblastically determined mesenchymal cell, a so-called endothelioblast, was thinkable and was discussed as the precursor cell of atypical vascular and spindle cell proliferation in Kaposi's sarcoma.

Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

PubMed Central

Trinkle-Mulcahy, Laura; Porter, Michael; Jeyaprakash, A. Arockia

2015-01-01

Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression. PMID:26124292

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

PubMed Central

Spiridonov, Ilia S.; McIntosh, J. Richard

2007-01-01

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end–directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole. PMID:17409356

Noninvasive three-dimensional live imaging methodology for the spindles at meiosis and mitosis

NASA Astrophysics Data System (ADS)

Zheng, Jing-gao; Huo, Tiancheng; Tian, Ning; Chen, Tianyuan; Wang, Chengming; Zhang, Ning; Zhao, Fengying; Lu, Danyu; Chen, Dieyan; Ma, Wanyun; Sun, Jia-lin; Xue, Ping

2013-05-01

The spindle plays a crucial role in normal chromosome alignment and segregation during meiosis and mitosis. Studying spindles in living cells noninvasively is of great value in assisted reproduction technology (ART). Here, we present a novel spindle imaging methodology, full-field optical coherence tomography (FF-OCT). Without any dye labeling and fixation, we demonstrate the first successful application of FF-OCT to noninvasive three-dimensional (3-D) live imaging of the meiotic spindles within the mouse living oocytes at metaphase II as well as the mitotic spindles in the living zygotes at metaphase and telophase. By post-processing of the 3-D dataset obtained with FF-OCT, the important morphological and spatial parameters of the spindles, such as short and long axes, spatial localization, and the angle of meiotic spindle deviation from the first polar body in the oocyte were precisely measured with the spatial resolution of 0.7 μm. Our results reveal the potential of FF-OCT as an imaging tool capable of noninvasive 3-D live morphological analysis for spindles, which might be useful to ART related procedures and many other spindle related studies.

Cell-cycle control in the face of damage--a matter of life or death.

PubMed

Clarke, Paul R; Allan, Lindsey A

2009-03-01

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

PubMed Central

Yukawa, Masashi; Kawakami, Tomoki; Okazaki, Masaki; Kume, Kazunori; Tang, Ngang Heok; Toda, Takashi

2017-01-01

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly. PMID:29021344

"Sarcomatoid" carcinomas of the lung: a clinicopathological study of 86 cases with a new perspective on tumor classification.

PubMed

Weissferdt, Annikka; Kalhor, Neda; Correa, Arlene M; Moran, Cesar A

2017-05-01

Pulmonary sarcomatoid carcinoma includes a heterogenous group of tumors difficult to diagnose and treat. We report the clinicopathological features of 86 such tumors, including 74 pleomorphic and 12 spindle cell carcinomas, and propose a novel approach to the classification of these neoplasms in an attempt to better guide patient management. The patients were 47 men and 39 women aged 36 to 87 years (mean, 63 years) who primarily presented with shortness of breath, cough, and chest pain. Eighty-six percent of patients had a smoking history. Histologically, the pleomorphic carcinomas consisted of spindle and/or giant cells with varying proportions of conventional non-small cell carcinoma in the form of adenocarcinoma (n=29), squamous cell carcinoma (n=10), or large cell carcinoma (n=18); 17 cases contained a mix of spindle and giant cells only. The 12 spindle cell carcinomas consisted of spindle cells only. Based on the combined histopathologic and immunohistochemical features of these tumors, we were able to reanalyze the spectrum of these lesions and reclassify them accordingly. Statistical analysis revealed an overall survival at 3, 5, and 10 years of 42.9%, 34.6%, and 23.5%, respectively, and a median survival of 15 months. Log-rank test showed that in multivariate analysis, only pathological T stage was a factor associated with prognosis. The current classification of pulmonary sarcomatoid carcinomas precludes optimal triaging of these tumors with the risk of denying patients access to novel treatment. Our proposal for a reclassification of these tumors would more accurately guide patient management and facilitate targeted therapies. Copyright © 2016 Elsevier Inc. All rights reserved.

Spontaneous extraskeletal osteosarcoma with various histological growth patterns in the abdominal wall of an ICR mouse

PubMed Central

Ito, Tsuyoshi; Katoh, Yoshitaka; Shimada, Yuko; Ohnuma-Koyama, Aya; Takahashi, Naofumi; Kuwahara, Maki; Harada, Takanori

2015-01-01

Extraskeletal osteosarcoma is extremely rare in mice. This case report demonstrates a spontaneous murine extraskeletal osteosarcoma that exhibited various histological growth patterns in an ICR mouse. At necropsy, the tumor mass was located in the abdominal wall and was 45 × 30 × 25 mm in size. Histopathologically, the tumor showed the following four growth patterns: a solid pattern of polygonal cells embedded in an osteoid eosinophilic matrix with calcification, an irregular sheet pattern of short spindle cells accompanying some eosinophilic multinucleated cells, a fascicular pattern of spindle cells and a cystic pattern lined by short spindle cells. Immunohistochemically, most of the tumor cells were positive for vimentin, proliferating cell nuclear antigen and osterix. The multinucleated cells mentioned above were desmin positive and were regarded as regenerative striated muscles but not tumor cells. Since no clear continuity with normal bone tissues was observed, the tumor was diagnosed as an “extraskeletal osteosarcoma.” PMID:26989300

Surgical and medical management of a uterine spindle cell tumor in an African hedgehog (Atelerix albiventris).

PubMed

Done, Lisa B; Deem, Sharon L; Fiorello, Christine V

2007-12-01

A 5-yr-old female African hedgehog (Ateleris albiventris) presented with hematuria. Vulvar culture results revealed a 4+ growth of Enterococcus sp. and gamma-Streptococcus sp. susceptible to trimethoprim sulfa and enrofloxacin. Ultrasound evaluation of the abdomen revealed an unidentifiable tubular structure in the region of the reproductive tract. An exploratory laparotomy and ovariohysterectomy were performed. Pathologic studies of the uterus showed a uterine spindle cell tumor, uterine endometrial polyp, uterine adenomyosis, and a possible acute infarct resulting in uterine wall necrosis. Hematuria did not reoccur, and the hedgehog lived for another 19 mo until she died from an oral squamous cell carcinoma. To date, this is the first report of a uterine spindle cell tumor in an African hedgehog.

Low-fat and fat-free pleomorphic lipomas: a diagnostic challenge.

PubMed

Sachdeva, Mandi P; Goldblum, John R; Rubin, Brian P; Billings, Steven D

2009-07-01

Pleomorphic lipomas are benign tumors that most commonly present as subcutaneous masses in the head and neck, shoulder, or back region of middle-aged to elderly men. They are related to spindle cell lipomas based on shared cytogenetic aberrations and histologic features. When little or no fat is present, the diagnosis can be challenging. A review of 38 pleomorphic lipomas seen in consultation revealed 7 cases in which fat was present in reduced (<5%) amounts (n = 5) or absent (n = 2). Six of 7 cases were from men with a mean age of 59 years. Excluding 1 case where the site was not specified, they all presented as solitary well-circumscribed subcutaneous masses in the head and neck (n = 3) or shoulder (n = 2) region. The seventh case was an intradermal tumor from the nose of a 48-year-old woman. All displayed pleomorphic and multinucleated floret cells interspersed among bland spindle cells and ropey collagen. They were diffusely immunoreactive for CD34. Referring diagnoses, when provided, included myxofibrosarcoma, giant cell fibroblastoma, and granulomatous rosacea for the tumor from the nose; none considered pleomorphic lipomas. When fat is absent or present in reduced amounts, clinical context and identification of classic nonlipogenic components are essential for the diagnosis of pleomorphic lipomas.

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

PubMed Central

Sanchez, Marco A.; Tran, Khoa D.; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M.

2016-01-01

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

BuGZ is required for Bub3 stability, Bub1 kinetochore function, and chromosome alignment

PubMed Central

Toledo, Chad M.; Herman, Jacob A.; Olsen, Jonathan B.; Ding, Yu; Corrin, Philip; Girard, Emily J.; Olson, James M.; Emili, Andrew; DeLuca, Jennifer G.; Paddison, Patrick J.

2014-01-01

Summary During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human Glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora kinase B activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes. PMID:24462187

An unusual and spectacular case of spindle cell lipoma of the posterior neck invading the spinal cervical canal and posterior cranial fossa.

PubMed

Petit, Damien; Menei, Philippe; Fournier, Henri-Dominique

2011-11-01

The authors describe the first case of spindle cell lipoma of the posterior neck invading the upper cervical spinal canal and the posterior cranial fossa. Spindle cell lipoma is an extremely rare variant of benign lipoma. It usually occurs as a solitary subcutaneous well-circumscribed lesion in the posterior neck or shoulders of adult men. Local aggressiveness is unusual. This 61-year-old man presented with an increased left cerebellar syndrome and headaches. He also had a posterior neck tumefaction, which had been known about for a long time. Computed tomography and MR imaging studies revealed a voluminous mass extending to the upper cervical canal and posterior cranial fossa and eroding the neighboring bones. The lesion was well delimited, and contrast enhancement was intense and heterogeneous. The tumor, which had initially developed under the muscles of the posterior neck, was totally resected. Histological assessment revealed numerous fat cells with spindle cells secreting collagen. The large size of the tumor and the submuscular location, bone erosion, and compression of the CNS were unusual in this rare subtype of benign adipose tumor. Its presentation could simulate a sarcoma.

Histological characterization of regression in acquired immunodeficiency syndrome-related Kaposi's sarcoma.

PubMed

Pantanowitz, Liron; Dezube, Bruce J; Pinkus, Geraldine S; Tahan, Steven R

2004-01-01

Kaposi's sarcoma (KS) is an angioproliferative lesion that may regress or progress. Progression is related to spindle cell proliferation and the expression of human herpes virus-8 latency genes, including latent nuclear antigen-1 (LNA-1), cyclin-D1, and bcl-2. KS regression has not been well characterized histologically. Therefore, this study was undertaken to characterize the histopathology of pharmacologically induced regressed cutaneous KS. Skin punch biopsies from eight patients with acquired immunodeficiency syndrome (AIDS)-related KS, that regressed following chemotherapy with paclitaxel or the angiogenesis inhibitor Col-3, were investigated by light microscopy. Comparative immunophenotyping on pre- and post-treatment specimens for CD31, LNA-1, cyclin-D1, bcl-2, and CD117 (c-kit) was performed. Clinical and histologic features of regression were similar for paclitaxel and Col-3 treatment. On clinical examination, lesions flattened, became smaller, and lost their purple-red appearance, resulting in an orange-brown macule. Histological regression was divided into partial (n = 3) and complete (n = 5) regression. Partially regressed lesions had a significant reduction of spindle cells in the dermal interstitium, with residual spindle cells arranged around superficial and mid-dermal capillaries. Complete regression was characterized by an absence of detectable spindle cells, with a slight increase in capillaries of the superficial plexus. All regressed samples exhibited a prominent, superficial, perivascular, lymphocytic infiltrate and abundant dermal hemosiderin-laden macrophages. This clinicopathologic picture resembled the findings of pigmented purpura. CD31 staining correlated with the reduction of spindle cells. Regression was accompanied by a quantitative and qualitative decrease in LNA-1 and cyclin-D1 immunoreactivity, but no change in bcl-2 or c-kit expression. Pharmacologically induced regression of AIDS-related cutaneous KS is characterized by a complete loss or decrease of spindle cells, increased lymphocytes, and prominent dermal siderophage deposition. Without any prior knowledge of the history of KS regression following therapy, regressed KS lesions may be misdiagnosed clinically and histologically as pigmented purpuric dermatitis.

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates

PubMed Central

Mahe, Karan; Ou, Tingyoung; Castro, Noemi M; Christensen, Lana N; Cheung, Lee; Jiang, Xueer; Yoon, Daniel; Huang, Bo

2018-01-01

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism. PMID:29708497

Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

PubMed Central

Skop, Ahna R.; Bergmann, Dominique; Mohler, William A.; White, John G.

2013-01-01

Background The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. Results We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. Conclusions We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell. PMID:11378383

Chromosome movement in lysed mitotic cells is inhibited by vanadate

PubMed Central

1978-01-01

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10-- 100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate. PMID:152767

TMAP/CKAP2 is essential for proper chromosome segregation.

PubMed

Hong, Kyung Uk; Kim, Eunhee; Bae, Chang-Dae; Park, Joobae

2009-01-15

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), is a novel mitotic spindle-associated protein which is frequently up-regulated in various malignances. However, its cellular functions remain unknown. Previous reports suggested that the cellular functions of TMAP/CKAP2 pertain to regulation of the dynamics and assembly of the mitotic spindle. To investigate its role in mitosis, we studied the effects of siRNA-mediated depletion of TMAP/CKAP2 in cultured mammalian cells. Unexpectedly, TMAP/CKAP2 knockdown did not result in significant alterations of the spindle apparatus. However, TMAP/CKAP2-depleted cells often exhibited abnormal nuclear morphologies, which were accompanied by abnormal organization of the nuclear lamina, and chromatin bridge formation between two daughter cell nuclei. Time lapse video microscopy revealed that the changes in nuclear morphology and chromatin bridge formations observed in TMAP/CKAP2-depleted cells are the result of defects in chromosome segregation. Consistent with this, the spindle checkpoint activity was significantly reduced in TMAP/CKAP2-depleted cells. Moreover, chromosome missegregation induced by depletion of TMAP/CKAP2 ultimately resulted in reduced cell viability and increased chromosomal instability. Our present findings demonstrate that TMAP/CKAP2 is essential for proper chromosome segregation and for maintaining genomic stability.

Benign Fibrous Histiocytoma: An Uncommon Presentation.

PubMed

Sarkar, Sagarika; Maiti, Moumita; Bhattacharyya, Palas; Sarkar, Ranu

2017-07-01

Intracranial fibrous histiocytomas are rare; Benign Fibrous Histiocytoma (BFH) being uncommon than its malignant counterpart. BFH comprises fibroblasts and histiocytes without any nuclear pleomorphism or atypia. We present a case of a 42-year-old male who had swelling over the occipital region for the past five years, which progressively increased in size. He developed headache, dizziness, and gait disturbance over the last six months. Computed tomographic scan revealed a posterior fossa space-occupying lesion. Fine-needle aspiration cytology from the swelling revealed spindled fibroblasts along with histiocytes and multinucleated giant cells. Later, histopathology showed presence of spindle-shaped cells in storiform pattern admixed with histiocytes and giant cells. The giant cells and histiocytes were immunopositive for CD68 and spindled cells were positive for vimentin, but immunonegative for CD34, epithelial membrane antigen, CD1a and S100. The final diagnosis was intracranial BFH. We present this case because of its extreme rarity and unusual location.

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

PubMed Central

Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

2010-01-01

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

The invariant cleavage pattern displayed by ascidian embryos depends on spindle positioning along the cell's longest axis in the apical plane and relies on asynchronous cell divisions

PubMed Central

Dumollard, Rémi; Minc, Nicolas; Salez, Gregory; Aicha, Sameh Ben; Bekkouche, Faisal; Hebras, Céline; Besnardeau, Lydia; McDougall, Alex

2017-01-01

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula. DOI: http://dx.doi.org/10.7554/eLife.19290.001 PMID:28121291

Spindle formation in the mouse embryo requires Plk4 in the absence of centrioles.

PubMed

Coelho, Paula A; Bury, Leah; Sharif, Bedra; Riparbelli, Maria G; Fu, Jingyan; Callaini, Giuliano; Glover, David M; Zernicka-Goetz, Magdalena

2013-12-09

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation

PubMed Central

Fennell, Alex; Fernández-Álvarez, Alfonso; Tomita, Kazunori

2015-01-01

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere–centrosome contact instead of telomere–centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks. PMID:25688135

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia, but is dispensable for pathogenicity in Magnaporthe oryzae.

PubMed

Li, Chao; Yang, Jun; Zhou, Wei; Chen, Xiao-Lin; Huang, Jin-Guang; Cheng, Zhi-Hua; Zhao, Wen-Sheng; Zhang, Yan; Peng, You-Liang

2014-11-01

Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria.

PubMed

Walsh, Charles J

2012-01-01

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.

Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement

PubMed Central

1978-01-01

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally. PMID:681451

Retention of Pax3 expression in satellite cells of muscle spindles.

PubMed

Kirkpatrick, Lisa J; Yablonka-Reuveni, Zipora; Rosser, Benjamin W C

2010-04-01

Intrafusal fibers within muscle spindles retain features characteristic of immaturity, unlike the larger and more numerous extrafusal fibers constituting the bulk of skeletal muscle. Satellite cells (SCs), myogenic progenitors, are detected on the surfaces of both intrafusal and extrafusal fibers, but little is known of spindle SCs. We have recently demonstrated that, like their extrafusal counterparts, SCs in muscle spindles of posthatch chickens express paired box transcription factor 7 (Pax7) protein. During vertebrate embryogenesis, myogenic progenitors express both Pax7 and Pax3 proteins. In postnatal mice, Pax3 appears in rare SC subsets, whereas Pax7 is expressed by all SCs within extrafusal fibers. Here we test the hypothesis that Pax3 protein maintains localized expression within SCs of muscle spindles. Immunohistochemical techniques were used to identify SCs by their Pax7 expression within anterior latissimus dorsi muscle excised from posthatch chickens of various ages. A greater percentage of SCs express Pax3 within intrafusal than extrafusal fibers at each age, and the proportion of SCs expressing Pax3 declines with aging. This is the first study to localize Pax3 expression in posthatch avian muscle and within SCs of muscle spindles. We suggest that Pax3-positive SCs are involved in fiber maintenance.

"Atypical" Pleomorphic Lipomatous Tumor: A Clinicopathologic, Immunohistochemical and Molecular Study of 21 Cases, Emphasizing its Relationship to Atypical Spindle Cell Lipomatous Tumor and Suggesting a Morphologic Spectrum (Atypical Spindle Cell/Pleomorphic Lipomatous Tumor).

PubMed

Creytens, David; Mentzel, Thomas; Ferdinande, Liesbeth; Lecoutere, Evelyne; van Gorp, Joost; Atanesyan, Lilit; de Groot, Karel; Savola, Suvi; Van Roy, Nadine; Van Dorpe, Jo; Flucke, Uta

2017-11-01

The classification of the until recently poorly explored group of atypical adipocytic neoplasms with spindle cell features, for which recently the term atypical spindle cell lipomatous tumor (ASLT) has been proposed, remains challenging. Recent studies have proposed ASLT as a unique entity with (in at least a significant subset of cases) a specific genetic background, namely deletions/losses of 13q14, including RB1 and its flanking genes RCBTB2, DLEU1, and ITM2B. Similar genetic aberrations have been reported in pleomorphic liposarcomas (PLSs). This prompted us to investigate a series of 21 low-grade adipocytic neoplasms with a pleomorphic lipoma-like appearance, but with atypical morphologic features (including atypical spindle cells, pleomorphic [multinucleated] cells, pleomorphic lipoblasts and poor circumscription), for which we propose the term "atypical" pleomorphic lipomatous tumor (APLT). Five cases of PLS were also included in this study. We used multiplex ligation-dependent probe amplification to evaluate genetic changes of 13q14. In addition, array-based comparative genomic hybridization was performed on 4 APLTs and all PLSs. Multiplex ligation-dependent probe amplification showed consistent loss of RB1 and its flanking gene RCBTB2 in all cases of APLT. This genetic alteration was also present in all PLSs, suggesting genetic overlap, in addition to morphologic overlap, with APLTs. However, array-based comparative genomic hybridization demonstrated more complex genetic alterations with more losses and gains in PLSs compared with APLTs. APLTs arose in the subcutis (67%) more frequently than in the deep (subfascial) soft tissues (33%). With a median follow-up of 42 months, recurrences were documented in 2 of 12 APLTs for which a long follow-up was available. Herein, we also demonstrate that APLTs share obvious overlapping morphologic, immunohistochemical, genetic and clinical characteristics with the recently defined ASLT, suggesting that they are related lesions that form a spectrum (atypical spindle cell/pleomorphic lipomatous tumor).

Clinicopathologic Diversity of Undifferentiated Sarcoma With BCOR-CCNB3 Fusion: Analysis of 11 Cases With a Reappraisal of the Utility of Immunohistochemistry for BCOR and CCNB3.

PubMed

Matsuyama, Atsuji; Shiba, Eisuke; Umekita, Yoshihisa; Nosaka, Kanae; Kamio, Takihiro; Yanai, Hiroyuki; Miyasaka, Chika; Watanabe, Reiko; Ito, Ichiro; Tamaki, Tomoko; Hayashi, Shinichi; Hisaoka, Masanori

2017-12-01

Undifferentiated sarcoma harboring the BCOR-CCNB3 fusion is characterized by its predilection to affect skeletons of adolescent males, cellular small round/spindle cell morphology, and CCNB3 immunoreactivity. We analyzed 11 cases of BCOR-CCNB3 sarcoma, 10 of which were identified in a reverse transcription-polymerase chain reaction-based screen of 85 patient samples recorded in our database as unclassified small round or spindle cell sarcomas. BCOR rearrangements were confirmed by fluorescence in situ hybridization in 8 tumors. All patients were males aged between 6 and 31 years. In addition to 5 tumors in soft tissue and 4 in the axial or appendicular skeletons, which are typical locations, a tumor was located in the paranasal sinus and another in the lung. Microscopically, the tumors comprised proliferating atypical spindle and/or small round cells with diverse morphologic features such as small concentric whorls, myxoid stroma, a hemangiopericytomatous appearance, and/or hyalinized collagen resembling a solitary fibrous tumor, and angiomatous or slit-like spaces containing extravasated erythrocytes. Tumor cells were immunoreactive to CCNB3 (9/11), BCOR (10/10), TLE1 (6/10), bcl-2 (9/11), CD99 (8/10), CD56 (8/10), c-kit (4/10), and cyclin D1 (10/10). In an immunohistochemical analysis of an additional 412 small round or spindle cell tumors, CCNB3 was detected in 6 (1.5%) and BCOR in 18 (4.4%). Our analysis highlights the varying clinicopathologic features of this tumor, which partially overlap with other small round or spindle cell tumors, including solitary fibrous tumor and vascular tumors. Because CCNB3 and BCOR immunohistochemistry lacks adequate sensitivity and specificity, a molecular genetic approach remains essential for diagnosis.

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

PubMed Central

Niikura, Yohei; Kitagawa, Katsumi

2016-01-01

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells. PMID:26967065

New frontiers: discovering cilia-independent functions of cilia proteins.

PubMed

Vertii, Anastassiia; Bright, Alison; Delaval, Benedicte; Hehnly, Heidi; Doxsey, Stephen

2015-10-01

In most vertebrates, mitotic spindles and primary cilia arise from a common origin, the centrosome. In non-cycling cells, the centrosome is the template for primary cilia assembly and, thus, is crucial for their associated sensory and signaling functions. During mitosis, the duplicated centrosomes mature into spindle poles, which orchestrate mitotic spindle assembly, chromosome segregation, and orientation of the cell division axis. Intriguingly, both cilia and spindle poles are centrosome-based, functionally distinct structures that require the action of microtubule-mediated, motor-driven transport for their assembly. Cilia proteins have been found at non-cilia sites, where they have distinct functions, illustrating a diverse and growing list of cellular processes and structures that utilize cilia proteins for crucial functions. In this review, we discuss cilia-independent functions of cilia proteins and re-evaluate their potential contributions to "cilia" disorders. © 2015 The Authors.

Cell biology: scaling and the emergence of evolutionary cell biology.

PubMed

Phillips, Patrick C; Bowerman, Bruce

2015-03-16

A new study investigating the origins of diversity in the structure of the mitotic spindle in nematode embryos, at timescales spanning a few generations to hundreds of millions of years, finds that most features of the spindle evolve via a scaling relationship generated by natural selection acting directly upon embryo size. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

PubMed

Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

2015-07-02

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Fibroblastic connective tissue nevus.

PubMed

Velez, Moises J; Billings, Steven D; Weaver, Joshua A

2016-01-01

Fibroblastic connective tissue nevus (FCTN) is a newly recognized, benign cutaneous mesenchymal lesion of fibroblasts/myofibroblastic lineage, which expands the classification of connective tissue nevi. We present three cases of FCTN and discuss significant clinical, morphologic and immunophenotypic overlap with dermatomyofibroma. Our cases were from young women, aged 32, 24 and 10, and presented as 1.2 and 1 cm nodules on the posterior neck and right upper flank, respectively while presenting as a linear plaque of the right posterior thigh in the latter case. The lesions showed a poorly circumscribed proliferation of hypercellular spindle cells arranged in short to longer intersecting fascicles entrapping adnexal structures. Superficial adipose tissue was also entrapped in one case. The spindle cells had fibroblastic features with pale eosinophilic cytoplasmic extensions and inconspicuous nucleoli. The spindle cells were positive for CD34 in two cases. One case was negative for CD34, smooth muscle actin (SMA), desmin and S100. The overall features were consistent with a diagnosis of FCTN. In two cases, we further elucidated the fibroblastic differentiation of the spindle cells in FCTN with electron microscopy, which has not been previously described. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hepatic carcinosarcoma: evidence of polyclonal origin based on microsatellite analysis.

PubMed

Gu, Yi-Jin; Zhu, Yu-Yao; Lu, Xin-Yuan; Zhao, Qian; Cong, Wen-Ming

2015-12-01

Hepatic carcinosarcoma (HCS) is an aggressive tumor for which a consensus regarding the clonal origin has not yet been reached. The aim of the study was to identify the origin of the hepatocellular carcinoma (HCC) and sarcoma components in HCS. We chose microsatellite technique containing loss of heterozygosity (LOH) and microsatellite instability (MSI) on three HCS patients who underwent curative resection confirmed by pathological examination. Tumors were firstly analyzed for Hep Par 1, CK18, CD10, CD117, SMA and vimentin expression by immunohistochemistry. LOH and MSI were then investigated. The incidence rate of LOH/MSI in all nine MS was calculated as the fractional allelic loss (FAL) index, which was internationally recognized standard. A FAL<30% was representative of a monoclonal origin and a FAL≥30% indicated a polyclonal origin. All patients were positive for HBsAg. Microscopic examination showed HCS containing two different cell types: a fibrosarcoma component with spindle cells and an HCC population of cells with a trabecular pattern. In the HCC tumor portions, Hep Par 1, CK18, CD10 were expressed while vimentin was not. In contrast, the spindle cell populations were positive for vimentin and negative for Hep Par 1, CK18, CD10. The highest frequencies of LOH and MSI were at the D16S505 (2/3; 66.7%), D17S831 (2/3; 66.7%) and D17S938 MS (2/3; 66.7%). The FALs for the three cases of HCS were 50% (4/8), 55.6% (5/9) and 33.3% (3/9), suggesting a polyclonal origin. Immunohistochemistry and analysis of LOH and MSI strongly demonstrated that the three HCS samples were consistent with a polyclonal origin for all three cases. Copyright © 2015 Elsevier GmbH. All rights reserved.

Inflammatory fibroid polyp in the duodenum of a common marmoset (Callithrix jacchus).

PubMed

Yokouchi, Yusuke; Imaoka, Masako; Sayama, Ayako; Jindo, Toshimasa; Sanbuissho, Atsushi

2013-01-01

A 32-month-old male common marmoset had a firm and white-colored mass in the duodenal wall. The cut surface was smooth and grayish white in color. Histologically, the mass consisted of a proliferation of spindle cells with an oval to spindle-shaped nucleus and scant eosinophilic cytoplasm in a loose myxoid or fibrotic background. Most of the lesion displayed no specific growth pattern whereas some of the cells concentrated around the vessels and created an onion-bulb structure. Additionally, marked inflammatory cellular infiltration, mainly eosinophils, was observed throughout the lesion. Immunohistochemically, the spindle cells were positive for vimentin, α-smooth muscle actin, fascin, and cyclin D1, and negative for S-100, factor VIII-related antigen, and c-kit. These histological and immunohistochemical features did not meet any differential diagnoses such as gastrointestinal stromal tumor, inflammatory myofibroblastic tumor, solitary fibrous tumor/hemangiopericytoma, smooth muscle tumor, schwannoma, and hemangiosarcoma. Collectively, the authors diagnosed the mass as a lesion that corresponded to an inflammatory fibroid polyp (IFP) in humans. IFP is defined as a mesenchymal proliferation composed of spindle stromal cells, small blood vessels, and inflammatory cells, particularly eosinophils, and is currently classified as a nonneoplastic lesion. To the best of our knowledge, this is the first case of spontaneous IFP in nonhuman primates.

TMBP200, a XMAP215 homologue of tobacco BY-2 cells, has an essential role in plant mitosis.

PubMed

Yasuhara, Hiroki; Oe, Yuki

2011-07-01

TMBP200 from tobacco BY-2 cells is a member of the highly conserved family of microtubule-associated proteins that includes Xenopus XMAP215, human TOGp, and Arabidopsis MOR1/GEM1. XMAP215 homologues have an essential role in spindle assembly and function in animals and yeast, but their role in plant mitosis is not fully clarified. Here, we show by immunoblot analysis that TMBP200 levels in synchronously cultured BY-2 cells increased when the cells entered mitosis, thus indicating that TMBP200 plays an important role in mitosis in tobacco. To investigate the role of TMBP200 in mitosis, we employed inducible RNA interference to silence TMBP200 expression in BY-2 cells. The resulting depletion of TMBP200 caused severe defects in bipolar spindle formation and resulted in the appearance of multinucleated cells with variable-sized nuclei. This finding indicates that TMBP200 has an essential role in bipolar spindle formation and function.

[Esophageal sarcomatoid carcinoma: report of a case with morphological, immunohistochemical and molecular study].

PubMed

Regragui, Asmaa; Lakhdar, Hind; Abderrahman Alaoui Belabbas, Moulay; Amrani, Meryem; Gamra, Lamia; Alaoui Belabbas, Mohamed

2004-05-01

Sarcomatoïd carcinoma is a rare tumor of the esophagus, characterized macroscopically by a polypoid aspect and histologically by the association of spindle cell carcinoma with sarcomatous pleomorphic component. We report here a case of esophagus sarcomatoïd carcinoma. Diagnosis was based on immunohistochemical analysis of tIssue samples. Human papillomavirus (HVP) detection by PCR amplification of DNA extracted from tumoral tIssue was negative, ruling out the role of HPV infection in this tumor.

Warthin tumor-like papillary thyroid carcinoma with a minor dedifferentiated component: report of a case with clinicopathologic considerations.

PubMed

Amico, Paolo; Lanzafame, Salvatore; Li Destri, Giovanni; Greco, Paolo; Caltabiano, Rosario; Vecchio, Giada Maria; Magro, Gaetano

2010-01-01

Warthin tumor-like papillary thyroid carcinoma is an uncommon variant of papillary thyroid carcinoma. We report a rare case of Warthin tumor-like variant of papillary thyroid carcinoma with a dedifferentiated component consisting of a solid tumor area composed of neoplastic cells with a spindle to tall cell morphology associated with marked nuclear pleomorphism, atypical mitoses, and foci of necrosis. Although our patient presented with a locally aggressive disease (T3 N1b Mo), she is disease-free without radioiodine therapy after a 23-month follow-up period. We emphasize that Warthin tumor-like papillary thyroid carcinoma, like other morphological variants of papillary carcinoma, may occasionally undergo dedifferentiation. As this component may be only focally detectable, we suggest an extensive sampling of all large-sized (>3 cm) papillary thyroid carcinoma. Recognition of any dedifferentiated component in a Warthin tumor-like papillary thyroid carcinoma should be reported, including its percentage, because it may reflect a more aggressive clinical course.

Warthin Tumor-Like Papillary Thyroid Carcinoma with a Minor Dedifferentiated Component: Report of a Case with Clinicopathologic Considerations

PubMed Central

Amico, Paolo; Lanzafame, Salvatore; Li Destri, Giovanni; Greco, Paolo; Caltabiano, Rosario; Vecchio, Giada Maria; Magro, Gaetano

2010-01-01

Warthin tumor-like papillary thyroid carcinoma is an uncommon variant of papillary thyroid carcinoma. We report a rare case of Warthin tumor-like variant of papillary thyroid carcinoma with a dedifferentiated component consisting of a solid tumor area composed of neoplastic cells with a spindle to tall cell morphology associated with marked nuclear pleomorphism, atypical mitoses, and foci of necrosis. Although our patient presented with a locally aggressive disease (T3 N1b Mo), she is disease-free without radioiodine therapy after a 23-month follow-up period. We emphasize that Warthin tumor-like papillary thyroid carcinoma, like other morphological variants of papillary carcinoma, may occasionally undergo dedifferentiation. As this component may be only focally detectable, we suggest an extensive sampling of all large-sized (>3 cm) papillary thyroid carcinoma. Recognition of any dedifferentiated component in a Warthin tumor-like papillary thyroid carcinoma should be reported, including its percentage, because it may reflect a more aggressive clinical course. PMID:20593036

Kinetic suppression of microtubule dynamic instability by griseofulvin: Implications for its possible use in the treatment of cancer

PubMed Central

Panda, Dulal; Rathinasamy, K.; Santra, Manas K.; Wilson, Leslie

2005-01-01

The antifungal drug griseofulvin inhibits mitosis strongly in fungal cells and weakly in mammalian cells by affecting mitotic spindle microtubule (MT) function. Griseofulvin also blocks cell-cycle progression at G2/M and induces apoptosis in human tumor cell lines. Despite extensive study, the mechanism by which the drug inhibits mitosis in human cells remains unclear. Here, we analyzed the ability of griseofulvin to inhibit cell proliferation and mitosis and to affect MT polymerization and organization in HeLa cells together with its ability to affect MT polymerization and dynamic instability in vitro. Griseofulvin inhibited cell-cycle progression at prometaphase/anaphase of mitosis in parallel with its ability to inhibit cell proliferation. At its mitotic IC50 of 20 μM, spindles in blocked cells displayed nearly normal quantities of MTs and MT organization similar to spindles blocked by more powerful MT-targeted drugs. Similar to previously published data, we found that very high concentrations of griseofulvin (>100 μM) were required to inhibit MT polymerization in vitro. However, much lower drug concentrations (1–20 μM) strongly suppressed the dynamic instability behavior of the MTs. We suggest that the primary mechanism by which griseofulvin inhibits mitosis in human cells is by suppressing spindle MT dynamics in a manner qualitatively similar to that of much more powerful antimitotic drugs, including the vinca alkaloids and the taxanes. In view of griseofulvin's lack of significant toxicity in humans, we further suggest that it could be useful as an adjuvant in combination with more powerful drugs for the treatment of cancer. PMID:15985553

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.

2008-07-15

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cellsmore » treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.« less

Fine-needle aspiration cytology of malignant hemangiopericytoma of the salivary gland: A case report.

PubMed

Shimizu, K; Ogura, S; Kobayashi, T K; Kushima, R; Toyokuni, S; Iwasa, Y; Sakurai, M

1999-12-01

A 79-yr-old woman presented with a 5-yr history of swelling of the left cheek. The fine-needle aspiration (FNA) smear showed a spindle-cell neoplasm with capillaries and benign endothelial cells. The spindle cells possessed pleomorphic, hyperchromatic elongated nuclei and a moderate amount of ill-defined cytoplasm. They also showed papillary arcades surrounded and encased by relatively small ovoid to short spindle cells. Subsequent surgical excision confirmed the presence of malignant hemangiopericytoma (HP). Immunohistochemical studies on the histologic section using vimentin were strongly positive, consistent with HP. To the best of our knowledge, this is the second published report of FNA cellular features of malignant HP of the salivary gland. Besides delineating the FNA cellular features of HP of the salivary gland, the present case illustrates the value of using immunohistochemical approaches. Diagn. Cytopathol. 1999;21:398-401. Copyright 1999 Wiley-Liss, Inc.

Modal identification of spindle-tool unit in high-speed machining

NASA Astrophysics Data System (ADS)

Gagnol, Vincent; Le, Thien-Phu; Ray, Pascal

2011-10-01

The accurate knowledge of high-speed motorised spindle dynamic behaviour during machining is important in order to ensure the reliability of machine tools in service and the quality of machined parts. More specifically, the prediction of stable cutting regions, which is a critical requirement for high-speed milling operations, requires the accurate estimation of tool/holder/spindle set dynamic modal parameters. These estimations are generally obtained through Frequency Response Function (FRF) measurements of the non-rotating spindle. However, significant changes in modal parameters are expected to occur during operation, due to high-speed spindle rotation. The spindle's modal variations are highlighted through an integrated finite element model of the dynamic high-speed spindle-bearing system, taking into account rotor dynamics effects. The dependency of dynamic behaviour on speed range is then investigated and determined with accuracy. The objective of the proposed paper is to validate these numerical results through an experiment-based approach. Hence, an experimental setup is elaborated to measure rotating tool vibration during the machining operation in order to determine the spindle's modal frequency variation with respect to spindle speed in an industrial environment. The identification of natural frequencies of the spindle under rotating conditions is challenging, due to the low number of sensors and the presence of many harmonics in the measured signals. In order to overcome these issues and to extract the characteristics of the system, the spindle modes are determined through a 3-step procedure. First, spindle modes are highlighted using the Frequency Domain Decomposition (FDD) technique, with a new formulation at the considered rotating speed. These extracted modes are then analysed through the value of their respective damping ratios in order to separate the harmonics component from structural spindle natural frequencies. Finally, the stochastic properties of the modes are also investigated by considering the probability density of the retained modes. Results show a good correlation between numerical and experiment-based identified frequencies. The identified spindle-tool modal properties during machining allow the numerical model to be considered as representative of the real dynamic properties of the system.

Synaptic and membrane mechanisms underlying synchronized oscillations in the ferret lateral geniculate nucleus in vitro.

PubMed Central

Bal, T; von Krosigk, M; McCormick, D A

1995-01-01

1. The cellular basis for generation of spindle waves and a slower synchronized oscillation resembling absence seizures was investigated with extracellular and intracellular recording techniques in slices of ferret dorsal lateral geniculate nucleus (LGNd) maintained in vitro. 2. Intracellular recording from LGNd relay cells in vitro revealed that spindle waves occurred once every 3-30 s and were associated with barrages of inhibitory postsynaptic potentials (IPSPs) occurring at a frequency of 6-10 Hz. These IPSPs resulted in the generation of rebound low threshold Ca2+ spikes at 2-4 Hz, owing to the intrinsic propensity of LGNd relay cells to generate oscillatory burst firing in this frequency range. These rebound bursts of action potentials were highly synchronized with local multiunit and single unit activity. 3. The spindle wave-associated IPSPs in LGNd relay cells exhibited a mean reversal potential of -86 mV. This reversal potential was shifted to more depolarized membrane potentials with the intracellular injection of Cl- through the use of KCl-filled microelectrodes. Simultaneous recording from the perigeniculate nucleus (PGN) and LGNd revealed the IPSPs to be synchronous with the occurrence of burst firing in the PGN. Excitation of PGN neurons with local electrical stimulation after pharmacological block of excitatory amino acid transmission resulted in bicuculline-sensitive IPSPs in relay neurons similar in amplitude and time course to those occurring during spindle waves. 4. Application of (-)-bicuculline methiodide resulted in the abolition of spindle wave-associated IPSPs or in the slowing of the rate of rise, an increase in amplitude and a prolongation of these IPSPs; this resulted in a synchronized 2-4 Hz oscillation, in which each relay cell strongly burst on nearly every cycle, thus forming a paroxysmal event. Bath application of the GABAB receptor antagonist 2-OH-saclofen blocked these slowed oscillations, indicating that they are mediated by the activation of GABAB receptors. In contrast, pharmacological antagonism of GABAB receptors did not block the generation of normal spindle waves. 5. These and other results indicate that spindle waves are generated in the ferret LGNd in vitro as a network phenomenon occurring through an interaction between the relay cells of the LGNd and the GABAergic neurons of the PGN. We propose that burst firing in PGN cells hyperpolarizes relay neurons through activation of GABAA receptors. These IPSPs result in rebound burst firing in LGNd cells, which then excite PGN neurons.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7776249

LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

PubMed Central

Price, Kari L.; Rose, Lesilee S.

2017-01-01

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule–dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells. PMID:28701343

Measuring and modeling polymer concentration profiles near spindle boundaries argues that spindle microtubules regulate their own nucleation

NASA Astrophysics Data System (ADS)

Kaye, Bryan; Stiehl, Olivia; Foster, Peter J.; Shelley, Michael J.; Needleman, Daniel J.; Fürthauer, Sebastian

2018-05-01

Spindles are self-organized microtubule-based structures that segregate chromosomes during cell division. The mass of the spindle is controlled by the balance between microtubule turnover and nucleation. The mechanisms that control the spatial regulation of microtubule nucleation remain poorly understood. While previous work found that microtubule nucleators bind to pre-existing microtubules in the spindle, it is still unclear whether this binding regulates the activity of those nucleators. Here we use a combination of experiments and mathematical modeling to investigate this issue. We measured the concentration of microtubules and soluble tubulin in and around the spindle. We found a very sharp decay in the concentration of microtubules at the spindle interface. This is inconsistent with a model in which the activity of nucleators is independent of their association with microtubules but consistent with a model in which microtubule nucleators are only active when bound to pre-existing microtubules. This argues that the activity of microtubule nucleators is greatly enhanced when bound to pre-existing microtubules. Thus, microtubule nucleators are both localized and activated by the microtubules they generate.

Dynein-mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

PubMed Central

Fink, Gero; Schuchardt, Isabel; Colombelli, Julien; Stelzer, Ernst; Steinberg, Gero

2006-01-01

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches ∼2 μm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex. PMID:17024185

Division of the intermediate compartment at the onset of mitosis provides a mechanism for Golgi inheritance.

PubMed

Marie, Michaël; Dale, Hege A; Kouprina, Nina; Saraste, Jaakko

2012-11-15

As mammalian cells prepare for mitosis, the Golgi ribbon is first unlinked into its constituent stacks and then transformed into spindle-associated, pleiomorphic membrane clusters in a process that remains enigmatic. Also, it remains unclear whether Golgi inheritance involves the incorporation of Golgi enzymes into a pool of coat protein I (COPI) vesicles, or their COPI-independent transfer to the endoplasmic reticulum (ER). Based on the observation that the intermediate compartment (IC) at the ER-Golgi boundary is connected to the centrosome, we examined its mitotic fate and possible role in Golgi breakdown. The use of multiple imaging techniques and markers revealed that the IC elements persist during the M phase, maintain their compositional and structural properties and remain associated with the mitotic spindle, forming circular arrays at the spindle poles. At G2/M transition, the movement of the pericentrosomal domain of the IC (pcIC) to the cell centre and its expansion coincide with the unlinking of the Golgi ribbon. At prophase, coupled to centrosome separation, the pcIC divides together with recycling endosomes, providing novel landmarks for mitotic entry. We provide evidence that the permanent IC elements function as way stations during the COPI-dependent dispersal of Golgi components at prometa- and metaphase, indicating that they correspond to the previously described Golgi clusters. In addition, they continue to communicate with the vesicular 'Golgi haze' and thus are likely to provide templates for Golgi reassembly. These results implicate the IC in mitotic Golgi inheritance, resulting in a model that integrates key features of the two previously proposed pathways.

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

PubMed Central

Bazzi, Hisham; Anderson, Kathryn V.

2014-01-01

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4−/− p53−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo. PMID:24706806

Malignant peripheral nerve sheath tumor of the uterine cervix expressing both S-100 protein and HMB-45.

PubMed

Kim, Na Rae; Chung, Dong-Hae; Park, Chan Yong; Ha, Seung Yeon

2009-12-01

A 50-year-old woman presented with a large cervical polypoid mass. Grossly, the mass occupied a substantial proportion of the cervical canal, measuring 6 cm. Histologically, the mass showed a spindle cell malignancy arranged in large fascicles that penetrated deeply into the fibromuscular wall of the cervix. The spindle cells were immunoreactive for both S-100 protein and HMB-45 antigen, but were negative for Melan-A. Electron microscopy showed that cytoplasmic processes of the spindle to oval tumor cells contained microtubules and were lined by basal lamina and abundant intercellular collagen spacing with no melanosomes in any stage. As far as we are aware, this is the ninth reported case of cervical malignant peripheral nerve sheath tumor (MPNST), and the second reported case of MPNST expressing HMB-45 antigen.

Direct Tumor Embolization of Sinonasal Unclassified Spindle Cell Sarcoma with Onyx.

PubMed

Kansal, Ankit; Srinet, Prateek; Manes, Richard Peter

2016-07-01

To evaluate the use of a new tumor embolization agent, Onyx (Covidien, Dublin, Ireland), for the use of intraoperative embolization of a sinonasal unclassified spindle cell sarcoma. A 45-year-old female patient presented to the rhinology clinic with a nasal mass. A biopsy revealed a highly vascular mass consistent with a sinonasal unclassified spindle cell sarcoma. Secondary to its extensive vascularity, the patient underwent preoperative transarterial embolization (TAE) before definitive resection. Due to complex vascular anatomy including feeding vessels emanating from intracranial circulation, incomplete embolization was achieved. Subsequently, intraoperative embolization with Onyx at the time of resection was performed. Intraoperative Onyx use resulted in almost complete devascularization of the tumor with decreased risk of intracranial embolization. Intraoperative embolization with Onyx after an incomplete TAE can be a safe and effective method of achieving near-total embolization of sinonasal tumors.

Isolated Cerebellar Spindle Cell Pseudotumor Caused by Mycobacterium Avium-Intracellulare Complex in a Patient without AIDS.

PubMed

Lim, Ming-Sheng; Bermingham, Niamh; O'Broin, Cathal; Khalil, Ayman; Keohane, Catherine; Lim, Chris

2016-06-01

Spindle cell pseudotumors are formed by histiocytes in response to infection by Mycobacterium avium-intracellulare complex (MAC) and are rare in patients without AIDS. A 66-year-old man presented with neck pain, ataxia, and a history of sarcoidosis. A cerebellar lesion was identified on magnetic resonance imaging and surgically excised. Histopathology revealed this to be a spindle cell pseudotumor and MAC was isolated by bacterial culture of cerebrospinal fluid. Hematology revealed cluster of differentiation 4 lymphocytopenia but human immunodeficiency virus serology was negative. The patient was commenced on antimicrobial treatment that included a macrolide and remained well at 1 year follow-up. This rare presentation of isolated intracranial MAC was treated with surgical excision and antimicrobials with a good outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

Free Vibration Analysis of a Spinning Flexible DISK-SPINDLE System Supported by Ball Bearing and Flexible Shaft Using the Finite Element Method and Substructure Synthesis

NASA Astrophysics Data System (ADS)

JANG, G. H.; LEE, S. H.; JUNG, M. S.

2002-03-01

Free vibration of a spinning flexible disk-spindle system supported by ball bearing and flexible shaft is analyzed by using Hamilton's principle, FEM and substructure synthesis. The spinning disk is described by using the Kirchhoff plate theory and von Karman non-linear strain. The rotating spindle and stationary shaft are modelled by Rayleigh beam and Euler beam respectively. Using Hamilton's principle and including the rigid body translation and tilting motion, partial differential equations of motion of the spinning flexible disk and spindle are derived consistently to satisfy the geometric compatibility in the internal boundary between substructures. FEM is used to discretize the derived governing equations, and substructure synthesis is introduced to assemble each component of the disk-spindle-bearing-shaft system. The developed method is applied to the spindle system of a computer hard disk drive with three disks, and modal testing is performed to verify the simulation results. The simulation result agrees very well with the experimental one. This research investigates critical design parameters in an HDD spindle system, i.e., the non-linearity of a spinning disk and the flexibility and boundary condition of a stationary shaft, to predict the free vibration characteristics accurately. The proposed method may be effectively applied to predict the vibration characteristics of a spinning flexible disk-spindle system supported by ball bearing and flexible shaft in the various forms of computer storage device, i.e., FDD, CD, HDD and DVD.

Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration

PubMed Central

DeZwaan, Todd M.; Ellingson, Eric; Pellman, David; Roof, David M.

1997-01-01

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus. PMID:9281581

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins.

PubMed

Maachani, Uday Bhanu; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J; Camphausen, Kevin; Tandle, Anita

2015-05-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell-specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. ©2015 American Association for Cancer Research.

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins

PubMed Central

Maachani, Uday B.; Kramp, Tamalee; Hanson, Ryan; Zhao, Shuping; Celiku, Orieta; Shankavaram, Uma; Colombo, Riccardo; Caplen, Natasha J.; Camphausen, Kevin; Tandle, Anita

2015-01-01

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells was analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor, NMS-P715 on radiosensitivity in multiple model systems including: GBM cell lines, a normal astrocyte and a normal fibroblast cell line. DNA double strand breaks (DSBs) were evaluated using γH2AX foci and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Further, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of post-radiation mitotic catastrophe. MNS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1 silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair and replication including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. PMID:25722303

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos

PubMed Central

Duan, Xunbao; Zhong, Zhisheng; Potireddy, Santhi; Moncada, Camilo; Merali, Salim; Latham, Keith E.

2015-01-01

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos. PMID:20883044

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

PubMed Central

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-01-01

ABSTRACT Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes. PMID:27433972

RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

PubMed

Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

2016-10-01

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

PubMed Central

Joseph, Nimesh; Cavazza, Tommaso; Vernos, Isabelle; Pfuhl, Mark; Gergely, Fanni; Bayliss, Richard

2015-01-01

The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation. PMID:26134678

Kaposiform hemangioendothelioma of the spleen in an adult: an initial case report.

PubMed

Yu, Lu; Yang, Shou Jing

2011-12-01

Kaposiform hemangioendothelioma (KHE) is a rare locally aggressive vascular neoplasm characterized by infiltrating nodules and sheets of spindle cells, and unmistakable resemblance to Kaposi's sarcoma. KHE occurs mainly in newborns and infants and presents most commonly in the skin, deep soft tissue, and bone. We report a case of KHE in a 36-year-old female who presented with a spleen mass and underwent splenectomy. Macroscopic examination revealed a large, dark-red, firm mass in the spleen. Histologically, the tumor consisted of irregular, infiltrating nodules of densely packed spindle-shaped tumor cells closely associated with small slit-like and sieve-like blood vessels, which were separated with hyalinized hypocellular fibrous stroma. Immunohistochemically, both spindle and epithelioid cells were positive for CD34, CD31, and vimentin, but negative for EMA, cytokeratin, CD21, CD35, CD1a, and S-100 protein. The well-formed capillaries and mature vessels but not spindle tumor cell showed reactivity for factor VIII- related antigen. Alpha-Smooth muscle actin was detected in pericytes surrounding small round or slit-like capillaries. The final histologic diagnosis was KHE. Follow-up 6 month after operation revealed no sign of recurrence or metastasis.To the best of our knowledge, this is the first report of KHE arising in the spleen.

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme

PubMed Central

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M.; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G.

2017-01-01

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches. PMID:29340013

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme.

PubMed

Abbruzzese, Claudia; Catalogna, Giada; Gallo, Enzo; di Martino, Simona; Mileo, Anna M; Carosi, Mariantonia; Dattilo, Vincenzo; Schenone, Silvia; Musumeci, Francesca; Lavia, Patrizia; Perrotti, Nicola; Amato, Rosario; Paggi, Marco G

2017-12-19

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro , as well as inhibiting tumor growth in vivo . We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

PubMed

Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

2016-06-02

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

Cutaneous amelanotic signet-ring cell malignant melanoma with interspersed myofibroblastic differentiation in a young cat.

PubMed

Hirz, Manuela; Herden, Christiane

2016-07-01

The diagnosis of malignant melanoma can be difficult because these tumors can be amelanotic and may contain diverse variants and divergent differentiations, of which the signet-ring cell subtype is very rare and has only been described in humans, dogs, cats, and a hamster. We describe herein histopathologic and immunohistochemical approaches taken to diagnose a case of signet-ring cell malignant melanoma with myofibroblastic differentiation in a cat. A tumor within the abdominal skin of a 2-year-old cat was composed of signet-ring cells and irregularly interwoven streams of spindle cells. Both neoplastic cell types were periodic-acid-Schiff, Fontana, and Sudan black B negative. Signet-ring cells strongly expressed vimentin and S100 protein. Spindle cells strongly expressed vimentin and smooth muscle actin; some cells expressed S100, moderately neuron-specific enolase, and others variably actin and desmin. A few round cells expressed melan A, and a few plump spindle cells expressed melan A and PNL2, confirming the diagnosis of amelanotic signet-ring cell malignant melanoma with myofibroblastic differentiation in a cat. Differential diagnoses were excluded, including signet-ring cell forms of adenocarcinomas, lymphomas, liposarcomas, leiomyosarcomas, squamous cell carcinomas, basal cell carcinomas, and adnexal tumors. © 2016 The Author(s).

The Concept of Electrically Assisted Friction Stir Welding (EAFSW) and Application to the Processing of Various Metals

DTIC Science & Technology

2008-09-01

unavailability of a precise load cell. This scale was set on the machine working table. Since the shaft spindle had been placed in fixed vertical...4 Figure 4. Mill Spindle with Slip Ring and Brush Assembly, Weld Tip is in W orking Position...areas . FSW of HSLA 65 and Type 304L Processing Parameter HSLA-65 304L Spindle Speed (RPM) 850 850 Travel Speed (ipm) 6 2 Z-Load (Ibs) 3500 3500

Cellular abundance of Mps1 and the role of its carboxyl terminal tail in substrate recruitment.

PubMed

Sun, Tingting; Yang, Xiaomei; Wang, Wei; Zhang, Xiaojuan; Xu, Quanbin; Zhu, Songcheng; Kuchta, Robert; Chen, Guanjun; Liu, Xuedong

2010-12-03

Mps1 is a protein kinase that regulates normal mitotic progression and the spindle checkpoint in response to spindle damage. The levels of Mps1 are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mps1 expression and the Mps1 catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mps1 kinase domain revealed that the carboxyl-terminal tail region of Mps1 is unstructured, raising the question of whether this region has any functional role in Mps1 catalysis. Here we first determined the cellular abundance of Mps1 during cell cycle progression and found that Mps1 levels vary between 60,000 per cell in early G(1) and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mps1 substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mps1 plays an essential role in substrate recruitment. Kinetics studies using the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mps1 but critical for trans-phosphorylation of substrates in vitro and in cultured cells. Mps1 mutant without the unstructured tail region is defective in mediating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mps1 in kinase activation.

Involvement of Vascular Endothelial Growth Factor in Kaposi's Sarcoma Associated with Acquired Immunodeficiency Syndrome

PubMed Central

Sakurada, Shinsaku; Kato, Tetsuji; Mashiba, Kohichi; Mori, Shigeo

1996-01-01

To examine the role of vascular endothelial growth factor (VEGF) in the development of edema associated with Kaposi's sarcoma (KS) in acquired immunodeficiency syndrome (AIDS), we exploited animal model systems to detect the activity that induces vascular hyper‐permeability (VHP) using cultured AIDS‐KS spindle cells. Cultured AIDS‐KS spindle cells and conditioned medium (AIDS‐KS‐CM) that had been semi‐purified through a heparin affinity column were tested for the ability to induce VHP in animals. The AIDS‐KS spindle cells and AIDS‐KS‐CM induced VHP that was histamine‐independent. The VHP‐inducing activity was detected in the 0.5 M NaCl fraction from the heparin affinity column and was blocked by anti‐VEGF neutralizing antibody. In addition, the production of VEGF was demonstrated in fresh AIDS‐KS tissue as well as in cultured AIDS‐KS cells, while control cells were negative for VEGF production. From these observations, we concluded that AIDS‐KS cells produce a factor(s) that promotes VHP, and this factor could be VEGF. PMID:9045943

Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain.

PubMed

Wong, Jim; Lerrigo, Robert; Jang, Chang-Young; Fang, Guowei

2008-05-01

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

[Clinicopathologic study of sinonasal teratocarcinosarcoma and its contrast with olfactory neuroblastoma].

PubMed

Li, Xue; Liu, Hong-Gang; Xie, Xin-Ji; Han, Yi-Ding; Li, Ming

2008-07-01

To study the clinicopathologic features, diagnosis and differential diagnosis of sinonasal teratocarcinosarcoma (SNTCS) and olfactory neuroblastoma (ONB), and to discuss the histogenesis and possible relationship between SNTCS and ONB. Seven cases of SNTCS and 34 cases of ONB were retrieved from the pathological archives together with one case each of malignant teratoma and immature embryonic tissue at 8 weeks were collected from Beijing Tongren Hospital. The clinicopathologic features were analyzed and immunohistochemical staining was performed on paraffin sections. Six of the SNTCS patients were male and one was female. The patients age range was 25 to 69 years (mean age 46). Four cases were initial presentation and three were recurrences. Histologically, the tumor shows multiple tissue components derived from three germ layers. There were mixture of teratoma-like tissue and carcinosarcoma. The components include fetal clear cell squamous epithelium derived from ectoderm. Glandular and tubular structures and ciliated columnar epithelium derived from endoderm. Fibroblasts, striated muscle, smooth muscle, cartilage and osteoid matrix derived from mesoderm. The carcinoma component exhibited mostly adenocarcinoma and squamous cell carcinoma, whereas the sarcoma component mostly exhibited rhabdomyosarcoma, leiomyosarcoma, and fibrosarcoma. In addition, carcinoid, and primitive mesenchymal tissue and the ONB component were also seen. The morphological characteristics of SNTCS comprised fetal clear cell squamous epithelium, carcinosarcoma and the ONB component. By immunohistochemistry, the epithelial component and cells with epithelium differentiation were positive for cytokeratin (pan) and EMA. The ONB component was positive for Syn, NSE, CD99, NF and CgA to different degrees. Neurofibril bundles were positive for S-100, and Flexner-Wintersteiner rosettes expressed cytokeratin (pan) and EMA. The spindle cells expressed vimentin, SMA, desmin, myosin and myoglobin. The primitive mesenchymal tissue expressed vimentin, and the mucoid materials and glycogen were positive for PAS. GFAP was negative in all cases. The 34 cases of ONB, included 18 men and 16 women, the age ranged from 12 to 72 years (mean 42.8 years). Microscopically, the tumor shows epithelial nests, net of angioma-like fibrous connective tissues, small round and spindle cells, glandular, squamous-like cells, and cells of rhabdomyoblastic differentiation, Homer-Wright and Flexner rosette, bundles of neurofibrils, etc. NSE and CgA were expressed in small cells. S-100 protein was positive in the areas of bunches of neurofibril. Cytokeratin (pan) was positive in epithelial cells. Myoglobin was positive in the cells of rhabdomyoblastic differentiation. The single case of immature malignant teratoma exhibited primitive nerve tissue, but fetal clear cell squamous epithelium was not found. In the immature embryonic tissue, rudimentary organs were formed, with fetal clear cell squamous epithelium lining present on the nasal and oral cavities surface. SNTCS is a rare and aggressive malignant neoplasm. Most of ONB are low-grade malignant tumors. Morphological differences are the most important basis to make differentiate SNTCS from ONB. As SNTCS may demonstrate a multiplicity of structures and pleomorphism, inadequate sampling at biopsy, therefore, may lead to errors in diagnosis. No evidence show that SNTCS are derived from germ cells and sinonasal teratoid carcinosarcoma may be a more proper name. SNTCS probably arises from primitive totipotential cells of olfactory/sinonasal membrane, and the relationship between SNTCS and ONB needs further study.

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies.

PubMed

Umemoto, M; Sakagami, M; Fukazawa, K; Ashida, K; Kubo, T; Senda, T; Yoneda, Y

1995-09-01

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.

Force-balance model of suppression of multipolar division in cancer cells with extra centrosomes

NASA Astrophysics Data System (ADS)

Zhu, Jie

2013-03-01

Cancer cells often possess extra centrosomes which have the potential to cause cell death due to catastrophic multipolar division. Many cancer cells, however, are able to escape multipolar mitosis by clustering the extra centrosomes to form bipolar spindles. The mechanism of centrosome clustering is therefore of great interest to the development of anti-cancer drugs because the de-clustering of extra centrosomes provides an appealing way to eliminate cancer cells while keeping healthy cells intact. We present a physical model assuming 1) dynamic centrosomal microtubules interact with chromosomes by both pushing on chromosome arms and pulling along kinetochores; 2) these microtubules interact with force generators associated with actin/adhesion structures at the cell boundary; and 3) motors act on anti-parallel microtubules from different centrosomes. We find via computer simulations that chromosomes tend to aggregate near the cell center while centrosomes can be either clustered to form bipolar spindles or scattered to form multipolar spindles, depending on the strengths of relative forces, cell shape and adhesion geometry. The model predictions agree with data from cells plated on adhesive micropatterns and from biochemically or genetically perturbed cells. Furthermore, our model is able to explain various microtubule distributions in interphase cells on patterned substrates. This work was supported by NSF

The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

PubMed Central

Govindaraghavan, Meera; Lad, Alisha A.

2014-01-01

The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

LOX is a novel mitotic spindle-associated protein essential for mitosis.

PubMed

Boufraqech, Myriem; Wei, Darmood; Weyemi, Urbain; Zhang, Lisa; Quezado, Martha; Kalab, Petr; Kebebew, Electron

2016-05-17

LOX regulates cancer progression in a variety of human malignancies. It is overexpressed in aggressive cancers and higher expression of LOX is associated with higher cancer mortality. Here, we report a new function of LOX in mitosis. We show that LOX co-localizes to mitotic spindles from metaphase to telophase, and p-H3(Ser10)-positive cells harbor strong LOX staining. Further, purification of mitotic spindles from synchronized cells show that LOX fails to bind to microtubules in the presence of nocodazole, whereas paclitaxel treated samples showed enrichment in LOX expression, suggesting that LOX binds to stabilized microtubules. LOX knockdown leads to G2/M phase arrest; reduced p-H3(Ser10), cyclin B1, CDK1, and Aurora B. Moreover, LOX knockdown significantly increased sensitivity of cancer cells to chemotherapeutic agents that target microtubules. Our findings suggest that LOX has a role in cancer cell mitosis and may be targeted to enhance the activity of microtubule inhibitors for cancer therapy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Era, Saho; Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501; Abe, Takuya

Highlights: Black-Right-Pointing-Pointer SENP1 knockout chicken DT40 cells are hypersensitive to spindle poisons. Black-Right-Pointing-Pointer Spindle poison treatment of SENP1{sup -/-} cells leads to increased mitotic slippage. Black-Right-Pointing-Pointer Mitotic slippage in SENP1{sup -/-} cells associates with apoptosis and endoreplication. Black-Right-Pointing-Pointer SENP1 counteracts sister chromatid separation during mitotic arrest. Black-Right-Pointing-Pointer Plk1-mediated cohesion down-regulation is involved in colcemid cytotoxicity. -- Abstract: SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockoutmore » chicken DT40 cells. SENP1{sup -/-} cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2{alpha}{sup +/-} mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2{alpha} is SUMOylated during mitosis, the TOP2{alpha}{sup +/-} mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1{sup -/-} cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.« less

Electro-Acoustic Behavior of the Mitotic Spindle: A Semi-Classical Coarse-Grained Model

PubMed Central

Havelka, Daniel; Kučera, Ondřej; Deriu, Marco A.; Cifra, Michal

2014-01-01

The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells—a strategy used in novel methods for cancer treatment. PMID:24497952

Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p

PubMed Central

Chua, Penelope R; Roof, David M; Lee, Yan; Sakowicz, Roman; Clarke, David; Pierce, Dan; Stephens, Thoryn; Hamilton, Matthew; Morgan, Brad; Morgans, David; Nakai, Takashi; Tomasi, Adam; Maxon, Mary E

2007-01-01

Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in ‘rigor-like’, tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery. PMID:17573815

Mitosis-Specific Mechanosensing and Contractile Protein Redistribution Control Cell Shape

PubMed Central

Effler, Janet C.; Kee, Yee-Seir; Berk, Jason M.; Tran, Minhchau N.; Iglesias, Pablo A.; Robinson, Douglas N.

2008-01-01

Summary Because cell division failure is deleterious, promoting tumorigenesis in mammals [1], cells utilize numerous mechanisms to control their cell-cycle progression [2–4]. Though cell division is considered a well-ordered sequence of biochemical events [5], cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Since cells respond to their mechanical environment [6, 7], we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin-II are vital to cell division [8, 9], we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins, myosin-II and cortexillin-I, redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, is independent of spindle orientation, but is dependent on myosin-II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis. PMID:17027494

Histological and immunohistochemical characteristics of undifferentiated small round cell sarcomas associated with CIC-DUX4 and BCOR-CCNB3 fusion genes.

PubMed

Yamada, Yuichi; Kuda, Masaaki; Kohashi, Kenichi; Yamamoto, Hidetaka; Takemoto, Junkichi; Ishii, Takeaki; Iura, Kunio; Maekawa, Akira; Bekki, Hirofumi; Ito, Takamichi; Otsuka, Hiroshi; Kuroda, Makoto; Honda, Yumi; Sumiyoshi, Shinji; Inoue, Takeshi; Kinoshita, Naoe; Nishida, Atsushi; Yamashita, Kyoko; Ito, Ichiro; Komune, Shizuo; Taguchi, Tomoaki; Iwamoto, Yukihide; Oda, Yoshinao

2017-04-01

CIC-DUX4 and BCOR-CCNB3 fusion-gene-associated small round cell sarcomas account for a proportion of pediatric small round cell sarcomas, but their pathological features have not been sufficiently clarified. We reviewed a large number of soft tissue tumors registered at our institution, retrieved the cases of unclassified tumors with a small round cell component, and subjected them to histopathological, immunohistochemical, and gene profile analysis. We reviewed 164 cases of unclassified tumors with a small round cell component and analyzed them by RT-PCR and FISH. Tumors positive for a specific fusion-gene were also subjected to histopathological and immunohistochemical examinations. We identified 16 cases of BCOR-CCNB3/CIC-associated (CIC-DUX4 or CIC gene rearrangement-positive) sarcomas. These included seven BCOR-CCNB3 sarcomas and nine CIC-associated sarcomas. Heterogeneous elements included a myxoid spindle cell component in three BCOR-CCNB3 sarcomas and an epithelioid cell component in two CIC-associated sarcomas (one CIC-DUX4-positive and one CIC-DUX4-negative sarcomas). Mitotic activity was low in both heterogeneous components. By immunohistochemistry, in seven BCOR-CCNB3 sarcomas expression of EMA was positive in two cases, of p63 in three, of CD56 in six, of TLE1 in seven, of NKX2.2 in two, of CCNB3 in seven, and of BCOR in six cases (one case could not be tested for BCOR). In nine cases of CIC-associated sarcoma, CD56 was expressed in five, alpha-smooth muscle actin in one, ERG in three, and CD99, WT1 and TLE1 each in eight cases. Both sarcoma types showed not only a small round cell component, but also a myxoid/epithelioid component with low mitotic activity.

A THERMODYNAMIC ANALYSIS OF MITOTIC SPINDLE EQUILIBRIUM AT ACTIVE METAPHASE

PubMed Central

Stephens, R. E.

1973-01-01

The mitotic apparatus of first-division metaphase eggs of the sea urchin Strongylocentrotus drobachiensis was observed by means of polarization microscopy under controlled temperature conditions. Eggs were fertilized and grown at two temperature extremes in order to produce two different sizes of available spindle pool. Slow division time allowed successive samples of such cells to be observed at the same point in metaphase but at different equilibrium temperatures, yielding curves of metaphase equilibrium birefringence vs. observational temperature. Using the plateau value of birefringence at higher temperatures as a measure of total available spindle pool and the observed birefringence at lower temperatures as a measure of polymerized material at equilibrium, the spindle protein association was evaluated according to the method of Inoué. Both pool conditions produced linear van't Hoff functions. Analysis of these functions yielded enthalpy and entropy changes of +55–65 kcal/mol and +197–233 entropy units (eu), respectively. These values for active mitotic metaphase are quite comparable to those obtained by Inoué and co-workers for arrested meiotic metaphase cells. When other equilibrium treatments were considered, the best fit to the experimental data was still that of Inoué, a treatment which theoretically involves first-order polymerization and dissociation kinetics. Treatment of metaphase cells with D2O by direct immersion drove the equilibrium to completion regardless of temperature, attaining or exceeding a birefringence value equal to the cell's characteristic pool size; perfusion with D2O appeared to erase the original temperature-determined pool size differences for the two growth conditions, attaining a maximum value characteristic of the larger pool condition. These data confirm Inoué's earlier contention that D2O treatment can modify the available spindle pool. PMID:4734864

Spindle epithelial tumor with thymus-like differentiation: a case report and review of literature.

PubMed

Misra, R K; Mitra, Shaila; Yadav, Rajesh; Bundela, Alpana

2013-01-01

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is an extremely rare type of thyroid tumor with fewer than 35 reported cases available in the literature so far, most of them having been diagnosed histologically after resection. The tumor is believed to be derived from branchial-pouch or thymic remnants, occurring in young adults, predominantly in males, with a male:female ratio 1.8:1. A 14-year-old girl presented with a nodular mass in her right thyroid that had been present for 1 year. Ultrasonological study revealed a heterogeneous solid mass (2.5 × 1.5 × 1.5 cm) in the right lobe of the thyroid. Fine-needle aspiration (FNA) smears were highly cellular and comprised of predominantly dissociated uniform spindle cells with naked oval nuclei along with some aggregates and groups. Occasional islands of epithelial cells were also present. Cytologically, the spindle cells had bland nuclear chromatin, with very scanty mitotic figures. Upon examination of the FNA smears, a provisional diagnosis of SETTLE was suggested along with a request for an incisional biopsy to rule out another differential diagnosis of medullary carcinoma thyroid. On the resected tissue specimen, diagnosis was histologically confirmed to be SETTLE. Immunohistochemical study revealed a strong and diffuse positivity for high-molecular-weight keratin and vimentin, and negativity for thyroglobulin, calcitonin, S-100 protein, desmin, chromogranin and synaptophysin. Cytologically, SETTLE can safely be considered, especially if spindle elements are observed along with the occasional group of epithelial cells in FNA smears from the thyroid of young adults. It can help in the preoperative recognition of lesions based on distinctive cytomorphological features and immunohistochemical characteristics, allowing a more sound therapeutic approach because these patients can present with delayed metastasis. Copyright © 2013 S. Karger AG, Basel.

LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring.

PubMed

Price, Kari L; Rose, Lesilee S

2017-09-01

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule-dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells. © 2017 Price and Rose. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Cytoskeletal mechanisms in positioning of the second-division spindles and meiotic restitution in tobacco (Nicotiana tabacum L.) microsporogenesis.

PubMed

Sidorchuk, Yuriy Vladimirovich; Deineko, Elena Victorovna

2017-06-01

Microsporogenesis patterns of the polyploid (2n = 4x = 96) and diploid (2n = 2x = 48) Nicotiana tabacum L. (cv. Havana Petit line SR1) plants have been analyzed and compared. Four types of abnormal positions of the second-division spindles-tripolar, parallel, proximal, and fused-have been observed. Of these abnormalities, only tripolar (2.4%) and parallel (1.4%) spindles are observable in diploid plants. As for polyploids, the increased ploidy is accompanied by an increase in the incidence of tripolar (22.8%) and parallel (8.1%) spindle orientations and emergence of two remaining abnormalities (proximal and fused spindles, 3.3%). As has been shown, the spindle position abnormalities in diploid plants have no effect on the meiotic products, whereas both dyads and triads are detectable among the tetrads in polyploid plants. Analysis of cytoskeletal remodeling has allowed for the insight into the role of interzonal radial microtubule system in spindle positioning during the second division. The reason underlying the change in spindle positioning is disturbed polymerization-depolymerization processes and interdigitation of microtubule plus ends within the interzonal cytoskeleton system in late telophase I-interkinesis and prophase II. As has been demonstrated, fused second-division spindles are formed as a result of fused cytoskeletal structures in prophase-prometaphase II in the case when the nuclei are drawn abnormally close to one another. © 2017 International Federation for Cell Biology.

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae.

PubMed

Wilmes, Anja; Hanna, Reem; Heathcott, Rosemary W; Northcote, Peter T; Atkinson, Paul H; Bellows, David S; Miller, John H

2012-04-15

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking. Copyright © 2012 Elsevier B.V. All rights reserved.

Modeling the temporal evolution of the spindle assembly checkpoint and role of Aurora B kinase

PubMed Central

Mistry, Hitesh B.; MacCallum, David E.; Jackson, Robert C.; Chaplain, Mark A. J.; Davidson, Fordyce A.

2008-01-01

Faithful separation of chromosomes prior to cell division at mitosis is a highly regulated process. One family of serine/threonine kinases that plays a central role in regulation is the Aurora family. Aurora B plays a role in the spindle assembly checkpoint, in part, by destabilizing the localization of BubR1 and Mad2 at centrosomes and responds to changes in tension caused by aberrant microtubule kinetochore attachments. Aurora B is overexpressed in a subset of cancers and is required for mitosis, making it an attractive anticancer target. Here, we use mathematical modeling to extend a current model of the spindle assembly checkpoint to incorporate all signaling kinetochores within a cell rather than just one and the role of Aurora B within the resulting model. We find that the current model of the spindle assembly checkpoint is robust to variation in its key diffusion-limited parameters. Furthermore, when Aurora B inhibition is considered within the model, for a certain range of inhibitor concentrations, a prolonged prometaphase/metaphase is observed. This level of inhibitor concentrations has not yet been studied experimentally, to the authors' best knowledge. Therefore, experimental verification of the results discussed here could provide a deeper understanding of how kinetochores and Aurora B cooperate in the spindle assembly checkpoint. PMID:19091947

Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis.

PubMed

Duan, Xing; Zhang, Hao-Lin; Pan, Meng-Hao; Zhang, Yu; Sun, Shao-Chen

2018-02-01

Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization. Copyright © 2017 Elsevier B.V. All rights reserved.

A Taz1- and Microtubule-Dependent Regulatory Relationship between Telomere and Centromere Positions in Bouquet Formation Secures Proper Meiotic Divisions

PubMed Central

Katsumata, Kazuhiro; Hirayasu, Ami; Miyoshi, Junpei; Nishi, Eriko; Ichikawa, Kento; Tateho, Kazuki; Wakuda, Airi; Matsuhara, Hirotada; Yamamoto, Ayumu

2016-01-01

During meiotic prophase, telomeres cluster, forming the bouquet chromosome arrangement, and facilitate homologous chromosome pairing. In fission yeast, bouquet formation requires switching of telomere and centromere positions. Centromeres are located at the spindle pole body (SPB) during mitotic interphase, and upon entering meiosis, telomeres cluster at the SPB, followed by centromere detachment from the SPB. Telomere clustering depends on the formation of the microtubule-organizing center at telomeres by the linker of nucleoskeleton and cytoskeleton complex (LINC), while centromere detachment depends on disassembly of kinetochores, which induces meiotic centromere formation. However, how the switching of telomere and centromere positions occurs during bouquet formation is not fully understood. Here, we show that, when impaired telomere interaction with the LINC or microtubule disruption inhibited telomere clustering, kinetochore disassembly-dependent centromere detachment and accompanying meiotic centromere formation were also inhibited. Efficient centromere detachment required telomere clustering-dependent SPB recruitment of a conserved telomere component, Taz1, and microtubules. Furthermore, when artificial SPB recruitment of Taz1 induced centromere detachment in telomere clustering-defective cells, spindle formation was impaired. Thus, detachment of centromeres from the SPB without telomere clustering causes spindle impairment. These findings establish novel regulatory mechanisms, which prevent concurrent detachment of telomeres and centromeres from the SPB during bouquet formation and secure proper meiotic divisions. PMID:27611693

FANCA safeguards interphase and mitosis during hematopoiesis in vivo

PubMed Central

Abdul-Sater, Zahi; Cerabona, Donna; Sierra Potchanant, Elizabeth; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W. Scott; Nalepa, Grzegorz

2015-01-01

Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in non-hematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material (PCM) to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA cross-linking and antimitotic chemotherapeutics in primary FANCA−/− cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that the FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers. PMID:26366677

The Aurora kinase A inhibitor TC-A2317 disrupts mitotic progression and inhibits cancer cell proliferation

PubMed Central

Min, Yoo Hong; Kim, Wootae; Kim, Ja-Eun

2016-01-01

Mitotic progression is crucial for the maintenance of chromosomal stability. A proper progression is ensured by the activities of multiple kinases. One of these enzymes, the serine/threonine kinase Aurora A, is required for proper mitosis through the regulation of centrosome and spindle assembly. In this study, we functionally characterized a newly developed Aurora kinase A inhibitor, TC-A2317. In human lung cancer cells, TC-A2317 slowed proliferation by causing aberrant formation of centrosome and microtubule spindles and prolonging the duration of mitosis. Abnormal mitotic progression led to accumulation of cells containing micronuclei or multinuclei. Furthermore, TC-A2317–treated cells underwent apoptosis, autophagy or senescence depending on cell type. In addition, TC-A2317 inactivated the spindle assembly checkpoint triggered by paclitaxel, thereby exacerbating mitotic catastrophe. Consistent with this, the expression level of Aurora A in tumors was inversely correlated with survival in lung cancer patients. Collectively, these data suggest that inhibition of Aurora kinase A using TC-A2317 is a promising target for anti-cancer therapeutics. PMID:27713168

INPP5E Preserves Genomic Stability through Regulation of Mitosis.

PubMed

Sierra Potchanant, Elizabeth A; Cerabona, Donna; Sater, Zahi Abdul; He, Ying; Sun, Zejin; Gehlhausen, Jeff; Nalepa, Grzegorz

2017-03-15

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development. Copyright © 2017 Sierra Potchanant et al.

Assays for the spindle assembly checkpoint in cell culture.

PubMed

Marcozzi, Chiara; Pines, Jonathon

2018-01-01

The spindle assembly checkpoint (SAC) is crucial to maintain genomic stability since it prevents premature separation of sister chromatids in mitosis and ensures the fidelity of chromosome segregation. The SAC arrests cells in mitosis and is not satisfied until all kinetochores are stably attached to the mitotic spindle. Improperly attached kinetochores activate the SAC and catalyze the formation of the mitotic checkpoint complex (MCC), containing Mad2, Cdc20, BubR1, and Bub3 proteins. The MCC binds and thereby inhibits the APC/C E3 ubiquitin ligase until the last kinetochore has attached to microtubules. Once the SAC is satisfied, the APC/C promptly activates and targets cyclin B1 and securin for degradation, thus allowing sister chromatids to separate and the cell to exit mitosis. Our understanding of SAC signaling has increased thanks to the development of new genetic, biochemical, molecular, and structural biology techniques. Here, we describe how live-cell imaging microscopy in combination with gene-targeting strategies and biochemical assays can be exploited to investigate the intrinsic properties of the SAC in mammalian cultured cells. © 2018 Elsevier Inc. All rights reserved.

Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Enzhi; Lei, Yan; Liu, Qian

2009-04-15

A novel protein that associates with interphase nucleus and mitotic apparatus (INMAP) was identified by screening HeLa cDNA expression library with an autoimmune serum followed by tandem mass spectrometry. Its complete cDNA sequence of 1.818 kb encodes 343 amino acids with predicted molecular mass of 38.2 kDa and numerous phosphorylation sites. The sequence is identical with nucleotides 1-1800 bp of an unnamed gene (GenBank accession no. (7022388)) and highly homologous with the 3'-terminal sequence of POLR3B. A monoclonal antibody against INMAP reacted with similar proteins in S. cerevisiae, Mel and HeLa cells, suggesting that it is a conserved protein. Confocalmore » microscopy using either GFP-INMAP fusion protein or labeling with the monoclonal antibody revealed that the protein localizes as distinct dots in the interphase nucleus, but during mitosis associates closely with the spindle. Double immunolabeling using specific antibodies showed that the INMAP co-localizes with {alpha}-tubulin, {gamma}-tubulin, and NuMA. INMAP also co-immunoprecipitated with these proteins in their native state. Stable overexpression of INMAP in HeLa cell lines leads to defects in the spindle, mitotic arrest, formation of polycentrosomal and multinuclear cells, inhibition of growth, and apoptosis. We propose that INMAP is a novel protein that plays essential role in spindle formation and cell-cycle progression.« less

Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

PubMed

Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

2014-02-01

During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly.

PubMed

Schlaitz, Anne-Lore; Srayko, Martin; Dammermann, Alexander; Quintin, Sophie; Wielsch, Natalie; MacLeod, Ian; de Robillard, Quentin; Zinke, Andrea; Yates, John R; Müller-Reichert, Thomas; Shevchenko, Andrei; Oegema, Karen; Hyman, Anthony A

2007-01-12

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.

Direct kinetochore–spindle pole connections are not required for chromosome segregation

PubMed Central

Sikirzhytski, Vitali; Magidson, Valentin; Steinman, Jonathan B.; He, Jie; Le Berre, Maël; Tikhonenko, Irina; Ault, Jeffrey G.; McEwen, Bruce F.; Chen, James K.; Sui, Haixin; Piel, Matthieu; Kapoor, Tarun M.

2014-01-01

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes′ kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells. PMID:25023516

Direct kinetochore-spindle pole connections are not required for chromosome segregation.

PubMed

Sikirzhytski, Vitali; Magidson, Valentin; Steinman, Jonathan B; He, Jie; Le Berre, Maël; Tikhonenko, Irina; Ault, Jeffrey G; McEwen, Bruce F; Chen, James K; Sui, Haixin; Piel, Matthieu; Kapoor, Tarun M; Khodjakov, Alexey

2014-07-21

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

The progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling.

PubMed Central

Katsanakis, Kostas D.; Gorgoulis, Vassilis; Papavassiliou, Athanasios G.; Zoumpourlis, Vassilis K.

2002-01-01

BACKGROUND: The ras family of proto-oncogenes encodes for small GTPases that play critical roles in cell-cycle progression and cellular transformation. ERK1/2 MAP kinases are major ras effectors. Tumors in chemically treated mouse skin contain mutations in the Ha-ras proto- oncogene. Amplification and mutation of Ha-ras has been shown to correlate with malignant progression of these tumors. Cell lines isolated from mouse skin tumors represent the stages of tumor development, such as the PDV:PDVC57 cell line pair and B9 squamous carcinoma and A5 spindle cells. PDVC57 cells were selected from PDV cells, which were transformed with dimethyl-benzanthracene (DMBA) in vitro and then transplanted in adult syngeneic mice. The PDV:PDVC57 pair contains ratio of normal:mutant Ha-ras 2:1 and 1:2, respectively. This genetic alteration correlates with more advanced tumorigenic characteristics of PDVC57 compared to PDV. The squamous carcinoma B9 cell clone was isolated from the same primary tumor as A5 spindle cell line. The mutant Ha-ras allele, also present in B9, is amplified and overexpressed in A5 cells. Therefore these cell line pairs represent an in vivo model for studies of Ha-ras and ERK1/2 signaling in mouse tumorigenesis. MATERIALS AND METHODS: The ERK1/2 status in the above mouse cell lines was examined by using various molecular techniques. For the study of the tumorigenic properties and the role of the ras/MEK/ERK1/2 pathway in the cell lines mentioned, phenotypic characteristics, colony formation assay, anchorage-independent growth, and gelatin zymography were assessed, after or without treatment with the MEK inhibitor, PD98059. RESULTS: ERK1/2 phosphorylation was found to be increased in PDVC57 when compared to PDV. This also applies to A5 spindle carcinoma cells when compared to squamous carcinoma and papilloma cells. The above finding was reproduced when transfecting human activated Ha-ras allele into PDV, thus demonstrating that Ha-ras enhances ERK1/2 signaling. To further test whether ERK1/2 activation was required for growth we used the MEK-1 inhibitor, PD98059. The latter inhibited cell proliferation and anchorage-independent growth of squamous and spindle cells. In addition, PD98059 treatment partially reverted the spindle morphology of A5 cells. CONCLUSIONS: These data suggest, for the first time, that oncogenicity and the degree of progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling. PMID:12477973

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis.

PubMed

D'Avino, Pier Paolo; Savoian, Matthew S; Capalbo, Luisa; Glover, David M

2006-11-01

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

Promyelocytic leukemia bodies tether to early endosomes during mitosis.

PubMed

Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

2014-01-01

During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

Loss and Rebirth of the Animal Microtubule Organizing Center: How Maternal Expression of Centrosomal Proteins Cooperates with the Sperm Centriole in Zygotic Centrosome Reformation.

PubMed

Inoue, Daigo; Wittbrodt, Joachim; Gruss, Oliver J

2018-04-01

Centrosomes are the main microtubule organizing centers in animal cells. In particular during embryogenesis, they ensure faithful spindle formation and proper cell divisions. As metazoan centrosomes are eliminated during oogenesis, they have to be reassembled upon fertilization. Most metazoans use the sperm centrioles as templates for new centrosome biogenesis while the egg's cytoplasm re-prepares all components for on-going centrosome duplication in rapidly dividing embryonic cells. We discuss our knowledge and the experimental challenges to analyze zygotic centrosome reformation, which requires genetic experiments to enable scrutinizing respective male and female contributions. Male and female knockout animals and mRNA injection to mimic maternal expression of centrosomal proteins could point a way to the systematic molecular dissection of the process. The most recent data suggest that timely expression of centrosome components in oocytes is the key to zygotic centrosome reformation that uses male sperm as coordinators for de novo centrosome production. © 2018 WILEY Periodicals, Inc.

Use of abnormal preprophase bands to decipher division plane determination

NASA Technical Reports Server (NTRS)

Granger, C.; Cyr, R.

2001-01-01

Many premitotic plant cells possess a cortical preprophase band of microtubules and actin filaments that encircles the nucleus. In vacuolated cells, the preprophase band is visibly connected to the nucleus by a cytoplasmic raft of actin filaments and microtubules termed the phragmosome. Typically, the location of the preprophase band and phragmosome corresponds to, and thus is thought to influence, the location of the cell division plane. To better understand the function of the preprophase band and phragmosome in orienting division, we used a green fluorescent protein-based microtubule reporter protein to observe mitosis in living tobacco bright yellow 2 cells possessing unusual preprophase bands. Observations of mitosis in these unusual cells support the involvement of the preprophase band/phragmosome in properly positioning the preprophase nucleus, influencing spindle orientation such that the cytokinetic phragmoplast initially grows in an appropriate direction, and delineating a region in the cell cortex that attracts microtubules and directs later stages of phragmoplast growth. Thus, the preprophase band/phragmosome appears to perform several interrelated functions to orient the division plane. However, functional information associated with the preprophase band is not always used or needed and there appears to be an age or distance-dependent character to the information. Cells treated with the anti-actin drug, latrunculin B, are still able to position the preprophase nucleus suggesting that microtubules may play a dominant role in premitotic positioning. Furthermore, in treated cells, spindle location and phragmoplast insertion are frequently abnormal suggesting that actin plays a significant role in nuclear anchoring and phragmoplast guidance. Thus, the microtubule and actin components of the preprophase band/phragmosome execute complementary activities to ensure proper orientation of the division plane.

The small molecule CS1 inhibits mitosis and sister chromatid resolution in HeLa cells.

PubMed

Wu, Xingkang; Li, Zhenyu; Shen, Yuemao

2018-05-01

Mitosis, the most dramatic event in the cell cycle, involves the reorganization of virtually all cellular components. Antimitotic agents are useful for dissecting the mechanism of this reorganization. Previously, we found that the small molecule CS1 accumulates cells in G2/M phase [1], but the mechanism of its action remains unknown. Cell cycle analysis, live cell imaging and nuclear staining were used. Chromosomal morphology was detected by chromosome spreading. The effects of CS1 on microtubules were confirmed by tubulin polymerization, colchicine tubulin-binding, cellular tubulin polymerization and immunofluorescence assays and by analysis of microtubule dynamics and molecular modeling. Histone phosphoproteomics was performed using mass spectrometry. Cell signaling cascades were analyzed using immunofluorescence, immunoprecipitation, immunoblotting, siRNA knockdown and chemical inhibition of specific proteins. The small molecule CS1 was shown to be an antimitotic agent. CS1 potently inhibited microtubule polymerization via interaction with the colchicine-binding pocket of tubulin in vitro and inhibited the formation of the spindle apparatus by reducing the bulk of growing microtubules in HeLa cells, which led to activation of the spindle assembly checkpoint (SAC) and mitotic arrest of HeLa cells. Compared with colchicine, CS1 impaired the progression of sister chromatid resolution independent of cohesin dissociation, and this was reversed by the removal of CS1. Additionally, CS1 induced unique histone phosphorylation patterns distinct from those induced by colchicine. CS1 is a unique antimitotic small molecule and a powerful tool with unprecedented value over colchicine that makes it possible to specifically and conditionally perturb mitotic progression. Copyright © 2018 Elsevier B.V. All rights reserved.

Fine needle aspiration of secondary synovial sarcoma of the thyroid gland.

PubMed

Murro, Diana; Slade, Jamie Macagba; Syed, Sahr; Gattuso, Paolo

2015-11-01

Synovial sarcomas (SS) of the head and neck region are extremely rare and arise in only 5% of cases. We present a case of secondary SS of the thyroid originally diagnosed as medullary carcinoma on fine needle aspiration (FNA). A 41-year-old man presented with several weeks of dysphonia and a left thyroid mass. FNA of the thyroid nodule showed a cellular smear composed of loosely cohesive oval to spindle-shaped cells with irregular nuclear borders, finely granular chromatin, and inconspicuous nucleoli. The patient was diagnosed with medullary carcinoma and underwent a total thyroidectomy. Intro-operatively, the mass was found to arise from the tracheoesophageal groove with spread to the left thyroid. Microscopic examination of the thyroid tumor revealed a dense spindle cell proliferation with abundant mitoses, scant cords and nests of epithelial cells and foci of necrosis. The spindle cells were positive for bcl2 and vimentin and the epithelial cells were positive for cytokeratin 8/18 and epithelial membrane antigen (EMA). Both spindle and epithelial cells were negative for thyroglobulin, calcitonin, synaptophysin and chromogranin. Fluorescence in situ hybridization (FISH) demonstrated translocation (X;18)(p11;q11), confirming the diagnosis of SS. The patient underwent a total laryngopharyngoesophagectomy with subsequent adjuvant therapy and is currently disease free. Only 6 cases of histologically confirmed primary SS of the thyroid have been reported. To the best of our knowledge, this is the first case of FISH-confirmed secondary SS of the thyroid and also the first case of SS arising from the tracheoesophageal groove. © 2015 Wiley Periodicals, Inc.

Withaferin A modulates the Spindle assembly checkpoint by degradation of Mad2-Cdc20 complex in colorectal cancer cell lines.

PubMed

Das, Tania; Roy, Kumar Singha; Chakrabarti, Tulika; Mukhopadhyay, Sibabrata; Roychoudhury, Susanta

2014-09-01

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability. Copyright © 2014 Elsevier Inc. All rights reserved.

Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

PubMed

Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

2013-08-01

Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

PubMed Central

Cahu, Julie; Olichon, Aurelien; Hentrich, Christian; Schek, Henry; Drinjakovic, Jovana; Zhang, Cunjie; Doherty-Kirby, Amanda; Lajoie, Gilles; Surrey, Thomas

2008-01-01

Background Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. Methodology/Principal Findings We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. Conclusions/Significance These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein. PMID:19079595

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals

PubMed Central

Hosseini, Sayyed-Morteza; Moulavi, Fariba; Tanhaie-Vash, Nima; Asgari, Vajihe; Ghanaei, Hamid-Reza; Abedi-Dorche, Maryam; Jafarzadeh, Naser; Gourabi, Hossein; Shahverdi, Abdol-Hossein; Dizaj, Ahmad Vosough; Shirazi, Abolfazl; Nasr-Esfahani, Mohammad-Hossein

2016-01-01

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages. PMID:27030988

Modeling Cytoskeletal Active Matter Systems

NASA Astrophysics Data System (ADS)

Blackwell, Robert

Active networks of filamentous proteins and crosslinking motor proteins play a critical role in many important cellular processes. One of the most important microtubule-motor protein assemblies is the mitotic spindle, a self-organized active liquid-crystalline structure that forms during cell division and that ultimately separates chromosomes into two daughter cells. Although the spindle has been intensively studied for decades, the physical principles that govern its self-organization and function remain mysterious. To evolve a better understanding of spindle formation, structure, and dynamics, I investigate course-grained models of active liquid-crystalline networks composed of microtubules, modeled as hard spherocylinders, in diffusive equilibrium with a reservoir of active crosslinks, modeled as hookean springs that can adsorb to microtubules and and translocate at finite velocity along the microtubule axis. This model is investigated using a combination of brownian dynamics and kinetic monte carlo simulation. I have further refined this model to simulate spindle formation and kinetochore capture in the fission yeast S. pombe. I then make predictions for experimentally realizable perturbations in motor protein presence and function in S. pombe.

SNM1B/Apollo interacts with astrin and is required for the prophase cell cycle checkpoint.

PubMed

Liu, Lingling; Akhter, Shamima; Bae, Jae-Bum; Mukhopadhyay, Sudit S; Richie, Christopher T; Liu, Xiaojun; Legerski, Randy

2009-02-15

Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-beta-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.

Progression of Mouse Skin Carcinogenesis Is Associated with Increased Erα Levels and Is Repressed by a Dominant Negative Form of Erα

PubMed Central

Michalopoulos, Ioannis; Sideridou, Maria; Tsimaratou, Katerina; Christodoulou, Ioannis; Pyrillou, Katerina; Gorgoulis, Vassilis; Vlahopoulos, Spiros; Zoumpourlis, Vassilis

2012-01-01

Estrogen receptors (ER), namely ERα and ERβ, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ERα in skin cancer, using chemically-induced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ERα is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ERβ levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ERα gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ERβ levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ERα (dnERα) resulted in reduced ERα levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ERα promoter through which dnERα decreased endogenous ERα levels. The dnERα-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ERα in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnERα. PMID:22870269

Error-prone meiotic division and subfertility in mice with oocyte-conditional knockdown of pericentrin.

PubMed

Baumann, Claudia; Wang, Xiaotian; Yang, Luhan; Viveiros, Maria M

2017-04-01

Mouse oocytes lack canonical centrosomes and instead contain unique acentriolar microtubule-organizing centers (aMTOCs). To test the function of these distinct aMTOCs in meiotic spindle formation, pericentrin (Pcnt), an essential centrosome/MTOC protein, was knocked down exclusively in oocytes by using a transgenic RNAi approach. Here, we provide evidence that disruption of aMTOC function in oocytes promotes spindle instability and severe meiotic errors that lead to pronounced female subfertility. Pcnt-depleted oocytes from transgenic (Tg) mice were ovulated at the metaphase-II stage, but show significant chromosome misalignment, aneuploidy and premature sister chromatid separation. These defects were associated with loss of key Pcnt-interacting proteins (γ-tubulin, Nedd1 and Cep215) from meiotic spindle poles, altered spindle structure and chromosome-microtubule attachment errors. Live-cell imaging revealed disruptions in the dynamics of spindle assembly and organization, together with chromosome attachment and congression defects. Notably, spindle formation was dependent on Ran GTPase activity in Pcnt-deficient oocytes. Our findings establish that meiotic division is highly error-prone in the absence of Pcnt and disrupted aMTOCs, similar to what reportedly occurs in human oocytes. Moreover, these data underscore crucial differences between MTOC-dependent and -independent meiotic spindle assembly. © 2017. Published by The Company of Biologists Ltd.

Drosophila Klp67A binds prophase kinetochores to subsequently regulate congression and spindle length.

PubMed

Savoian, Matthew S; Glover, David M

2010-03-01

The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolymerising motor activity of Klp67A exclusively when located at kinetochores, but not along the spindle. These data reveal that, unlike other metazoan kinesin-8 proteins, Klp67A binds the nascent prophase and mature metaphase kinetochore. From this location, Klp67A uses its motor activity to ensure chromosome alignment and proper spindle length.

Clathrin heavy chain 1 is required for spindle assembly and chromosome congression in mouse oocytes.

PubMed

Zhao, Jie; Wang, Lu; Zhou, Hong-Xia; Liu, Li; Lu, Angeleem; Li, Guang-Peng; Schatten, Heide; Liang, Cheng-Guang

2013-10-01

Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.

A unilateral hemothorax as a presenting manifestation of mediastinal spindle cell sarcoma.

PubMed

Chabowski, M; Szymanska-Chabowska, A; Szolkowska, M; Janczak, D

2015-01-01

The article presents the case of a 73-year-old female injured in a bicycle accident, who was diagnosed with a left hemothorax. Initially, a chest drain was inserted and the pleural hematoma was evacuated. Then a thoracotomy was performed. A hematoma debridement and decortication with a subsequent tissue biopsy was carried out and a final diagnosis of spindle cell sarcoma was made. There is a brief discussion on the differential diagnosis of spontaneous hemothorax and its management.

Topological defects in confined populations of spindle-shaped cells

NASA Astrophysics Data System (ADS)

Duclos, Guillaume; Erlenkämper, Christoph; Joanny, Jean-François; Silberzan, Pascal

2017-01-01

Most spindle-shaped cells (including smooth muscles and sarcomas) organize in vivo into well-aligned `nematic’ domains, creating intrinsic topological defects that may be used to probe the behaviour of these active nematic systems. Active non-cellular nematics have been shown to be dominated by activity, yielding complex chaotic flows. However, the regime in which live spindle-shaped cells operate, and the importance of cell-substrate friction in particular, remains largely unexplored. Using in vitro experiments, we show that these active cellular nematics operate in a regime in which activity is effectively damped by friction, and that the interaction between defects is controlled by the system’s elastic nematic energy. Due to the activity of the cells, these defects behave as self-propelled particles and pairwise annihilate until all displacements freeze as cell crowding increases. When confined in mesoscopic circular domains, the system evolves towards two identical +1/2 disclinations facing each other. The most likely reduced positions of these defects are independent of the size of the disk, the cells’ activity or even the cell type, but are well described by equilibrium liquid crystal theory. These cell-based systems thus operate in a regime more stable than other active nematics, which may be necessary for their biological function.

Remote drill bit loader

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dokos, J.A.

1996-12-31

A drill bit loader is described for loading a tapered shank of a drill bit into a similarly tapered recess in the end of a drill spindle. The spindle has a transverse slot at the inner end of the recess. The end of the tapered shank of the drill bit has a transverse tang adapted to engage in the slot so that the drill bit will be rotated by the spindle. The loader is in the form of a cylinder adapted to receive the drill bit with the shank projecting out of the outer end of the cylinder. Retainer pinsmore » prevent rotation of the drill bit in the cylinder. The spindle is lowered to extend the shank of the drill bit into the recess in the spindle and the spindle is rotated to align the slot in the spindle with the tang on the shank. A spring unit in the cylinder is compressed by the drill bit during its entry into the recess of the spindle and resiliently drives the tang into the slot in the spindle when the tang and slot are aligned. In typical remote drilling operations, whether in hot cells or water pits, drill bits have been held using a collet or end mill type holder with set screws. In either case, to load or change a drill bit required the use master-slave manipulators to position the bits and tighten the collet or set screws. This requirement eliminated many otherwise useful work areas because they were not equipped with slaves, particularly in water pits.« less

Centrosome Linker-induced Tetraploid Segregation Errors Link Rhabdoid Phenotypes and Lethal Colorectal Cancers.

PubMed

Remo, Andrea; Manfrin, Erminia; Parcesepe, Pietro; Ferrarini, Alberto; Han, Hye Seung; Ugnius, Mickys; Laudanna, Carmelo; Simbolo, Michele; Malanga, Donatella; Mendes Oliveira, Duarte; Baritono, Elisabetta; Colangelo, Tommaso; Sabatino, Lina; Giuliani, Jacopo; Molinari, Enrico; Garonzi, Marianna; Xumerle, Luciano; Delledonne, Massimo; Giordano, Guido; Ghimenton, Claudio; Lonardo, Fortunato; D'angelo, Fulvio; Grillo, Federica; Mastracci, Luca; Viglietto, Giuseppe; Ceccarelli, Michele; Colantuoni, Vittorio; Scarpa, Aldo; Pancione, Massimo

2018-05-21

Centrosome anomalies contribute to tumorigenesis but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAF(V600E) mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosomal-linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAF(V600E) mutant and microsatellite stable (MSS) rhabdoid colorectal cancers but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAF(V600E) mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors resulting in a highly aggressive rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability. Mis-segregation of chromosomes is a prominent feature of chromosome instability and intra-tumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. The present study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Copyright ©2018, American Association for Cancer Research.

A role for the Rab6A′ GTPase in the inactivation of the Mad2-spindle checkpoint

PubMed Central

Miserey-Lenkei, Stéphanie; Couëdel-Courteille, Anne; Del Nery, Elaine; Bardin, Sabine; Piel, Matthieu; Racine, Victor; Sibarita, Jean-Baptiste; Perez, Franck; Bornens, Michel; Goud, Bruno

2006-01-01

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A′, regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A′ function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150Glued, a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A′ likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A′, through its interaction with p150Glued and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition. PMID:16395330

Jacalin and peanut agglutinin (PNA) bindings in the taste bud cells of the rat: new reliable markers for type IV cells of the rat taste buds.

PubMed

Taniguchi, Ryo; Shi, Lei; Fujii, Masae; Ueda, Katsura; Honma, Shiho; Wakisaka, Satoshi

2005-12-01

Lectin histochemistry of Jacalin (Artocarpus integrifolia) and peanut agglutinin (PNA), specific lectins for galactosyl (beta-1, 3) N-acetylgalactosamine (galactosyl (beta-1, 3) GalNAc), was applied to the gustatory epithelium of the adult rat. In the ordinary lingual epithelium, Jacalin and PNA labeled the cell membrane from the basal to granular cell layer. They also bound membranes of rounded-cells at the basal portion of taste buds, but the number of PNA labeled cells was smaller than that of Jacalin labeled cells. There was no apparent difference in the binding patterns of Jacalin and PNA among the taste buds of the lingual papillae and those of the palatal epithelium. Occasionally, a few spindle-shaped cells were labeled with Jacalin, but not with PNA. Double labeling of Jacalin and alpha-gustducin, a specific marker for type II cells, revealed that Jacalin-labeled spindle-shaped taste cells were immunonegative for alpha-gustducin. Spindle-shaped cells expressing protein gene product 9.5 (PGP 9.5) immunoreactivity lacked Jacalin labeling. During the development of taste buds in circumvallate papillae, the binding pattern of Jacalin became almost identical from postnatal day 5. The present results indicate that rounded cells at the basal portion of the taste buds cells (type IV cells) bind to Jacalin and PNA, and these lectins are specific markers for type IV cells of the rat taste cells.

Cortical dendritic activity correlates with spindle-rich oscillations during sleep in rodents.

PubMed

Seibt, Julie; Richard, Clément J; Sigl-Glöckner, Johanna; Takahashi, Naoya; Kaplan, David I; Doron, Guy; de Limoges, Denis; Bocklisch, Christina; Larkum, Matthew E

2017-09-25

How sleep influences brain plasticity is not known. In particular, why certain electroencephalographic (EEG) rhythms are linked to memory consolidation is poorly understood. Calcium activity in dendrites is known to be necessary for structural plasticity changes, but this has never been carefully examined during sleep. Here, we report that calcium activity in populations of neocortical dendrites is increased and synchronised during oscillations in the spindle range in naturally sleeping rodents. Remarkably, the same relationship is not found in cell bodies of the same neurons and throughout the cortical column. Spindles during sleep have been suggested to be important for brain development and plasticity. Our results provide evidence for a physiological link of spindles in the cortex specific to dendrites, the main site of synaptic plasticity.Different stages of sleep, marked by particular electroencephalographic (EEG) signatures, have been linked to memory consolidation, but underlying mechanisms are poorly understood. Here, the authors show that dendritic calcium synchronisation correlates with spindle-rich sleep phases.

Late-Stage Erythema Elevatum Diutinum Mimicking a Fibroblastic Tumor: A Potential Pitfall.

PubMed

Llamas-Velasco, Mar; Stengel, Berhard; Pérez-González, Yosmar Carolina; Mentzel, Thomas

2018-06-01

Erythema elevatum diutinum (EED) is a rare dermatosis with evolving histopathological features that vary according to the age of the lesions, with a variable fibrosis and a fascicled proliferation of spindle cells in late phases. The authors present an otherwise healthy 57-year-old woman with multiple indurated nodules on the inner aspect of both feet. Skin biopsy showed storiform interlacing bundles of spindled cells with plump nuclei and some areas with neutrophils and leukocytoclasia. CD34 and S100 were negative. This case is noteworthy clinically due to its location and its histopathological presentation that comprises a wide differential diagnosis, including inflammatory pseudotumor, dermatofibrosarcoma protuberans, superficial nodular fasciitis, hyalinized leiomyoma, sclerosing spindle cell perineuroma, and sclerotic fibroma. The authors have reviewed the main histopathological and immunohistochemical features that help in the differential diagnosis of this rare variant of EED. A careful search for leukocytoclasia and neutrophilic vasculitis is mandatory to establish the right diagnosis of nodular or late-stage EED and avoid the pitfall of considering this a neoplastic process.

A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

PubMed Central

Kern, David M.; Nicholls, Peter K.; Page, David C.

2016-01-01

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. PMID:27138257

Geometric shapes inversion method of space targets by ISAR image segmentation

NASA Astrophysics Data System (ADS)

Huo, Chao-ying; Xing, Xiao-yu; Yin, Hong-cheng; Li, Chen-guang; Zeng, Xiang-yun; Xu, Gao-gui

2017-11-01

The geometric shape of target is an effective characteristic in the process of space targets recognition. This paper proposed a method of shape inversion of space target based on components segmentation from ISAR image. The Radon transformation, Hough transformation, K-means clustering, triangulation will be introduced into ISAR image processing. Firstly, we use Radon transformation and edge detection to extract space target's main body spindle and solar panel spindle from ISAR image. Then the targets' main body, solar panel, rectangular and circular antenna are segmented from ISAR image based on image detection theory. Finally, the sizes of every structural component are computed. The effectiveness of this method is verified using typical targets' simulation data.

Apparatus and method for forming a workpiece surface into a non-rotationally symmetric shape

DOEpatents

Dow, Thomas A.; Garrard, Kenneth P.; Moorefield, II, George M.; Taylor, Lauren W.

1995-11-21

A turning machine includes a controller for generating both aspherical and non-symmetrical shape components defining the predetermined shape, and a controller for controlling a spindle and a positionable cutting blade to thereby form a predetermined non-rotationally symmetric shape in a workpiece surface. The apparatus includes a rotatable spindle for rotatably mounting the workpiece about an axis, a spindle encoder for sensing an angular position of the rotating workpiece, the cutting blade, and radial and transverse positioners for relatively positioning the cutting blade and workpiece along respective radial and transverse directions. The controller cooperates with a fast transverse positioner for positioning the cutting blade in predetermined varying transverse positions during a revolution of the workpiece.

Single Site α-Tubulin Mutation Affects Astral Microtubules and Nuclear Positioning during Anaphase in Saccharomyces cerevisiae: Possible Role for Palmitoylation of α-Tubulin

PubMed Central

Caron, Joan M.; Vega, Leticia R.; Fleming, James; Bishop, Robert; Solomon, Frank

2001-01-01

We generated a strain of Saccharomyces cerevisiae in which the sole source of α-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S α-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377S tub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15°C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in α-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated α-tubulin in TUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated α-tubulin in C377S tub1 cells. Our results suggest that cys-377 of α-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle. PMID:11553707

Spindle checkpoint–independent inhibition of mitotic chromosome segregation by Drosophila Mps1

PubMed Central

Althoff, Friederike; Karess, Roger E.; Lehner, Christian F.

2012-01-01

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation. PMID:22553353

Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1.

PubMed

Althoff, Friederike; Karess, Roger E; Lehner, Christian F

2012-06-01

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.

Measuring mitotic forces.

PubMed

Ye, Anna A; Maresca, Thomas J

2018-01-01

Productive chromosome movements require that a large multiprotein complex called the kinetochore assemble on sister centromeres. The kinetochore fulfills two critical functions as (1) the physical linkage between chromosomes and spindle microtubules and (2) a mechanomolecular sensor that relays a spindle assembly checkpoint signal delaying anaphase onset until chromosomes are attached to spindle microtubules and bioriented. Given its central roles in such a vital process, the kinetochore is one of the most important force-transducing structures in cells; yet it has been technically challenging to measure kinetochore forces. Barriers to measuring cellular forces have begun to be broken by the development of fluorescence-based tension sensors. In this chapter, two methods will be described for measuring kinetochore forces in living cells and strategies for applying these sensors to other force-transducing processes and molecules will be discussed. © 2018 Elsevier Inc. All rights reserved.

The fake fat phenomenon in organizing pleuritis: a source of confusion with desmoplastic malignant mesotheliomas.

PubMed

Churg, Andrew; Cagle, Philip; Colby, Thomas V; Corson, Joseph M; Gibbs, Allen R; Hammar, Samuel; Ordonez, Nelson; Roggli, Victor L; Tazelaar, Henry D; Travis, William D; Wick, Mark

2011-12-01

We report 9 patients with pleural biopsies referred because of concern about infiltration of what appeared to be chest wall fat by pan-keratin-positive spindled cells, a finding that led to a consideration of desmoplastic mesothelioma. All patients showed pleural effusions/pleural thickening on computed tomographic scan. Pleural biopsy showed a greatly thickened and fibrotic paucicellular pleura with circular fat-like spaces and, sometimes, adjacent oblate spaces mostly deep in the fibrotic area. Indistinct, keratin-positive, spindle cells arranged parallel to the pleural surface coursed between these fat-like spaces. S-100 stains were negative around the fat-like spaces. Vimentin stains showed that the spaces did not have a cellular lining of any kind. Sometimes the spaces contained faintly hematoxyphilic material that was Alcian blue positive, and similar material was seen in the fibrotic stroma. Follow-up with periods ranging from 6 to 30 months revealed that 8 cases had stable disease on chest imaging or by clinical findings. One case had slowly progressive pleural thickening. These observations suggest that spaces resembling fat may be encountered in fibrotic pleurae and that horizontally oriented keratin-positive spindled cells between the fat-like spaces deep in the fibrotic portion of a thickened pleura represent a benign finding seen in some cases of organizing pleuritis/fibrothorax. The spaces themselves are probably artifacts derived from the biopsy procedure and/or cutting artifacts. In contrast, in true desmoplastic mesotheliomas there is downward, rather than horizontal, growth of keratin-positive spindled cells running between clearly definable fat cells.

Does the peritumoral stroma of basal cell carcinoma recapitulate the follicular connective tissue sheath?

PubMed

Sellheyer, Klaus; Krahl, Dieter

2011-07-01

There are compelling embryologic and anatomic relationships within adnexal tumors. However, these are mostly perceived within the epithelial component while the stromal component of the tumors is frequently overlooked. In postnatal skin, nestin is almost exclusively expressed in the perifollicular mesenchyme. This study examines the expression of this neuroepithelial stem cell protein in trichoblastoma/trichoepithelioma and in basal cell carcinoma (BCC), which is increasingly being viewed as follicular in nature. We employed standard immunohistochemical methods with three different antibodies to examine the expression of nestin in 25 BCCs and compared the staining pattern with that of 7 trichoblastomas and 11 trichoepitheliomas. Nestin is expressed in the peritumoral stroma of all tumors examined and is limited to the immediate layer of mesenchymal cells surrounding the tumor epithelium. In BCC, nestin-immunoreactive cells are found as a sheath of thin, spindled fibroblasts, while reactive cells are plump in trichoepitheliomas/trichoblastomas. We postulate that the peritumoral stroma of BCC imitates the perifollicular connective tissue sheath, while in contrast that of trichoepithelioma/trichoblastoma is similar to the papillary and immediate peripapillary follicular mesenchyme. Further functional and animal experimental studies are needed to test this hypothesis. Copyright © 2011 John Wiley & Sons A/S.

Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G(2)/M transition and sustained spindle checkpoint responses.

PubMed

Zhang, Xiaojuan; Yin, Qingqing; Ling, Youguo; Zhang, Yanhong; Ma, Runlin; Ma, Qingjun; Cao, Cheng; Zhong, Hui; Liu, Xuedong; Xu, Quanbin

2011-08-15

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G 2/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.

Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G2/M transition and sustained spindle checkpoint responses

PubMed Central

Zhang, Xiaojuan; Yin, Qingqing; Ling, Youguo; Zhang, Yanhong; Ma, Runlin; Ma, Qingjun; Cao, Cheng; Zhong, Hui

2011-01-01

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G2/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses. PMID:21778823

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Per; Sellin, Mikael E.; Gullberg, Martin, E-mail: Martin.Gullberg@molbiol.umu.se

2010-07-15

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18{yields}E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis,more » conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.« less

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

PubMed Central

Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

2013-01-01

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis.

PubMed

Lee, Seung Joon; Langhans, Sigrid A

2012-01-26

Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin.

Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

PubMed Central

2012-01-01

Background Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Methods Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. Results We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. Conclusions We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin. PMID:22280307

Malignant solitary fibrous tumor in the extremity: Cytopathologic findings

PubMed Central

Khanchel, Fatma; Driss, Maha; Mrad, Karima; Romdhane, Khaled Ben

2012-01-01

Malignant solitary fibrous tumor (SFT) is an extremely rare neoplasm. There are only rare published accounts of the cytopathologic features of this tumor. We report a case of a 59-year-old woman presented with a 10-year history of a right thigh mass. A preoperative fine needle aspiration (FNA) was performed. Smears were hypercellular, with cohesive and crowded tissue fragments, haphazard cell arrangements and many single cells. The tumor cells were polymorphous, plump spindled or round with often indented or bare nuclei. A differential diagnosis of low grade sarcoma was favored. The diagnosis of malignant SFT is extremely difficult on FNA and must be included in the differential diagnosis of spindle cell neoplasms. PMID:22787298

Structure-Based Design of Orally Bioavailable 1H-Pyrrolo[3,2-c]pyridine Inhibitors of Mitotic Kinase Monopolar Spindle 1 (MPS1)

PubMed Central

2013-01-01

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition. PMID:24256217

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

PubMed

Naud, Sébastien; Westwood, Isaac M; Faisal, Amir; Sheldrake, Peter; Bavetsias, Vassilios; Atrash, Butrus; Cheung, Kwai-Ming J; Liu, Manjuan; Hayes, Angela; Schmitt, Jessica; Wood, Amy; Choi, Vanessa; Boxall, Kathy; Mak, Grace; Gurden, Mark; Valenti, Melanie; de Haven Brandon, Alexis; Henley, Alan; Baker, Ross; McAndrew, Craig; Matijssen, Berry; Burke, Rosemary; Hoelder, Swen; Eccles, Suzanne A; Raynaud, Florence I; Linardopoulos, Spiros; van Montfort, Rob L M; Blagg, Julian

2013-12-27

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts

PubMed Central

Maresca, Thomas J.; Freedman, Benjamin S.; Heald, Rebecca

2005-01-01

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis. PMID:15967810

Control of cleavage spindle orientation in Caenorhabditis elegans: The role of the genes par-2 and par-3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, N.N.; Kirby, C.M.; Kemphues, K.J.

1995-02-01

Polarized asymmetric divisions play important roles in the development of plants and animals. The first two embryonic cleavages of Caenorhabditis elegans provide an opportunity to study the mechanisms controlling polarized asymmetric divisions. The first cleavage is unequal, producing daughters with different sizes and fates. The daughter blastomeres divide with different orientations at the second cleavage; the anterior blastomere divides equally across the long axis of the egg, whereas the posterior blastomere divides unequally along the long axis. We report here the results of our analysis of the genes par-2 and par-3 with respect to their contribution to the polarity ofmore » these divisions. Strong loss-of-function mutations in both genes lead to an equal first cleavage and an altered second cleavage. Interestingly, the mutations exhibit striking gene-specific differences at the second cleavage. The par-2 mutations lead to transverse spindle orientations in both blastomeres, whereas par-3 mutations lead to longitudinal spindle orientations in both blastomeres. The spindle orientation defects correlate with defects in centrosome movements during both the first and the second cell cycle. Temperature shift experiments with par-2 (it5ts) indicate that the par-2(+) activity is not required after the two-cell stage. Analysis of double mutants shows that par-3 is epistatic to par-2. We propose a model wherein par-2(+) and par-3(+) act in concert during the first cell cycle to affect asymmetric modification of the cytoskeleton. This polar modification leads to different behaviors of centrosomes in the anterior and posterior and leads ultimately to blastomere-specific spindle orientations at the second cleavage. 44 refs., 5 figs., 5 tabs.« less

Heat Control via Torque Control in Friction Stir Welding

NASA Technical Reports Server (NTRS)

Venable, Richard; Colligan, Kevin; Knapp, Alan

2004-01-01

In a proposed advance in friction stir welding, the torque exerted on the workpiece by the friction stir pin would be measured and controlled in an effort to measure and control the total heat input to the workpiece. The total heat input to the workpiece is an important parameter of any welding process (fusion or friction stir welding). In fusion welding, measurement and control of heat input is a difficult problem. However, in friction stir welding, the basic principle of operation affords the potential of a straightforward solution: Neglecting thermal losses through the pin and the spindle that supports it, the rate of heat input to the workpiece is the product of the torque and the speed of rotation of the friction stir weld pin and, hence, of the spindle. Therefore, if one acquires and suitably processes data on torque and rotation and controls the torque, the rotation, or both, one should be able to control the heat input into the workpiece. In conventional practice in friction stir welding, one uses feedback control of the spindle motor to maintain a constant speed of rotation. According to the proposal, one would not maintain a constant speed of rotation: Instead, one would use feedback control to maintain a constant torque and would measure the speed of rotation while allowing it to vary. The torque exerted on the workpiece would be estimated as the product of (1) the torque-multiplication ratio of the spindle belt and/or gear drive, (2) the force measured by a load cell mechanically coupled to the spindle motor, and (3) the moment arm of the load cell. Hence, the output of the load cell would be used as a feedback signal for controlling the torque (see figure).

Sarcomatoid carcinoma associated with small cell carcinoma of the urinary bladder: a series of 28 cases.

PubMed

Urrea, Yuly Ramirez; Epstein, Jonathan I

2017-09-01

The association of sarcomatoid carcinoma (SC) with small cell carcinoma (SCC) has not been systematically studied. We identified 39 consult cases between 2001 and 2016 with available slides for review in 28 cases. There were 19 men and 9 women (mean age: 78 years [51-89]). In 26 (92.8%) cases, the sarcomatoid component had nonspecific malignant spindle cells, 4 (14%) chondrosarcoma, 2 (7%) myxoid sarcomatous, 1 (3.5%) osteosarcoma, and 1 (3.5%) rhabdomyosarcoma. The predominant component was SCC in 11 (39%) cases, urothelial carcinoma in 6 (21%), sarcomatoid in 3 (10%), and equal sarcomatoid and SCC in 8 (29%). There were 3 morphological groups: group 1 (18/28 [64%]) showed a gradual transition from SCC to other components; group 2 (5/28 [18%]) had an abrupt transition from SCC to other components; and in group 3 (5/28 [18%]), the SCC was separate from other components. In group 1, 12 (66%) cases of SCC showed a gradual transition to sarcomatoid areas; 3 (17%) to urothelial carcinoma; and 3 (17%) to multiple components including squamous cell carcinoma, urothelial carcinoma, and sarcomatoid. Mortality did not differ based on pathological groups. The 36-month actuarial risk of death was 64.3%. The multitude of different components in these tumors is further evidence of the remarkable ability of carcinoma of the bladder to show divergent differentiation with, in some cases, gradual transition between SCC and other elements including sarcomatoid. Greater recognition of this entity with chemotherapy targeted to the various histological elements may have important therapeutic implications. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of Wind Tunnel and Scaling Effects with the UH-60A Airloads Rotor

DTIC Science & Technology

2011-05-01

V! free-stream velocity, ft/s x chordwise distance from leading edge, ft #c, #s corrected/geometric shaft angles, deg $1c, $1s cos/sin components...attached to spindles that were retained by elastomeric bearings to a one-piece titanium hub. These bearings permitted blade flap, lead-lag, and...Figure 3. UH-60A small-scale rotor installed in DNW. Main rotor dampers were installed between each of the main rotor spindles and the hub to

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

PubMed

Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

PubMed Central

Jordan, Shawn N.; Davies, Tim; Zhuravlev, Yelena; Dumont, Julien; Shirasu-Hiza, Mimi

2016-01-01

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation. PMID:26728855

Cytogenetic analyses of four solid tumours in dogs.

PubMed

Mayr, B; Reifinger, M; Weissenböck, H; Schleger, W; Eisenmenger, E

1994-07-01

Four solid tumours (one haemangiopericytoma, one haemangioendothelioma, one spindle-cell sarcoma and one mammary carcinoma) in dogs were analysed cytogenetically. In the haemangiopericytoma, an additional small chromosomal segment was present. Very complex changes including centric fusions and symmetric meta-centrics 1, 6, 10 and 12 were conspicuous in the highly unbalanced karyotype of the haemangioendothelioma. Complex changes, particularly many centric fusions and a tandem translocation 4/14, were features of the spindle-cell sarcoma. One centric fusion and a symmetric metacentric 13 were present in the mammary carcinoma.

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

PubMed

Goshima, Gohta; Nédélec, François; Vale, Ronald D

2005-10-24

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

Natural Loss of Mps1 Kinase in Nematodes Uncovers a Role for Polo-like Kinase 1 in Spindle Checkpoint Initiation.

PubMed

Espeut, Julien; Lara-Gonzalez, Pablo; Sassine, Mélanie; Shiau, Andrew K; Desai, Arshad; Abrieu, Ariane

2015-07-07

The spindle checkpoint safeguards against chromosome loss during cell division by preventing anaphase onset until all chromosomes are attached to spindle microtubules. Checkpoint signal is generated at kinetochores, the primary attachment site on chromosomes for spindle microtubules. Mps1 kinase initiates checkpoint signaling by phosphorylating the kinetochore-localized scaffold protein Knl1 to create phospho-docking sites for Bub1/Bub3. Mps1 is widely conserved but is surprisingly absent in many nematode species. Here, we show that PLK-1, which targets a substrate motif similar to that of Mps1, functionally substitutes for Mps1 in C. elegans by phosphorylating KNL-1 to direct BUB-1/BUB-3 kinetochore recruitment. This finding led us to re-examine checkpoint initiation in human cells, where we found that Plk1 co-inhibition significantly reduced Knl1 phosphorylation and Bub1 kinetochore recruitment relative to Mps1 inhibition alone. Thus, the finding that PLK-1 functionally substitutes for Mps1 in checkpoint initiation in C. elegans uncovered a role for Plk1 in species that have Mps1. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization

PubMed Central

Okada, Naoyuki; Sato, Masamitsu

2015-01-01

Spindle microtubules capture and segregate chromosomes and, therefore, their assembly is an essential event in mitosis. To carry out their mission, many key players for microtubule formation need to be strictly orchestrated. Particularly, proteins that assemble the spindle need to be translocated at appropriate sites during mitosis. A small GTPase (hydrolase enzyme of guanosine triphosphate), Ran, controls this translocation. Ran plays many roles in many cellular events: nucleocytoplasmic shuttling through the nuclear envelope, assembly of the mitotic spindle, and reorganization of the nuclear envelope at the mitotic exit. Although these events are seemingly distinct, recent studies demonstrate that the mechanisms underlying these phenomena are substantially the same as explained by molecular interplay of the master regulator Ran, the transport factor importin, and its cargo proteins. Our review focuses on how the transport machinery regulates mitotic progression of cells. We summarize translocation mechanisms governed by Ran and its regulatory proteins, and particularly focus on Ran-GTP targets in fission yeast that promote spindle formation. We also discuss the coordination of the spatial and temporal regulation of proteins from the viewpoint of transport machinery. We propose that the transport machinery is an essential key that couples the spatial and temporal events in cells. PMID:26308057

Evolution of an ancient protein function involved in organized multicellularity in animals.

PubMed

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-07

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

The Spindle Positioning Protein Kar9p Interacts With the Sumoylation Machinery in Saccharomyces cerevisiae

PubMed Central

Meednu, Nida; Hoops, Harold; D'Silva, Sonia; Pogorzala, Leah; Wood, Schuyler; Farkas, David; Sorrentino, Mark; Sia, Elaine; Meluh, Pam; Miller, Rita K.

2008-01-01

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes. PMID:18832349

Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint-restricted Cdc20.

PubMed

Boekhout, Michiel; Wolthuis, Rob

2015-04-15

Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid being stabilized by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity. © 2015. Published by The Company of Biologists Ltd.

[Rapidly-growing nodular pseudoangiomatous stromal hyperplasia of the breast: case report].

PubMed

Elıyatkin, Nuket; Karasu, Başak; Selek, Elif; Keçecı, Yavuz; Postaci, Hakan

2011-01-01

Pseudoangiomatous stromal hyperplasia is a benign proliferative lesion of the mammary stroma that rarely presents as a localized mass. Pseudoangiomatous stromal hyperplasia is characterized by a dense, collagenous proliferation of the mammary stroma, associated with capillary-like spaces. Pseudoangiomatous stromal hyperplasia can be mistaken with fibroadenoma on radiological examination or with low-grade angiosarcoma on histological examination. Its main importance is its distinction from angiosarcoma. The presented case was a 40-year-old woman who was admitted with a rapidly growing breast tumor. Physical examination revealed an elastic-firm, well-defined, mobile and painless mass in her right breast. Mammograms revealed a 6.7 x 3.7 cm, lobulated, well-circumscribed mass in her right breast but no calcification. Sonographic examination showed a well-defined and homogenous mass, not including any cyst. Based on these findings, a provisional diagnosis of fibroadenoma was made. Considering the rapid growth history of the mass, tumor excision was performed. The excised tumor was well demarcated and had a smooth external surface. Histological examination revealed the tumor to be composed of markedly increased fibrous stroma and scattered epithelial components (cystic dilatation of the ducts, blunt duct adenosis). The fibrous stroma contained numerous anastomosing slit-like spaces. Isolated spindle cells appeared intermittently at the margins of the spaces resembled endothelial cells. Immunohistochemical staining showed that the spindle cells were positive for CD34 and negative for Factor VIII-related antigen. The lesion was diagnosed as nodular pseudoangiomatous stromal hyperplasia.

Dedifferentiated Liposarcoma With Epithelioid/Epithelial Features.

PubMed

Makise, Naohiro; Yoshida, Akihiko; Komiyama, Motokiyo; Nakatani, Fumihiko; Yonemori, Kan; Kawai, Akira; Fukayama, Masashi; Hiraoka, Nobuyoshi

2017-11-01

Dedifferentiated liposarcoma (DDLPS) demonstrates a variety of growth patterns, and their histologic resemblance to other spindle cell mesenchymal tumors has been widely recognized. However, epithelioid morphology in DDLPS has only rarely been documented. Here, we report 6 cases of DDLPS with striking epithelioid/epithelial features. The patients were 5 men and 1 woman with a median age of 61 years. All tumors were located in the internal trunk. During follow-up of 1 to 41 months, local recurrence, distant metastases, and tumor-related death occurred in 4, 2, and 4 patients, respectively. Beside well-differentiated liposarcoma component and conventional high-grade spindle cell morphology, all tumors focally exhibited growth comprising small or large epithelioid cells in diffuse or sheet-like proliferation. Rhabdoid cells were present in 2 cases. All 5 tumors tested harbored MDM2 amplification. Cytokeratin and/or epithelial membrane antigen were at least focally positive in all 5 tumors tested. One case contained a small focus of novel heterologous epithelial differentiation with acinar structures, wherein cytokeratin, MOC31, and claudin-4 were diffusely expressed and H3K27me3 expression was lost. DDLPS with epithelioid/epithelial features may lead to misdiagnosis of carcinoma or mesothelioma, and their diagnosis should be based on correlation with clinicopathologic and molecular findings. The epithelioid morphology in DDLPS may suggest an aggressive behavior based on this small series. In addition, we document 2 cases of MDM2-amplified undifferentiated neoplasm with epithelioid features in the internal trunk that lacked association with well-differentiated liposarcoma histology and showed rapid clinical course. Whether these latter tumors belong to DDLPS with epithelioid features requires further study.

Thermal Error Test and Intelligent Modeling Research on the Spindle of High Speed CNC Machine Tools

NASA Astrophysics Data System (ADS)

Luo, Zhonghui; Peng, Bin; Xiao, Qijun; Bai, Lu

2018-03-01

Thermal error is the main factor affecting the accuracy of precision machining. Through experiments, this paper studies the thermal error test and intelligent modeling for the spindle of vertical high speed CNC machine tools in respect of current research focuses on thermal error of machine tool. Several testing devices for thermal error are designed, of which 7 temperature sensors are used to measure the temperature of machine tool spindle system and 2 displacement sensors are used to detect the thermal error displacement. A thermal error compensation model, which has a good ability in inversion prediction, is established by applying the principal component analysis technology, optimizing the temperature measuring points, extracting the characteristic values closely associated with the thermal error displacement, and using the artificial neural network technology.

Sinonasal inflammatory myofibroblastic pseudotumor (plasma cell granuloma).

PubMed

Arpacı, Rabia Bozdoğan; Kara, Tuba; Özyedek, Esen; Serinsöz, Ebru; Vayısoğlu, Yusuf; Özgür, Anıl; Arpacı, Taner; Özcan, Cengiz

2015-01-01

Inflammatory myofibroblastic pseudotumor (plasma cell granuloma) is a soft tissue lesion consisting of myofibroblasts, mature lymphocytes, histiocytes, plasma cells, eosinophils, and extracellular collagen. Various sites in the body may harbor these lesions. Lungs, omentum, intestines, mesentery, and urinary system are the most susceptible areas. It is usually seen in children and young adults. The lesion is rarely detected in the head and neck region. The orbit and the upper respiratory system are the most common localizations in the head and neck region. Sinonasal tract is a rare site of involvement. The differential diagnosis includes squamous cell carcinoma (spindle cell variant), inflammatory fibrosarcoma, leiomyosarcoma, schwannoma, and nonspecific inflammation. Our patient who had a sinonasal mass showed a benign tumor consisting of spindle tumor cells and inflammatory cells histopathologically. This case was presented due to its rare existence to this site.

Recent advances in understanding oogenesis: interactions with the cytoskeleton, microtubule organization, and meiotic spindle assembly in oocytes

PubMed Central

Marlow, Florence L.

2018-01-01

Maternal control of development begins with production of the oocyte during oogenesis. All of the factors necessary to complete oocyte maturation, meiosis, fertilization, and early development are produced in the transcriptionally active early oocyte. Active transcription of the maternal genome is a mechanism to ensure that the oocyte and development of the early embryo begin with all of the factors needed for successful embryonic development. To achieve the maximum maternal store, only one functional cell is produced from the meiotic divisions that produce the oocyte. The oocyte receives the bulk of the maternal cytoplasm and thus is significantly larger than its sister cells, the tiny polar bodies, which receive a copy of the maternal genome but essentially none of the maternal cytoplasm. This asymmetric division is accomplished by an enormous cell that is depleted of centrosomes in early oogenesis; thus, meiotic divisions in oocytes are distinct from those of mitotic cells. Therefore, these cells must partition the chromosomes faithfully to ensure euploidy by using mechanisms that do not rely on a conventional centrosome-based mitotic spindle. Several mechanisms that contribute to assembly and maintenance of the meiotic spindle in oocytes have been identified; however, none is fully understood. In recent years, there have been many exciting and significant advances in oogenesis, contributed by studies using a myriad of systems. Regrettably, I cannot adequately cover all of the important advances here and so I apologize to those whose beautiful work has not been included. This review focuses on a few of the most recent studies, conducted by several groups, using invertebrate and vertebrate systems, that have provided mechanistic insight into how microtubule assembly and meiotic spindle morphogenesis are controlled in the absence of centrosomes.

Centralspindlin and Chromosomal Passenger Complex Behavior During Normal and Rappaport Furrow Specification in Echinoderm Embryos

PubMed Central

Argiros, Haroula; Henson, Lauren; Holguin, Christiana; Foe, Victoria; Shuster, Charles Bradley

2014-01-01

The chromosomal passenger (CPC) and Centralspindlin complexes are essential for organizing the anaphase central spindle and providing cues that position the cytokinetic furrow between daughter nuclei. However, echinoderm zygotes are also capable of forming “Rappaport furrows” between asters positioned back-to-back without intervening chromosomes. To understand how these complexes contribute to normal and Rappaport furrow formation, we studied the localization patterns of Survivin and mitotic-kinesin-like-protein1 (MKLP1), members respectively of the CPC and the Centralspindlin complex, and the effect of CPC inhibition on cleavage in mono- and binucleate echinoderm zygotes. In zygotes, Survivin initially localized to metaphase chromosomes, upon anaphase onset relocalized to the central spindle and then, together with MKLP1 spread towards the equatorial cortex in an Aurora-dependent manner. Inhibition of Aurora kinase activity resulted in disruption of central spindle organization and furrow regression, although astral microtubule elongation and furrow initiation were normal. In binucleate cells containing two parallel spindles MKLP1 and Survivin localized to the plane of the former metaphase plate, but were not observed in the secondary cleavage plane formed between unrelated spindle poles, except when chromosomes were abnormally present there. However, the secondary furrow was sensitive to Aurora inhibition, indicating that Aurora kinase may still contribute to furrow ingression without chromosomes nearby. Our results provide insights that reconcile classic micromanipulation studies with current molecular understanding of furrow specification in animal cells. PMID:22887753

On the robustness of SAC silencing in closed mitosis

NASA Astrophysics Data System (ADS)

Ruth, Donovan; Liu, Jian

Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

p600 regulates spindle orientation in apical neural progenitors and contributes to neurogenesis in the developing neocortex.

PubMed

Belzil, Camille; Asada, Naoyuki; Ishiguro, Kei-Ichiro; Nakaya, Takeo; Parsons, Kari; Pendolino, Valentina; Neumayer, Gernot; Mapelli, Marina; Nakatani, Yoshihiro; Sanada, Kamon; Nguyen, Minh Dang

2014-05-08

Apical neural progenitors (aNPs) drive neurogenesis by means of a program consisting of self-proliferative and neurogenic divisions. The balance between these two manners of division sustains the pool of apical progenitors into late neurogenesis, thereby ensuring their availability to populate the brain with terminal cell types. Using knockout and in utero electroporation mouse models, we report a key role for the microtubule-associated protein 600 (p600) in the regulation of spindle orientation in aNPs, a cellular event that has been associated with cell fate and neurogenesis. We find that p600 interacts directly with the neurogenic protein Ndel1 and that aNPs knockout for p600, depleted of p600 by shRNA or expressing a Ndel1-binding p600 fragment all display randomized spindle orientation. Depletion of p600 by shRNA or expression of the Ndel1-binding p600 fragment also results in a decreased number of Pax6-positive aNPs and an increased number of Tbr2-positive basal progenitors destined to become neurons. These Pax6-positive aNPs display a tilted mitotic spindle. In mice wherein p600 is ablated in progenitors, the production of neurons is significantly impaired and this defect is associated with microcephaly. We propose a working model in which p600 controls spindle orientation in aNPs and discuss its implication for neurogenesis. © 2014. Published by The Company of Biologists Ltd.

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte-to-embryo transition in gibel carp.

PubMed

Sun, Min; Li, Zhi; Gui, Jian-Fang

2010-10-01

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.

Method and apparatus for suppressing regenerative instability and related chatter in machine tools

DOEpatents

Segalman, Daniel J.; Redmond, James M.

2001-01-01

Methods of and apparatuses for mitigating chatter vibrations in machine tools or components thereof. Chatter therein is suppressed by periodically or continuously varying the stiffness of the cutting tool (or some component of the cutting tool), and hence the resonant frequency of the cutting tool (or some component thereof). The varying of resonant frequency of the cutting tool can be accomplished by modulating the stiffness of the cutting tool, the cutting tool holder, or any other component of the support for the cutting tool. By periodically altering the impedance of the cutting tool assembly, chatter is mitigated. In one embodiment, a cyclic electric (or magnetic) field is applied to the spindle quill which contains an electro-rheological (or magneto-rheological) fluid. The variable yield stress in the fluid affects the coupling of the spindle to the machine tool structure, changing the natural frequency of oscillation. Altering the modal characteristics in this fashion disrupts the modulation of current tool vibrations with previous tool vibrations recorded on the workpiece surface.

Method and apparatus for suppressing regenerative instability and related chatter in machine tools

DOEpatents

Segalman, Daniel J.; Redmond, James M.

1999-01-01

Methods of and apparatuses for mitigating chatter vibrations in machine tools or components thereof. Chatter therein is suppressed by periodically or continuously varying the stiffness of the cutting tool (or some component of the cutting tool), and hence the resonant frequency of the cutting tool (or some component thereof). The varying of resonant frequency of the cutting tool can be accomplished by modulating the stiffness of the cutting tool, the cutting tool holder, or any other component of the support for the cutting tool. By periodically altering the impedance of the cutting tool assembly, chatter is mitigated. In one embodiment, a cyclic electric (or magnetic) field is applied to the spindle quill which contains an electro-rheological (or magneto-rheological) fluid. The variable yield stress in the fluid affects the coupling of the spindle to the machine tool structure, changing the natural frequency of oscillation. Altering the modal characteristics in this fashion disrupts the modulation of current tool vibrations with previous tool vibrations recorded on the workpiece surface.

Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes.

PubMed

Yi, Kexi; Rubinstein, Boris; Unruh, Jay R; Guo, Fengli; Slaughter, Brian D; Li, Rong

2013-03-04

Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes.

Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes

PubMed Central

Yi, Kexi; Rubinstein, Boris; Unruh, Jay R.; Guo, Fengli; Slaughter, Brian D.

2013-01-01

Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes. PMID:23439682

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes.

PubMed

Sugawara, Atsushi; Sugimura, Satoshi; Hoshino, Yumi; Sato, Eimei

2009-08-01

Cloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

Spindle Cell Carcinoma of Nasal Cavity- A Case Report

PubMed Central

Mittal, Abhishek; Nagpal, Tapan

2016-01-01

Spindle Cell Carcinoma (SpCC), also known as Sarcomatoid Carcinoma, is a rare and peculiar biphasic malignant neoplasm that occurs mainly in the upper aero-digestive tract, mostly in larynx. SCC accounts for 3% of all squamous cell carcinomas (SCCs) in the head and neck region. It is a rare variant of SCC which shows spindled or pleomorphic tumour cells simulating a true sarcoma. We present a case report of SpCC nasal cavity in a 50-year-old female patient, presented with intermittent epistaxis from left nasal cavity. On physical examination, the patient had an ulcero-exophytic type of mass in the left nasal cavity and a smooth bulge on the left side of anterior hard palate. Patient underwent excision of nasal mass along with partial palatectomy by facial degloving approach and reconstruction of palate with naso-labial flap. The postoperative histopathological report showed SCC. Surgery forms the mainstay of treatment. Radiotherapy and Chemotherapy is warranted in order to improve treatment results. As only few cases have been reported, we report a case of this rare entity to contribute for better understanding and awareness of this rare malignancy. PMID:27190843

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

PubMed Central

Petry, Sabine; Groen, Aaron C.; Ishihara, Keisuke; Mitchison, Timothy J.; Vale, Ronald D.

2013-01-01

Summary The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance. PMID:23415226

Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

PubMed Central

McKenzie, Callum; Bassi, Zuni I.; Debski, Janusz; Gottardo, Marco; Callaini, Giuliano; Dadlez, Michal; D'Avino, Pier Paolo

2016-01-01

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K's coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins. PMID:27009191

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

PubMed

Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

2013-10-25

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Evidence for an association between increased oxidative stress and derangement of FOXO1 signaling in tumorigenesis of a cellular angiofibroma with monoallelic 13q14: a case report

PubMed Central

Arakaki, Kazunari; Chinen, Katsuya; Kamiya, Masuzo; Tanabe, Yasuka; Tawata, Natsumi; Ikehara, Fukino; Uehara, Karina; Shimabukuro, Hiroichi; Kinjo, Takao

2014-01-01

Cellular angiofibroma (CAF) is a rare soft tissue tumor characterized by random arrangement of spindle tumor cells in the stroma with short collagen bundles and thick- and hyalinized small vessels. CAFs share histological characteristics with spindle cell lipomas and mammary type myofibroblastomas. Because these tumors harbor monoallelic 13q14, common genetic and molecular mechanism for tumorigenesis is presumed. In this study, we reported a case of CAF in a 69-year-old man with monoallelic 13q14. Immunohistochemical analysis revealed that FOXO1, which is located in chromosome 13q14, was not expressed in the tumor. We also detected oxidative stress markers and found p38 MAPK activation, which is often induced by cellular stressors such as reactive oxygen species (ROS). Because FOXO1 induces the expression of genes encoding enzymes that generate antioxidants, oxidative stress induced by loss of FOXO1 expression may be common among CAFs, spindle cell lipomas, and mammary type myofibroblastomas. PMID:25674275

Evidence for an association between increased oxidative stress and derangement of FOXO1 signaling in tumorigenesis of a cellular angiofibroma with monoallelic 13q14: a case report.

PubMed

Arakaki, Kazunari; Chinen, Katsuya; Kamiya, Masuzo; Tanabe, Yasuka; Tawata, Natsumi; Ikehara, Fukino; Uehara, Karina; Shimabukuro, Hiroichi; Kinjo, Takao

2014-01-01

Cellular angiofibroma (CAF) is a rare soft tissue tumor characterized by random arrangement of spindle tumor cells in the stroma with short collagen bundles and thick- and hyalinized small vessels. CAFs share histological characteristics with spindle cell lipomas and mammary type myofibroblastomas. Because these tumors harbor monoallelic 13q14, common genetic and molecular mechanism for tumorigenesis is presumed. In this study, we reported a case of CAF in a 69-year-old man with monoallelic 13q14. Immunohistochemical analysis revealed that FOXO1, which is located in chromosome 13q14, was not expressed in the tumor. We also detected oxidative stress markers and found p38 MAPK activation, which is often induced by cellular stressors such as reactive oxygen species (ROS). Because FOXO1 induces the expression of genes encoding enzymes that generate antioxidants, oxidative stress induced by loss of FOXO1 expression may be common among CAFs, spindle cell lipomas, and mammary type myofibroblastomas.

Do centrioles generate a polar ejection force?

PubMed

Wells, Jonathan

2005-01-01

A microtubule-dependent polar ejection force that pushes chromosomes away from spindle poles during prometaphase is observed in animal cells but not in the cells of higher plants. Elongating microtubules and kinesin-like motor molecules have been proposed as possible causes, but neither accounts for all the data. In the hypothesis proposed here a polar ejection force is generated by centrioles, which are found in animals but not in higher plants. Centrioles consist of nine microtubule triplets arranged like the blades of a tiny turbine. Instead of viewing centrioles through the spectacles of molecular reductionism and neo-Darwinism, this hypothesis assumes that they are holistically designed to be turbines. Orthogonally oriented centriolar turbines could generate oscillations in spindle microtubules that resemble the motion produced by a laboratory vortexer. The result would be a microtubule-mediated ejection force tending to move chromosomes away from the spindle axis and the poles. A rise in intracellular calcium at the onset of anaphase could regulate the polar ejection force by shutting down the centriolar turbines, but defective regulation could result in an excessive force that contributes to the chromosomal instability characteristic of most cancer cells.

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

PubMed Central

Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

2015-01-01

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

Morphologic expression of glandular differentiation in the epidermoid nasal carcinomas induced by phenylglycidyl ether inhalation.

PubMed Central

Lee, K. P.; Schneider, P. W.; Trochimowicz, H. J.

1983-01-01

Charles River-CD Sprague-Dawley rats in 3 equal groups of 100 males and 100 females each were exposed to 12, 1, and 0 ppm of phenylglycidyl ether vapor for 24 months. Nasal tumors were first detected after 621 days' exposure at 12 ppm with an incidence of 11% in males and 4.4% in females. No nasal tumors were found at 1 ppm in rats exposed for 24 months. The nasal tumors, mostly epidermoid carcinomas, were derived from the respiratory epithelium and nasal glands, both of which revealed squamous metaplasia or dysplasia in the anterior nasal cavity. Most nasal tumors were confined to the anterior nasal cavity and occasionally invaded the dorsonasal bones and posterior nasal cavity. The undifferentiated glandular cells appear to differentiate to neoplastic squamous cells, because the ultrastructure of epidermoid carcinoma revealed traits of glandular cell differentiation in the neoplastic squamous cells. The features of glandular cell differentiation in the neoplastic squamous cells were intercellular or intracellular glandular lumens, secretory vesicles, mucus droplets, and intermediate cells showing both glandular and squamous differentiation. Squamous cells in the well-differentiated epidermoid carcinomas revealed abundant tonofibrils, desmosomes, glycogen particulates, and interdigitated cytoplasmic processes. These markers of squamous-cell differentiation were markedly reduced in the undifferentiated epidermoid carcinomas. The spindle-cell squamous carcinoma showed both squamous and fibroblastic-like differentiations. Some spindle cells had only fibroblastic-like differentiation, suggesting spindle-cell metaplasia of the squamous cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:6846500

PLK1 has tumor-suppressive potential in APC-truncated colon cancer cells.

PubMed

Raab, Monika; Sanhaji, Mourad; Matthess, Yves; Hörlin, Albrecht; Lorenz, Ioana; Dötsch, Christina; Habbe, Nils; Waidmann, Oliver; Kurunci-Csacsko, Elisabeth; Firestein, Ron; Becker, Sven; Strebhardt, Klaus

2018-03-16

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

PubMed Central

Bernis, Cyril; Swift-Taylor, Beth; Nord, Matthew; Carmona, Sarah; Chook, Yuh Min; Forbes, Douglass J.

2014-01-01

The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel. PMID:24478460

Assignment of the human caltractin gene (CALT) to Xq28 by fluorescence in situ hybridization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, Tanaka; Okui, Keiko; Nakamura, Yusuke

1994-12-01

The centrosome is the major microtubule-organizing center of interphase eukaryotic cells, an its duplication is essential to eukaryotic cell division. Caltractin, a structural component of centrosomes, is highly homologous in amino acid sequence to the product of the CDC31 gene of Saccharomyces cerevisiae. In S. cerevisiae, an important role for CDC31 in duplication of the spindle pole body (SPB), a kind of microtubule-organizing center, has been demonstrated by an experiment in which mutant CDC31 prevented SPB duplication and led to formation of a monopolar spindle. In view of the localization of human caltractin in centrosomes and the sequence homology itmore » bears to yeast CDC31, it is reasonable to assume that caltractin functions in humans as CDC31 does in yeast. As a part of the Human Genome Project, we have been determining nucleotide sequences of DNA clones randomly selected from a directionally cloned cDNA library constructed from fetal brain mRNA obtained from Clontech (La Jolla, CA). By comparing 5{prime} partial DNA sequences of these cDNA clones with known DNA sequences in the database, we found one clone that was highly homologous to the caltractin gene of Chlamydomonas, which turned out to be the same as a human gene identified recently. 4 refs., 1 fig.« less

Enhanced polarizing microscopy as a new tool in aneuploidy research in oocytes.

PubMed

Shen, Ying; Betzendahl, Ilse; Tinneberg, Hans-Rudolf; Eichenlaub-Ritter, Ursula

2008-03-12

Chromosomal non-disjunction in female meiosis gives rise to reduced fertility and trisomy in humans. Human oocytes, especially from aged women, appear especially susceptible to non-disjunction. The oocyte spindle is crucial for high fidelity of chromosome segregation at meiotic divisions, and alterations in spindle morphology are therefore indicators of adverse conditions during oocyte development that may result in meiotic aneuploidy. In the past, oocytes had to be fixed for spindle analysis, precluding direct non-invasive identification of aneugens and adverse maturation conditions that affect spindle integrity and chromosome behaviour. Aneuploidy research for detection of spindle aberrations was therefore mainly focused on in vivo or in vitro exposed, fixed animal oocytes or cytogenetic analysis of spread oocytes. Orientation independent enhanced polarizing microscopy with nearly circularly polarized light and electronically controlled liquid crystal compensator optics is a new tool to study spindle morphology non-invasively in vivo for qualitative as well as quantitative analysis. Image generation by polarization microscopy depends on the intrinsic optical properties of the spindle with its paracrystalline microtubule lattice. When polarized light passes through such a lattice it induces a splitting of the beam and shift in the plane of vibration and retardation of light (termed birefringence and retardance). Studies of animal oocytes and follicle-cell denuded human oocytes fertilized by intracytoplasmic sperm injection for assisted conception have demonstrated the safety and efficacy of enhanced polarization microscopy. The method can be employed in aneuploidy research for non-invasive dose-response studies to detect spindle aberrations, for instance, in combination with cytogenetic analysis. Due to the non-invasive nature of the technique it may be employed in routine analysis of human oocytes to assess risks by lifestyle factors, and occupational and adverse environmental exposures.

Modulation of jaw muscle spindle afferent activity following intramuscular injections with hypertonic saline.

PubMed

Ro, J Y; Capra, N F

2001-05-01

Transient noxious chemical stimulation of small diameter muscle afferents modulates jaw movement-related responses of caudal brainstem neurons. While it is likely that the effect is mediated from the spindle afferents in the mesencephalic nucleus (Vmes) via the caudally projecting Probst's tract, the mechanisms of pain induced modulations of jaw muscle spindle afferents is not known. In the present study, we tested the hypothesis that jaw muscle nociceptors gain access to muscle spindle afferents in the same muscle via central mechanisms and alter their sensitivity. Thirty-five neurons recorded from the Vmes were characterized as muscle spindle afferents based on their responses to passive jaw movements, muscle palpation, and electrical stimulation of the masseter nerve. Each cell was tested by injecting a small volume (250 microl) of either 5% hypertonic and/or isotonic saline into the receptor-bearing muscle. Twenty-nine units were tested with 5% hypertonic saline, of which 79% (23/29) showed significant modulation of mean firing rates (MFRs) during one or more phases of ramp-and-hold movements. Among the muscle spindle primary-like units (n = 12), MFRs of 4 units were facilitated, five reduced, two showed mixed responses and one unchanged. In secondary-like units (n = 17), MFRs of 9 were facilitated, three reduced and five unchanged. Thirteen units were tested with isotonic saline, of which 77% showed no significant changes of MFRs. Further analysis revealed that the hypertonic saline not only affected the overall output of muscle spindle afferents, but also increased the variability of firing and altered the relationship between afferent signal and muscle length. These results demonstrated that activation of muscle nociceptors significantly affects proprioceptive properties of jaw muscle spindles via central neural mechanisms. The changes can have deleterious effects on oral motor function as well as kinesthetic sensibility.

On the Synchronization of EEG Spindle Waves

NASA Astrophysics Data System (ADS)

Long, Wen; Zhang, ChengFu; Zhao, SiLan; Shi, RuiHong

2000-06-01

Based on recently sleeping cellular substrates, a network model synaptically coupled by N three-cell circuits is provided. Simulation results show that: (i) the dynamic behavior of every circuit is chaotic; (ii) the synchronization of the network is incomplete; (iii) the incomplete synchronization can integrate burst firings of cortical cells into waxing-and-wanning EEG spindle waves. These results enlighten us that this kind of incomplete synchronization may integrate microscopic, electrical activities of neurons in billions into macroscopic, functional states in human brain. In addition, the effects of coupling strength, connectional mode and noise to the synchronization are discussed.

Cutaneous fibroma in a captive common snapping turtle (Chelydra serpentina).

PubMed

Gonzales-Viera, O; Bauer, G; Bauer, A; Aguiar, L S; Brito, L T; Catão-Dias, J L

2012-11-01

An adult female common snapping turtle (Chelydra serpentina) had a mass on the plantar surface of the right forelimb that was removed surgically. Microscopical examination revealed many spindle cells with mild anisocytosis and anisokaryosis and a surrounding collagenous stroma. There were no mitoses. Immunohistochemistry showed that the spindle cells expressed vimentin, but not desmin. A diagnosis of cutaneous fibroma was made. Tumours are reported uncommonly in chelonian species. Cutaneous fibroma has been diagnosed in an alligator snapping turtle (Macrochelys temminckii), but not previously in a common snapping turtle. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Meiosis: An Overview of Key Differences from Mitosis

PubMed Central

Ohkura, Hiroyuki

2015-01-01

Meiosis is the specialized cell division that generates gametes. In contrast to mitosis, molecular mechanisms and regulation of meiosis are much less understood. Meiosis shares mechanisms and regulation with mitosis in many aspects, but also has critical differences from mitosis. This review highlights these differences between meiosis and mitosis. Recent studies using various model systems revealed differences in a surprisingly wide range of aspects, including cell-cycle regulation, recombination, postrecombination events, spindle assembly, chromosome–spindle interaction, and chromosome segregation. Although a great degree of diversity can be found among organisms, meiosis-specific processes, and regulation are generally conserved. PMID:25605710

Malignant mast cell tumor of the thymus in an Royal College of Surgeons (RCS) rat.

PubMed

Terayama, Yui; Matsuura, Tetsuro; Ozaki, Kiyokazu

2017-01-01

A 152-week-old male Royal College of Surgeons (RCS) rat kept as a non-treated animal in a long-term animal study presented with a soft mass in the anterior mediastinum, which adhered to the pleura of the lung. Histopathologically, the mass mainly consisted of round to short spindle-shaped tumor cells that had infiltrated through the hyperplastic thymic tissue. The tumor cells were arranged in loose to dense sheets. Nuclei were moderate in size and round to spindle-shaped, with small nucleoli. Almost all tumor cells exhibited abundant eosinophilic cytoplasm, including eosinophilic granules of a range of sizes. The granules of tumor cells exhibited metachromasia with toluidine blue stain and were positive for c-kit and mast cell protease II. These findings indicate that the tumor described here represents a rare case of spontaneous malignant mast cell tumor with thymic epithelial hyperplasia.

Machinability evaluation of titanium alloys (Part 2)--Analyses of cutting force and spindle motor current.

PubMed

Kikuchi, Masafumi; Okuno, Osamu

2004-12-01

To establish a method of determining the machinability of dental materials for CAD/CAM systems, the machinability of titanium, two titanium alloys (Ti-6Al-4V and Ti-6Al-7Nb), and free-cutting brass was evaluated through cutting force and spindle motor current. The metals were slotted using a milling machine and square end mills at four cutting conditions. Both the static and dynamic components of the cutting force represented well the machinability of the metals tested: the machinability of Ti-6Al-4V and Ti-6Al-7Nb was worse than that of titanium, while that of free-cutting brass was better. On the other hand, the results indicated that the spindle motor current was not sensitive enough to detect the material difference among the titanium and its alloys.

Drosophila Mgr, a Prefoldin subunit cooperating with von Hippel Lindau to regulate tubulin stability

PubMed Central

Delgehyr, Nathalie; Wieland, Uta; Rangone, Hélène; Pinson, Xavier; Mao, Guojie; Dzhindzhev, Nikola S.; McLean, Doris; Riparbelli, Maria G.; Llamazares, Salud; Callaini, Giuliano; Gonzalez, Cayetano; Glover, David M.

2012-01-01

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not. PMID:22451918

Fibroblastic osteosarcoma with epithelioid and squamous differentiation in a dog.

PubMed

Jenkins, Tiffany L; Agnew, Dalen; Rissi, Daniel R

2018-04-01

A fibroblastic osteosarcoma with epithelioid and squamous differentiation in the distal femur of a 9-y-old spayed female Greyhound dog is described. Grossly, the tumor consisted of a pale-white, firm-to-hard mass that replaced the medullary and cortical areas of the distal end of the right femur. Histologically, the mass was composed predominantly of spindle cells admixed with areas of mineralized and non-mineralized osteoid matrix that were surrounded by stellate osteoblasts and scattered multinucleate giant cells, consistent with the diagnosis of a fibroblastic osteosarcoma. In addition, well-demarcated clusters of neoplastic epithelioid cells and foci of squamous differentiation with keratin pearls were present throughout the neoplasm. The spindle cells, epithelioid cells, and areas of squamous differentiation expressed cytoplasmic immunostaining for osteocalcin and osteonectin. The spindle cells and epithelioid cells were also immunopositive for vimentin. Epithelioid cells also expressed occasional cytoplasmic immunostaining for pancytokeratin (PCK) Lu-5, and areas of squamous differentiation were immunoreactive for PCK Lu-5 and high molecular weight CK; these areas were inconsistently immunoreactive for CK 5-6 and immunonegative for low molecular weight CK. Foci of squamous differentiation were not located within blood or lymphatic vessels, given that no immunoreactivity for factor VIII-related antigen was observed around these areas. A thorough autopsy and an evaluation of the medical history excluded a primary carcinoma or other neoplasm elsewhere in the dog. The findings were consistent with a diagnosis of fibroblastic osteosarcoma with epithelioid and squamous differentiation.

Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro.

PubMed

Mosesso, Pasquale; Bohm, Lothar; Pepe, Gaetano; Fiore, Mario; Carpinelli, Alice; Gäde, Gerd; Nagini, Siddavaram; Ottavianelli, Alessandro; Degrassi, Francesca

2012-09-18

In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2'-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals. These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Preliminary Development of Real Time Usage-Phase Monitoring System for CNC Machine Tools with a Case Study on CNC Machine VMC 250

NASA Astrophysics Data System (ADS)

Budi Harja, Herman; Prakosa, Tri; Raharno, Sri; Yuwana Martawirya, Yatna; Nurhadi, Indra; Setyo Nogroho, Alamsyah

2018-03-01

The production characteristic of job-shop industry at which products have wide variety but small amounts causes every machine tool will be shared to conduct production process with dynamic load. Its dynamic condition operation directly affects machine tools component reliability. Hence, determination of maintenance schedule for every component should be calculated based on actual usage of machine tools component. This paper describes study on development of monitoring system to obtaining information about each CNC machine tool component usage in real time approached by component grouping based on its operation phase. A special device has been developed for monitoring machine tool component usage by utilizing usage phase activity data taken from certain electronics components within CNC machine. The components are adaptor, servo driver and spindle driver, as well as some additional components such as microcontroller and relays. The obtained data are utilized for detecting machine utilization phases such as power on state, machine ready state or spindle running state. Experimental result have shown that the developed CNC machine tool monitoring system is capable of obtaining phase information of machine tool usage as well as its duration and displays the information at the user interface application.

Design and analysis of drum lathe for manufacturing large-scale optical microstructured surface and load characteristics of aerostatic spindle

NASA Astrophysics Data System (ADS)

Wu, Dongxu; Qiao, Zheng; Wang, Bo; Wang, Huiming; Li, Guo

2014-08-01

In this paper, a four-axis ultra-precision lathe for machining large-scale drum mould with microstructured surface is presented. Firstly, because of the large dimension and weight of drum workpiece, as well as high requirement of machining accuracy, the design guidelines and component parts of this drum lathe is introduced in detail, including control system, moving and driving components, position feedback system and so on. Additionally, the weight of drum workpiece would result in the structural deformation of this lathe, therefore, this paper analyses the effect of structural deformation on machining accuracy by means of ANSYS. The position change is approximately 16.9nm in the X-direction(sensitive direction) which could be negligible. Finally, in order to study the impact of bearing parameters on the load characteristics of aerostatic journal bearing, one of the famous computational fluid dynamics(CFD) software, FLUENT, is adopted, and a series of simulations are carried out. The result shows that the aerostatic spindle has superior performance of carrying capacity and stiffness, it is possible for this lathe to bear the weight of drum workpiece up to 1000kg since there are two aerostatic spindles in the headstock and tailstock.

Establishment and characterization of a human uterine endometrial undifferentiated carcinoma cell line, TMG-L.

PubMed

Hasegawa, Kiyoshi; Suzuki, Machiko; Ishikawa, Kunimi; Yasue, Akira; Kato, Rina; Nakamura, Azumi; Kuroki, Jun; Udagawa, Yasuhiro

2003-03-01

A new cell line of human uterine endometrial undifferentiated carcinoma, designated as TMG-L, was established from the metastatic lymph node of 56-year-old patient TMG-L cells have been cultured with Ham's F-12 medium supplemented with 10% FCS and grew as a loosely adherent monolayer with polygonal or spindle-shaped cells exhibiting poor cell-cell contact and piled up against each other, showing a tendency to grow as floating cells. The doubling time of this cell line was about 48 hours, and chromosomal analysis revealed aneuploidy at passage 25. The cells formed tumors in SCID mouse, the histology of which was similar to that of undifferentiated carcinoma component of primary tumor. TMG-L cells showed the loss of expression and membranous localization of either E-cadherin or alpha-catenin, implied corresponding loss of their adhesive function. And this dysfunction implicated the biological aggressive behavior of uterine endometrial undifferentiated carcinoma. This cell line appears to provide a useful system for studying uterine undifferentiated carcinoma in vivo and in vitro.

Stopped in its tracks: negative regulation of the dynein motor by the yeast protein She1

PubMed Central

Moore, Jeffrey K.

2013-01-01

Summary How do cells direct the microtubule motor protein dynein to move cellular components to the right place at the right time? Recent studies in budding yeast shed light on a new mechanism for directing dynein, involving the protein She1. She1 restricts where and when dynein moves the nucleus and mitotic spindle. Experiments with purified proteins show that She1 binds to microtubules and inhibits dynein by stalling the motor on its track. Here I describe what we have learned so far about She1, based on a combination of genetic, cell biology, and biophysical approaches. These findings set the stage for further interrogation of the She1 mechanism, and raise the question of whether similar mechanisms exist in other species. PMID:23666903

Imaging, morphologic, and immunohistochemical correlation in gastrointestinal stromal tumors.

PubMed

Logrono, Roberto; Bhanot, Punam; Chaya, Charles; Cao, Li; Waxman, Irving; Bhutani, Manoop S

2006-08-25

Gastrointestinal stromal tumors (GISTs) recently have been distinguished morphologically, immunohistochemically, and genetically from other gastrointestinal-tract spindle cell neoplasms. The objective of this study was to correlate clinical and imaging findings with morphology and immunohistochemistry to diagnose GISTs and to differentiate them from other spindle cell lesions in the gastrointestinal tract. The authors reviewed 9 patients who had tumors that were diagnosed as GIST by image-guided and endosonographic-guided fine-needle aspiration (FNA) with or without core biopsy (7 stomach tumors and 2 intraabdominal tumors). The male:female ratio was 3:6, and the patients ranged in age from 38 years to 80 years. Onsite evaluation, preliminary cytologic evaluation, and immunohistochemistry were provided for 6 patients. Immunostains were performed, depending on sample size, on aspirates and/or core biopsies. On imaging studies, most tumors were smooth and homogenous, consistent with GIST. Tumors ranged in size from 1.8 cm to 22 cm. The largest neoplasm showed solid/cystic and necrotic components. Aspirates consisted of spindle cell, neoplastic proliferation arranged in fascicles that exhibited focal, nuclear palisading; indistinct, cytoplasmic borders; and no significant atypia or mitosis. Focal epithelioid changes or cytologic atypia and mitoses were observed in 2 tumors. Immunostains revealed tumor expression of CD117 and/or CD34 in 5 of 6 tumors, expression of actin in 3 of 6 tumors, and expression of desmin in 1 of 6 tumors. All tumors were diagnosed as GIST (or consistent with GIST for tumors that lacked immunochemical analysis). Five patients underwent surgical excision, and the GIST diagnosis was confirmed in 3 patients, whereas 1 tumor proved to be neurofibroma, and another tumor was leiomyoma. No surgical follow-up was available for the remaining 4 patients, who had imaging and morphologic findings consistent with GIST. In the setting of consistent clinical and radiologic findings, the combined use of cytomorphology and immunohistochemistry on FNA and/or core biopsy in most instances provides a reliable pathologic diagnosis of GIST. The need of sufficient material for performing ancillary studies and the usual impossibility of excluding malignancy are limitations of FNA cytology of GIST. Copyright 2006 American Cancer Society.

Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

PubMed Central

Holden, Jennifer M.; Koreny, Ludek; Obado, Samson; Ratushny, Alexander V.; Chen, Wei-Ming; Chiang, Jung-Hsien; Kelly, Steven; Chait, Brian T.; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

2014-01-01

The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. PMID:24600046

Fibrocartilage tissue engineering: the role of the stress environment on cell morphology and matrix expression.

PubMed

Thomopoulos, Stavros; Das, Rosalina; Birman, Victor; Smith, Lester; Ku, Katherine; Elson, Elliott L; Pryse, Kenneth M; Marquez, Juan Pablo; Genin, Guy M

2011-04-01

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development.

Fibrocartilage Tissue Engineering: The Role of the Stress Environment on Cell Morphology and Matrix Expression

PubMed Central

Das, Rosalina; Birman, Victor; Smith, Lester; Ku, Katherine; Elson, Elliott L.; Pryse, Kenneth M.; Marquez, Juan Pablo; Genin, Guy M.

2011-01-01

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development. PMID:21091338

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas.

PubMed

Arai, Eri; Gotoh, Masahiro; Tian, Ying; Sakamoto, Hiromi; Ono, Masaya; Matsuda, Akio; Takahashi, Yoriko; Miyata, Sayaka; Totsuka, Hirohiko; Chiku, Suenori; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Matsumoto, Kenji; Yamada, Tesshi; Yoshida, Teruhiko; Kanai, Yae

2015-12-01

CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p = 1.427 × 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10(-6))" and "Spindle assembly and chromosome separation (p = 9.260 × 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

PubMed Central

Arai, Eri; Gotoh, Masahiro; Tian, Ying; Sakamoto, Hiromi; Ono, Masaya; Matsuda, Akio; Takahashi, Yoriko; Miyata, Sayaka; Totsuka, Hirohiko; Chiku, Suenori; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Matsumoto, Kenji; Yamada, Tesshi; Yoshida, Teruhiko

2015-01-01

CpG‐island methylator phenotype (CIMP)‐positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP‐positive renal carcinogenesis. Genome (whole‐exome and copy number), transcriptome and proteome (two‐dimensional image converted analysis of liquid chromatography‐mass spectrometry) analyses were performed using tissue specimens of 87 CIMP‐negative and 14 CIMP‐positive clear cell RCCs and corresponding specimens of non‐cancerous renal cortex. Genes encoding microtubule‐associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non‐synonymous single‐nucleotide mutations and insertions/deletions) in CIMP‐positive RCCs, whereas CIMP‐negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP‐positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the “The metaphase checkpoint (p = 1.427 × 10−6),” “Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10−6)” and “Spindle assembly and chromosome separation (p = 9.260 × 10−6)” pathways. Quantitative RT‐PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP‐positive than in CIMP‐negative RCCs. All CIMP‐positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP‐positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP‐positive RCCs. PMID:26061684

Omental leiomyosarcoma with unusual giant cells in a Beagle dog - Short communication.

PubMed

Sasaki, Jun; Toyoshima, Megumi; Okamura, Yasuhiko; Goryo, Masanobu

2016-06-01

A 10-year-old castrated male Beagle dog was presented with a 2-month history of intermittent vomiting and abdominal pain. The dog was referred to the Veterinary Teaching Hospital at Iwate University for further evaluation, and a splenic tumour was suspected on the basis of ultrasonography and computed tomography. Surgery identified a large, solid, light-pink mass on the greater omentum with blood-coloured ascites in the abdominal cavity, and resection was performed. Microscopically, the mass comprised spindle-shaped tumour cells and scattered osteoclast-like giant cells. Most spindle-shaped cells were positive for vimentin, desmin, and smooth muscle actin (α-SMA), whereas osteoclast-like giant cells were positive only for vimentin. On the basis of histopathological and immunohistochemical findings, a diagnosis of leiomyosarcoma was made. To the best of our knowledge, this represents the first report of leiomyosarcoma associated with osteoclast-like giant cells developing from the greater omentum in a dog.

Design, Synthesis, and Biological Evaluation of an Allosteric Inhibitor of HSET that Targets Cancer Cells with Supernumerary Centrosomes

PubMed Central

Watts, Ciorsdaidh A.; Richards, Frances M.; Bender, Andreas; Bond, Peter J.; Korb, Oliver; Kern, Oliver; Riddick, Michelle; Owen, Paul; Myers, Rebecca M.; Raff, Jordan; Gergely, Fanni; Jodrell, Duncan I.; Ley, Steven V.

2013-01-01

Summary Centrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics. PMID:24210220

Life Cycle Characterization of Sulfolobus Monocaudavirus 1, an Extremophilic Spindle-Shaped Virus with Extracellular Tail Development

PubMed Central

Uldahl, Kristine B.; Jensen, Signe B.; Bhoobalan-Chitty, Yuvaraj; Martínez-Álvarez, Laura; Papathanasiou, Pavlos

2016-01-01

ABSTRACT We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length. IMPORTANCE Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea. This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field. PMID:27053548

Life Cycle Characterization of Sulfolobus Monocaudavirus 1, an Extremophilic Spindle-Shaped Virus with Extracellular Tail Development.

PubMed

Uldahl, Kristine B; Jensen, Signe B; Bhoobalan-Chitty, Yuvaraj; Martínez-Álvarez, Laura; Papathanasiou, Pavlos; Peng, Xu

2016-06-15

We provide here, for the first time, insights into the initial infection stages of a large spindle-shaped archaeal virus and explore the following life cycle events. Our observations suggest that Sulfolobus monocaudavirus 1 (SMV1) exhibits a high adsorption rate and that virions adsorb to the host cells via three distinct attachment modes: nosecone association, body association, and body/tail association. In the body/tail association mode, the entire virion, including the tail(s), aligns to the host cell surface and the main body is greatly flattened, suggesting a possible fusion entry mechanism. Upon infection, the intracellular replication cycle lasts about 8 h, at which point the virions are released as spindle-shaped tailless particles. Replication of the virus retarded host growth but did not cause lysis of the host cells. Once released from the host and at temperatures resembling that of its natural habitat, SMV1 starts developing one or two tails. This exceptional property of undergoing a major morphological development outside, and independently of, the host cell has been reported only once before for the related Acidianus two-tailed virus. Here, we show that SMV1 can develop tails of more than 900 nm in length, more than quadrupling the total virion length. Very little is known about the initial life cycle stages of viruses infecting hosts of the third domain of life, Archaea This work describes the first example of an archaeal virus employing three distinct association modes. The virus under study, Sulfolobus monocaudavirus 1, is a representative of the large spindle-shaped viruses that are frequently found in acidic hot springs. The results described here will add valuable knowledge about Archaea, the least studied domain in the virology field. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

PubMed

Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

2010-11-01

The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

Occurrence of maternal and paternal spindles in unfertilized human oocytes: possible relationship to nucleation defects after silent fertilization.

PubMed

Van Blerkom, Jonathan; Davis, Patrick; Alexander, Samuel

2004-04-01

Experience with conventional clinical IVF indicates that a first cleavage can occur in the absence of detectable pronuclear formation (so-called silent fertilization). In these instances, the first division is often asymmetrical and delayed when compared with normally fertilized siblings. In this study, DNA configurations and spindle organization were examined by fluorescence microscopy in metaphase II human oocytes that remained unfertilized after conventional IVF and were considered likely candidates for silent fertilization. The results show comparatively high frequencies of penetration in the absence of detectable pronuclear evolution, and that both a maternal meiotic and a sperm-derived mitotic-like spindle can coexist in the same oocyte. Patterns of cell division and blastomere nucleation in silent fertilizations suggest the possibility that this division may involve uniparental chromosomal segregation in which maternal and paternal DNA is differentially partitioned into daughter blastomeres. This pattern of inheritance may generate certain types of ploidy and nucleation defects detected at the 2- to 4-cell stage.

Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds.

PubMed

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-08-10

Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin "clouds" are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10's role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. Copyright © 2015 Elsevier Inc. All rights reserved.

Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds

PubMed Central

Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz; Pellman, David

2015-01-01

SUMMARY Positioning of centrosomes is vital for cell division and development. In metazoan cells, spindle positioning is controlled by a dynamic pool of subcortical actin that organizes in response to the position of retraction fibers. These actin “clouds” are proposed to generate pulling forces on centrosomes and mediate spindle orientation. However, the motors that pull astral microtubules toward these actin structures are not known. Here, we report that the unconventional myosin, Myo10, couples actin-dependent forces from retraction fibers and subcortical actin clouds to centrosomes. Myo10-mediated centrosome positioning requires its direct microtubule binding. Computational image analysis of large microtubule populations reveals a direct effect of Myo10 on microtubule dynamics and microtubule-cortex interactions. Myo10’s role in centrosome positioning is distinct from, but overlaps with, that of dynein. Thus, Myo10 plays a key role in integrating the actin and microtubule cytoskeletons to position centrosomes and mitotic spindles. PMID:26235048

Evolution of an ancient protein function involved in organized multicellularity in animals

PubMed Central

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-01

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. DOI: http://dx.doi.org/10.7554/eLife.10147.001 PMID:26740169

A giant mesentery malignant solitary fibrous tumor recurring as dedifferentiated liposarcoma- a report of a very rare case and literature review.

PubMed

Liu, Yang; Ishibashi, Haruaki; Sako, Shozou; Takeshita, Kazuyoshi; Li, Yan; Elnemr, Ayman; Yonemura, Yutaka

2013-11-01

We report a case of a 59-year-old woman with a very rare giant mesentery malignant solitary fibrous tumor that recurred as dedifferentiated liposarcoma. The woman was admitted to the hospital because of low abdominal pain. Radiological and biopsy findings revealed a multi-lobulated giant malignant solitary fibrous tumor that had invaded the inferior vena cava, abdominal aorta, and superior mesentery vessels. The tumor was completely removed during the first cytoreductive surgery. Histopathologically, tumor had a heterogeneous cell population, composed of spindle cells with fibrous collagen proliferation. The spindle cells were not arranged in a specific pattern. Immunohistochemistry revealed that the tumor cells were positive for CD34, CD99, Bcl-2, and smooth muscle actin( SMA) and negative for CD117, epithelial membrane antigen (EMA), CAM5.7, S100, desmin, and caldesmon. The tumor recurred 9 months after surgery, and another cytoreductive surgery was then performed. The postoperative histopathological appearance of the invaded area indicated a well-differentiated liposarcoma. Formation of tumorous bone was also noted in the same area, in addition to atypical mesenchymal cells and multi-vacuolated lipoblasts in the area of the well-differentiated liposarcoma. Proliferated spindle cells arranged in a storiform pattern were found in the area adjacent to the tumor. Immunohistochemical analysis revealed that the tumors cells were positive for SMA, HHF-35, and caldesmon and negative for CD117, CD34, and S100. A diagnosis of dedifferentiated liposarcoma was made.

Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells.

PubMed Central

Kawamura, K. I.; Grabowski, D.; Weizer, K.; Bukowski, R.; Ganapathi, R.

1996-01-01

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies. Images Figure 6 PMID:8546904

Protein kinase C zeta suppresses low‐ or high‐grade colorectal cancer (CRC) phenotypes by interphase centrosome anchoring

PubMed Central

Deevi, Ravi Kiran; Javadi, Arman; McClements, Jane; Vohhodina, Jekaterina; Savage, Kienan; Loughrey, Maurice Bernard; Evergren, Emma

2018-01-01

Abstract Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi‐lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low‐grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high‐grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high‐grade morphology in formalin‐fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of mitotic slippage that shape the development of low‐ or high‐grade CRC phenotypes. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:29520890

Protein kinase C zeta suppresses low- or high-grade colorectal cancer (CRC) phenotypes by interphase centrosome anchoring.

PubMed

Deevi, Ravi Kiran; Javadi, Arman; McClements, Jane; Vohhodina, Jekaterina; Savage, Kienan; Loughrey, Maurice Bernard; Evergren, Emma; Campbell, Frederick Charles

2018-04-01

Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi-lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low-grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high-grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high-grade morphology in formalin-fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of mitotic slippage that shape the development of low- or high-grade CRC phenotypes. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Structural and functional characterization of the protein kinase Mps1 in Arabidopsis thaliana.

PubMed

de Oliveira, Eduardo Alves Gamosa; Romeiro, Nelilma Correia; Ribeiro, Elane da Silva; Santa-Catarina, Claudete; Oliveira, Antônia Elenir Amâncio; Silveira, Vanildo; de Souza Filho, Gonçalo Apolinário; Venancio, Thiago Motta; Cruz, Marco Antônio Lopes

2012-01-01

In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes.

Malignant Mesothelioma—Health Professional Version

Cancer.gov

Epithelial mesothelioma is the most common type of malignant mesothelioma, which forms in the cells that line organs. The other types begin in spindle-shaped cells called sarcomatoid cells or are a mixture of both cell types. Find evidence-based information on malignant mesothelioma treatment.

Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy.

PubMed

Gibeaux, Romain; Politi, Antonio Z; Nédélec, François; Antony, Claude; Knop, Michael

2013-02-01

Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.

Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy

PubMed Central

Gibeaux, Romain; Politi, Antonio Z.; Nédélec, François; Antony, Claude; Knop, Michael

2013-01-01

Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule–microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion. PMID:23388829

A rare case of anal carcinosarcoma with human papilloma virus infection in both biphasic tumor elements: An immunohistochemical, molecular and ultrastructural study.

PubMed

Hickman, Richard A; Bradshaw, Azore-Dee; Cassai, Nicholas; Neto, Antonio Galvao; Zhou, David; Fu, Tinghao; Lee, Peng; Pei, Zhiheng; Wieczorek, Rosemary

2016-12-01

Carcinosarcoma of the anus is rare and has yet to be reportedly associated with the keratinocyte-specific Human Papilloma Virus (HPV). We describe a case of anal carcinosarcoma with HPV infection in both the epithelial and mesenchymal components of the tumor by immunohistochemistry, chromogenic in-situ hybridization (CISH) and further supported by electron microscopy (EM). Microscopic examination of the tumor showed nests of poorly-differentiated invasive squamous cell carcinoma with basaloid features intermixed with a hypercellular, atypical spindle cell proliferation. Immunohistochemistry demonstrated that the epithelial component was positive for AE1/AE3, p63, CK5/6 and p16, whilst the mesenchymal component was positive for smooth muscle actin, vimentin, and focally positive for desmin and p16, consistent with carcinosarcoma. The tumor was negative for GATA-3, CK7 and CK20. CISH demonstrated that the tumor was positive for high risk HPV (subtype 16/18) in both tumor components. EM further supported the presence of intracellular virus particles (~50 nm) that is compatible with HPV infection. Infection of both epithelial and mesenchymal tumor components by HPV has not been previously observed in the gastrointestinal tract. This finding may represent initial epithelial HPV infection with subsequent divergent tumoral differentiation and suggests the presence of viral replication in both biphasic tumor components.

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

PubMed

Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

2015-11-01

Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions.

γ-Tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly.

PubMed

Zhou, Qing; Li, Ziyin

2015-11-01

γ-Tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, gamma-tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex. © 2015 John Wiley & Sons Ltd.

The γ-tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly

PubMed Central

Zhou, Qing; Li, Ziyin

2015-01-01

The γ-tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, GCP2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex. PMID:26224545

[A rare pulmonary benign bi-phasic tumor: a case report of pulmonary adenofibroma and literature review].

PubMed

Mei, F; Zhao, T T; Gao, F; Zheng, J

2017-12-18

Pulmonary adenofibroma is an extremely rare benign primary tumor of the lung, with characteristic bi-phasic differential pattern. They are usually sub-pleural solid pulmonary nodules with clear margins. The tumor is composed of glands and peri-gland stroma. The glands are often quite simple, forming long and narrow tubules, with uniformly monolayer lining cells. Combined papillary or phyllodes structures were reported in some cases. The stromal cells are spindle-shaped and look mild, mixed with the collagen bands. Up till now, only a few cases of pulmonary adenofibroma have been reported all over the world. And because of the limited recognition, this tumor is easily misinterpreted as malignancy in frozen section or biopsy specimens. We reported a new case of pulmonary adenofibroma. The mass peripherally located in the left lobe of the lung, found by chance in a 74-year-old woman. The patient underwent a wedge resection of the left lung by the thoracoscope, because of the slowly gradual enlargement of the mass annually. An oval grayish-white nodule was sub-pleural located in the specimen, with solid and rubbery texture, but without a distinct capsule. Two distinct components of simple glands and mild spindle cell stroma were found to mix together uniformly under the microscope. Collagen bands of various widths evenly surrounded each stromal cell. A few small coarse papillae or phyllodes structures were randomly distributed in some area. The immunohistochemical staining pattern of the glandular cells was accordant with typeIIalveolar epithelium. Stromal cells were positive with CD34, B-cell lymphoma-2 (Bcl-2), CD99 and estrogen receptor (ER), while S-100, smooth muscle actin (SMA) and all the mesothe-lium markers were negative. The patient was disease free after the surgery, although the follow-up time was only one year. Besides the new case above, we also reviewed all the reported cases, and tentatively discussed the probable histological origin of pulmonary adenofibroma.

The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle

PubMed Central

1994-01-01

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein. PMID:8138567

TECHNICAL NOTE: High-speed grinding using thin abrasive disks for microcomponents

NASA Astrophysics Data System (ADS)

Yeo, S. H.; Balon, S. A. P.

2002-01-01

This paper introduces the development of a high-speed grinding device for cylindrical grinding of microcomponents made of hard and brittle materials. The study made use of an ultraprecision diamond turning machine tool as a basic platform. The novelty of the device is based on the high-speed air bearing spindle with a thin grinding wheel, similar to the dicing technology for silicon wafer fabrication. The spindle attachment is inclined at an angle to the main spindle which holds the precision fixture mechanism via the vacuum chuck. Experiments have been conducted to verify the design and implementation of the grinding methodology. A feature size as small as 31 μm in diameter and average surface roughness of 98 nm were obtained in the experimental work. It is found that the work done is capable of manufacturing miniature components, such as microcylindrical stepped shafts.

Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

PubMed

Edamatsu, M

2016-02-11

Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro.

Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint.

PubMed

London, Nitobe; Biggins, Sue

2014-01-15

The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.

Inhibition of the spindle assembly checkpoint kinase Mps-1 as a novel therapeutic strategy in malignant mesothelioma

PubMed Central

Szymiczek, Agata; Carbone, Michele; Pastorino, Sandra; Napolitano, Andrea; Tanji, Mika; Minaai, Michael; Pagano, Ian; Mason, Jacqueline M.; Pass, Harvey I.; Bray, Mark R.; Mak, Tak W.; Yang, Haining

2017-01-01

Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. TTK/Mps-1 (monopolar spindle 1 kinase) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients’ outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients. PMID:28759042

Polyoma small T antigen triggers cell death via mitotic catastrophe

PubMed Central

Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

2014-01-01

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

Radiation noise of the bearing applied to the ceramic motorized spindle based on the sub-source decomposition method

NASA Astrophysics Data System (ADS)

Bai, X. T.; Wu, Y. H.; Zhang, K.; Chen, C. Z.; Yan, H. P.

2017-12-01

This paper mainly focuses on the calculation and analysis on the radiation noise of the angular contact ball bearing applied to the ceramic motorized spindle. The dynamic model containing the main working conditions and structural parameters is established based on dynamic theory of rolling bearing. The sub-source decomposition method is introduced in for the calculation of the radiation noise of the bearing, and a comparative experiment is adopted to check the precision of the method. Then the comparison between the contribution of different components is carried out in frequency domain based on the sub-source decomposition method. The spectrum of radiation noise of different components under various rotation speeds are used as the basis of assessing the contribution of different eigenfrequencies on the radiation noise of the components, and the proportion of friction noise and impact noise is evaluated as well. The results of the research provide the theoretical basis for the calculation of bearing noise, and offers reference to the impact of different components on the radiation noise of the bearing under different rotation speed.

XMAP310: A Xenopus Rescue-promoting Factor Localized to the Mitotic Spindle

PubMed Central

Andersen, Søren S.L.; Karsenti, Eric

1997-01-01

To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289–1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5–10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985–993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation. PMID:9362515

EGF Induced Centrosome Separation Promotes Mitotic Progression and Cell Survival

PubMed Central

Mardin, Balca R.; Isokane, Mayumi; Cosenza, Marco R.; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M.; Schiebel, Elmar

2014-01-01

Summary Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy since cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362

The functions of the cytoskeleton and associated proteins during mitosis and cytokinesis in plant cells

PubMed Central

Li, Shanwei; Sun, Tiantian; Ren, Haiyun

2015-01-01

In higher plants, microtubule (MT)-based, and actin filament (AF)-based structures play important roles in mitosis and cytokinesis. Besides the mitotic spindle, the evolution of a band comprising cortical MTs and AFs, namely, the preprophase band (PPB), is evident in plant cells. This band forecasts a specific division plane before the initiation of mitosis. During cytokinesis, another plant-specific cytoskeletal structure called the phragmoplast guides vesicles in the creation of a new cell wall. In addition, a number of cytoskeleton-associated proteins are reportedly involved in the formation and function of the PPB, mitotic spindle, and phragmoplast. This review summarizes current knowledge on the cytoskeleton-associated proteins that mediate the cytoskeletal arrays during mitosis and cytokinesis in plant cells and discusses the interaction between MTs and AFs involved in mitosis and cytokinesis. PMID:25964792

Depletion of Aurora-A in zebrafish causes growth retardation due to mitotic delay and p53-dependent cell death.

PubMed

Jeon, Hee-Yeon; Lee, Hyunsook

2013-03-01

Aurora-A is a serine/threonine mitotic kinase that is required for centrosome maturation. Many cancer cells over-express Aurora-A, and several reports have suggested that Aurora-A has prognostic value in the clinical treatment of cancer. Therefore, inhibitors for Aurora-A kinase have been developed. However, studies on Aurora-A are largely performed in cancer cell lines and are sometimes controversial. For effective evaluation of Aurora-A inhibitors in cancer treatment, it is essential to understand its function at the organism level. Here, we report the crucial functions of Aurora-A in homeostasis of spindle organization in mitosis using zebrafish embryogenesis as a model system. Using morpholino technology, we show that depletion of Aurora-A in zebrafish embryogenesis results in short bent trunks, accompanied by growth retardation and eventual cell death. Live-imaging and immunofluorescence analyses of the embryos revealed that the developmental defects are due to problems in mitosis, manifested through monopolar and disorganized spindle formation. Aurora-A-depleted cells exhibited mitotic arrest with congression failure, leading to activation of the spindle assembly checkpoint. Cell death in the absence of Aurora-A was partially rescued by co-injection of the p53 morpholino, suggesting that apoptosis after Aurora-A depletion is p53-dependent. The clinical implications of these results relate to the indication that Aurora-A inhibitors may be effective towards cancers with intact p53. © 2013 The Authors Journal compilation © 2013 FEBS.

Cenp-E inhibitor GSK923295: Novel synthetic route and use as a tool to generate aneuploidy.

PubMed

Bennett, Ailsa; Bechi, Beatrice; Tighe, Anthony; Thompson, Sarah; Procter, David J; Taylor, Stephen S

2015-08-28

Aneuploidy is a common feature of cancer, with human solid tumour cells typically harbouring abnormal chromosome complements. The aneuploidy observed in cancer is often caused by a chromosome instability phenotype, resulting in genomic heterogeneity. However, the role aneuploidy and chromosome instability play in tumour evolution and chemotherapy response remains poorly understood. In some contexts, aneuploidy has oncogenic effects, whereas in others it is anti-proliferative and tumour-suppressive. Dissecting fully the role aneuploidy plays in tumourigenesis requires tools and facile assays that allow chromosome missegregation to be induced experimentally in cells that are otherwise diploid and chromosomally stable. Here, we describe a chemical biology approach that induces low-level aneuploidy across a large population of cells. Specifically, cells are first exposed to GSK923295, an inhibitor targeting the mitotic kinesin Cenp-E; while the majority of chromosomes align at the cell's equator, a small number cluster near the spindle poles. By then driving these cells into anaphase using AZ3146, an inhibitor targeting the spindle checkpoint kinase Mps1, the polar chromosomes are missegregated. This results in, on average, two chromosome missegregation events per division, and avoids trapping chromosomes in the spindle midzone, which could otherwise lead to DNA damage. We also describe an efficient route for the synthesis of GSK923295 that employs a novel enzymatic resolution. Together, the approaches described here open up new opportunities for studying cellular responses to aneuploidy.

The p90 ribosomal S6 kinase 2 specifically affects mitotic progression by regulating the basal level, distribution and stability of mitotic spindles

PubMed Central

Park, Yun Yeon; Nam, Hyun-Ja; Do, Mihyang; Lee, Jae-Ho

2016-01-01

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles. PMID:27491410

Meet Spinky: An Open-Source Spindle and K-Complex Detection Toolbox Validated on the Open-Access Montreal Archive of Sleep Studies (MASS).

PubMed

Lajnef, Tarek; O'Reilly, Christian; Combrisson, Etienne; Chaibi, Sahbi; Eichenlaub, Jean-Baptiste; Ruby, Perrine M; Aguera, Pierre-Emmanuel; Samet, Mounir; Kachouri, Abdennaceur; Frenette, Sonia; Carrier, Julie; Jerbi, Karim

2017-01-01

Sleep spindles and K-complexes are among the most prominent micro-events observed in electroencephalographic (EEG) recordings during sleep. These EEG microstructures are thought to be hallmarks of sleep-related cognitive processes. Although tedious and time-consuming, their identification and quantification is important for sleep studies in both healthy subjects and patients with sleep disorders. Therefore, procedures for automatic detection of spindles and K-complexes could provide valuable assistance to researchers and clinicians in the field. Recently, we proposed a framework for joint spindle and K-complex detection (Lajnef et al., 2015a) based on a Tunable Q-factor Wavelet Transform (TQWT; Selesnick, 2011a) and morphological component analysis (MCA). Using a wide range of performance metrics, the present article provides critical validation and benchmarking of the proposed approach by applying it to open-access EEG data from the Montreal Archive of Sleep Studies (MASS; O'Reilly et al., 2014). Importantly, the obtained scores were compared to alternative methods that were previously tested on the same database. With respect to spindle detection, our method achieved higher performance than most of the alternative methods. This was corroborated with statistic tests that took into account both sensitivity and precision (i.e., Matthew's coefficient of correlation (MCC), F1, Cohen κ). Our proposed method has been made available to the community via an open-source tool named Spinky (for spindle and K-complex detection). Thanks to a GUI implementation and access to Matlab and Python resources, Spinky is expected to contribute to an open-science approach that will enhance replicability and reliable comparisons of classifier performances for the detection of sleep EEG microstructure in both healthy and patient populations.

The reaction of Lupinus angustifolius L. root meristematic cell nucleoli to lead.

PubMed

Balcerzak, Lucja; Glińska, Sława; Godlewski, Mirosław

2011-04-01

The effect of 2-48 h treatment of Lupinus angustifolius L. roots with lead nitrate at the concentration of 10(-4) M on the nucleoli in meristematic cells was investigated. In the lead presence the number of ring-shaped as well as segregated nucleoli increased especially after 12-48 h of treatment, while spindle-shaped nucleoli appeared after 24 h and 48 h. Lead presence also increased the frequency of cells with silver-stained particles in the nucleus and the number of these particles especially from the 12th hour of treatment. It was accompanied by significant decline of nucleolar area. Analysis of these cells in transmission electron microscope confirmed the presence of ring-shaped and segregated nucleoli. Moreover, electron microscopy revealed compact structure nucleoli without granular component. Additionally, one to three oval-shaped fibrillar structures attached to nucleolus or lying free in the nucleoplasm were visible. The possible mechanism of lead toxicity to the nucleolus is briefly discussed.

Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish

NASA Technical Reports Server (NTRS)

Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.

Cdk1-cyclin B1-mediated phosphorylation of tumor-associated microtubule-associated protein/cytoskeleton-associated protein 2 in mitosis.

PubMed

Hong, Kyung Uk; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-06-12

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis*

PubMed Central

Uk Hong, Kyung; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-01-01

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis. PMID:19369249

WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes.

PubMed

Sun, Shao-Chen; Xu, Yong-Nan; Li, Ying-Hua; Lee, Seung-Eun; Jin, Yong-Xun; Cui, Xiang-Shun; Kim, Nam-Hyung

2011-06-01

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altenfeld, Anika; Wohlgemuth, Sabine; Wehenkel, Annemarie

The 800 kDa complex of the human Rod, Zwilch and ZW10 proteins (the RZZ complex) was reconstituted in insect cells, purified, crystallized and subjected to preliminary X-ray diffraction analysis. The spindle-assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three-subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted bymore » co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3{sub 1} (No. 144) or P3{sub 2} (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°.« less

Fine needle aspiration cytology of hepatic metastasis from a meningeal hemangiopericytoma. A case report.

PubMed

Ghaffar, Hasan; Parwani, Anil; Rosenthal, Dorothy L

2003-01-01

Hemangiopericytomas (HPCs) are rare spindle cell tumors, constituting 2.5% of soft tissue neoplasms. Few reports have addressed the fine needle aspiration (FNA) cytology of HPC. We describe the FNA biopsy (FNAB) findings in a 44-year-old patient with a previously resected meningeal hemangiopericytoma. The patient underwent ultrasound-guided FNAB of a 16.0-cm, radiographically heterogeneous density in the liver. The FNA smear showed crowded, ovoid to spindle-shaped cells with poorly defined, scant cytoplasm. The neoplastic cells were positive for CD34 and negative for CD31, factor VIII, glial fibrillary acid protein and cytokeratin AE1/AE3, supporting a diagnosis of HPC and compatible with metastasis from the patient's cerebral tumor. This case documents the role of FNA cytology in confirming HPC.

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Toshihide, E-mail: toshi-su@pharm.teikyo-u.ac.j; Miyazaki, Koichi; Kita, Kayoko

2009-12-15

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvementmore » of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.« less

The GTPase SPAG-1 orchestrates meiotic program by dictating meiotic resumption and cytoskeleton architecture in mouse oocytes

PubMed Central

Huang, Chunjie; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiaofei; Guan, Kaifeng; Huo, Lijun

2016-01-01

In mammals, a finite population of oocytes is generated during embryogenesis, and proper oocyte meiotic divisions are crucial for fertility. Sperm-associated antigen 1 (SPAG-1) has been implicated in infertility and tumorigenesis; however, its relevance in cell cycle programs remains rudimentary. Here we explore a novel role of SPAG-1 during oocyte meiotic progression. SPAG-1 associated with meiotic spindles and its depletion severely compromised M-phase entry (germinal vesicle breakdown [GVBD]) and polar body extrusion. The GVBD defect observed was due to an increase in intraoocyte cAMP abundance and decrease in ATP production, as confirmed by the activation of AMP-dependent kinase (AMPK). SPAG-1 RNA interference (RNAi)–elicited defective spindle morphogenesis was evidenced by the dysfunction of γ-tubulin, which resulted from substantially reduced phosphorylation of MAPK and irregularly dispersed distribution of phospho-MAPK around spindles instead of concentration at spindle poles. Significantly, actin expression abruptly decreased and formation of cortical granule–free domains, actin caps, and contractile ring disrupted by SPAG-1 RNAi. In addition, the spindle assembly checkpoint remained functional upon SPAG-1 depletion. The findings broaden our knowledge of SPAG-1, showing that it exerts a role in oocyte meiotic execution via its involvement in AMPK and MAPK signaling pathways. PMID:27053660

Ultrastructural study on dynamics of plastids and mitochondria during microgametogenesis in watermelon.

PubMed

Liu, Lin

2012-02-01

Dynamics of plastids and mitochondria during microgametogenesis in watermelon were examined by means of transmission electron microscopy. Plastids are present as proplastids in the microspore and as amyloplasts in the vegetative cell of the bicellular pollen grain, whereas the generative cell is completely devoid of plastids, suggesting that microspore plastids are excluded from the generative cell during the microspore mitotic division. Therefore, watermelon is classified as Lycopersicon type, where plastids exclusion from the generative cell leads to purely maternal plastid inheritance. Mitochondria in the generative cell show noticeable alterations in size and cristae during microgametogenesis. The diameter of mitochondria is about 0.5 μm in the newly born generative cell, while only about 0.16 μm in the spindle-shaped generative cell. Numerous cristae are present in mitochondria in the spherical generative cell, but, in contrast, mere two or three cristae retain in the spindle-shaped generative cell in the mature pollen grain. In conclusion, the size and cristae number of mitochondria in the generative cell are reduced significantly during microgametogenesis in watermelon. Copyright © 2011 Elsevier Ltd. All rights reserved.

Discovery of (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent.

PubMed

Theoclitou, Maria-Elena; Aquila, Brian; Block, Michael H; Brassil, Patrick J; Castriotta, Lillian; Code, Erin; Collins, Michael P; Davies, Audrey M; Deegan, Tracy; Ezhuthachan, Jayachandran; Filla, Sandra; Freed, Ellen; Hu, Haiqing; Huszar, Dennis; Jayaraman, Muthusamy; Lawson, Deborah; Lewis, Paula M; Nadella, Murali V P; Oza, Vibha; Padmanilayam, Maniyan; Pontz, Timothy; Ronco, Lucienne; Russell, Daniel; Whitston, David; Zheng, Xiaolan

2011-10-13

Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.

Novel p53 tumour suppressor mutations in cases of spindle cell sarcoma, pleomorphic sarcoma and fibrosarcoma in cats.

PubMed

Mayr, B; Reifinger, M; Alton, K; Schaffner, G

1998-06-01

Twenty feline neoplasms were sequenced in the region from exons 5 to 8 for the presence of tumour suppressor gene p53 mutations. In a spindle cell sarcoma of the bladder, a missense mutation (codon 164 AAG-->GAG, lysine-->glutamic acid) in exon 5 was detected. In a pleomorphic sarcoma, a 23 bp deletion involving the splicing junction between intron 5 and exon 6 was observed. In a fibrosarcoma, a 6 bp deletion of p53 covering 2 bp of exon 7 and 4 bp of intron 7, including the splicing junction, was found. The study demonstrates three new p53 mutations in different types of sarcomas in cats.

Ligand- and structure-based in silico studies to identify kinesin spindle protein (KSP) inhibitors as potential anticancer agents.

PubMed

Balakumar, Chandrasekaran; Ramesh, Muthusamy; Tham, Chuin Lean; Khathi, Samukelisiwe Pretty; Kozielski, Frank; Srinivasulu, Cherukupalli; Hampannavar, Girish A; Sayyad, Nisar; Soliman, Mahmoud E; Karpoormath, Rajshekhar

2017-11-29

Kinesin spindle protein (KSP) belongs to the kinesin superfamily of microtubule-based motor proteins. KSP is responsible for the establishment of the bipolar mitotic spindle which mediates cell division. Inhibition of KSP expedites the blockade of the normal cell cycle during mitosis through the generation of monoastral MT arrays that finally cause apoptotic cell death. As KSP is highly expressed in proliferating/cancer cells, it has gained considerable attention as a potential drug target for cancer chemotherapy. Therefore, this study envisaged to design novel KSP inhibitors by employing computational techniques/tools such as pharmacophore modelling, virtual database screening, molecular docking and molecular dynamics. Initially, the pharmacophore models were generated from the data-set of highly potent KSP inhibitors and the pharmacophore models were validated against in house test set ligands. The validated pharmacophore model was then taken for database screening (Maybridge and ChemBridge) to yield hits, which were further filtered for their drug-likeliness. The potential hits retrieved from virtual database screening were docked using CDOCKER to identify the ligand binding landscape. The top-ranked hits obtained from molecular docking were progressed to molecular dynamics (AMBER) simulations to deduce the ligand binding affinity. This study identified MB-41570 and CB-10358 as potential hits and evaluated these experimentally using in vitro KSP ATPase inhibition assays.

Modulation of miR-21 signaling by MPS1 in human glioblastoma

PubMed Central

Maachani, Uday B.; Tandle, Anita; Shankavaram, Uma; Kramp, Tamalee; Camphausen, Kevin A.

2016-01-01

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy. PMID:25991676

Modulation of miR-21 signaling by MPS1 in human glioblastoma.

PubMed

Maachani, Uday B; Tandle, Anita; Shankavaram, Uma; Kramp, Tamalee; Camphausen, Kevin

2016-08-16

Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPAs), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor PDCD4 and MSH2 genes, by down regulating micro RNA-21 (miR-21). In GBMs miR-21 expression is significantly elevated and is associated with chemo and radioresistance. Both MPS1 and miR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind miR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via miR-21 regulation. We also, show the prognostic effect of miR-21, PDCD4 and MSH2 levels to patient survival across different GBM molecular subtypes. This scenario in which miR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy.

Effect of MPS1 Inhibition on Genotoxic Stress Responses in Murine Tumour Cells.

PubMed

Suzuki, Motofumi; Yamamori, Tohru; Yasui, Hironobu; Inanami, Osamu

2016-06-01

The monopolar spindle 1 (MPS1) is a serine/threonine kinase that plays an important role in spindle assembly checkpoint signaling. To determine the possible relationship between MPS1 inhibition and genotoxic stress responses, herein we examined whether MPS1 inhibition influences cellular susceptibility towards two genotoxic treatments, etoposide and ionizing radiation (IR). Two murine tumour cell lines, SCCVII and EMT6, were used. The effect of genotoxic treatments with or without two novel MPS1 inhibitors, NMS-P715 and AZ3146, on cellular survival, cell-cycle distribution, centrosome status and mitotic catastrophe (MC) was evaluated. MPS1 inhibition sensitized murine tumour cells to etoposide but not to IR. In addition, MPS1 inhibition altered cell-cycle progression and exacerbated centrosome abnormalities, resulting in enhanced MC induced by etoposide but not by IR. MPS1 inhibition promotes the etoposide-induced aberrant mitosis and, consequently, the induction of tumour cell death. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Angiofibroma of soft tissue: clinicopathologic study of 2 cases of a recently characterized benign soft tissue tumor.

PubMed

Zhao, Ming; Sun, Ke; Li, Changshui; Zheng, Jiangjiang; Yu, Jingjing; Jin, Jie; Xia, Wenping

2013-01-01

Angiofibroma of soft tissue is a very recently characterized, histologically distinctive benign mesenchymal neoplasm of unknown cellular origin composed of 2 principal components, the spindle cell component and very prominent stromal vasculatures. It usually occurs in middle-aged adults, with a female predominance. Herein, we describe the clinical and pathologic details of 2 other examples of this benign tumor. Both patients were middle-aged male and presented with a slow-growing, painless mass located in the deep-seated soft tissue of thigh and left posterior neck region, respectively. Grossly, both tumors were well-demarcated, partial encapsulated of a grayish-white color with firm consistence. Histologically, one case showed morphology otherwise identical to those have been described before, whereas the other case showed in areas being more cellular than most examples of this subtype tumor had, with the lesional cells frequently exhibiting short fascicular, vaguely storiform and occasionally swirling arrangements, which posed a challenging differential diagnosis. Immunostains performed on both tumors did not confirm any specific cell differentiation with lesional cells only reactive for vimentin and focally desmin and negative for all the other markers tested. This report serves to broaden the morphologic spectrum of angiofibroma of soft tumor. Awareness of this tumor is important to prevent misdiagnosis as other more aggressive soft tissue tumor.

Myopericytoma proliferating in an unusual anastomosing multinodular fashion.

PubMed

Inoue, Takuya; Misago, Noriyuki; Asami, Akihiko; Tokunaga, Osamu; Narisawa, Yutaka

2016-05-01

We herein describe a case of myopericytoma that proliferated in an unusual fashion. Myopericytoma is described as a group of rare, benign, dermal or subcutaneous tumors that are characterized histologically by a striking, concentric, perivascular proliferation of spindle cells and showing apparent differentiation towards perivascular myoid cells. Myopericytoma forms a morphological continuum with myofibroma/myofibromatosis, glomus tumor and angioleiomyoma. The patient was a 64-year-old woman who demonstrated a recurrent ulcer on an atrophic plaque on her left shin. A histopathological examination of the plaque demonstrated that tumor cells proliferated in an anastomosing multinodular fashion along the vessels in the dermis and subcutaneous tissue. In those nodules, there were numerous, small, concentric proliferations of myoid-appearing spindle cells around small vascular lumina. The present case is an unusual example of myopericytoma, manifesting in a characteristic anastomosing, multinodular, infiltrating fashion. © 2015 Japanese Dermatological Association.

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

PubMed Central

St Johnston, Daniel

2016-01-01

Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside of the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

In vitro culture of various typed meningiomas and characterization of a human malignant meningioma cell line (HKBMM).

PubMed

Ishiwata, Isamu; Ishiwata, Chieko; Ishiwata, Emiko; Sato, Yoshiro; Kiguchi, Kazushige; Tachibana, Toshiaki; Ishikawa, Hiroshi

2004-12-01

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

Study designs to investigate Nox1 acceleration of neoplastic progression in immortalized human epithelial cells by selection of differentiation resistant cells.

PubMed

Sattayakhom, Apsorn; Chunglok, Warangkana; Ittarat, Wanida; Chamulitrat, Walee

2014-01-01

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines - FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.

Oncologic outcomes in patients with nonurothelial bladder cancer.

PubMed

Patel, Sanjay G; Weiner, Adam Benjamin; Keegan, Kirk; Morgan, Todd

2018-01-01

We aimed to evaluate the relative prognostic impact of the most common variant histologies on disease-specific survival (DSS) in patients undergoing radical cystectomy. The Surveillance, Epidemiology, and End Result database was used to identify patients who underwent radical cystectomy for bladder cancer from 1990 to 2007. Patients with urothelial cell carcinoma (UCC), squamous cell carcinoma (SCC), adenocarcinoma (AC), sarcoma, small cell carcinoma, signet ring carcinoma, and spindle cell carcinoma were included in the study. Multivariable analysis was performed using Cox proportional hazards model to assess independent predictors of disease-specific survival (DSS). Mortality rates were estimated using Kaplan-Meier analyses. A total of 14,130 patients met inclusion criteria with the following histologies: UCC (90.1%), SCC (4.6%), AC, (2.3%), sarcoma (0.8%), small cell carcinoma (0.8%), signet ring carcinoma (0.5%), and spindle cell carcinoma (0.9%). Three-year DSS was most favorable in patients with UCC (63.7%; 95% confidence interval [62.9%-64.8%]) and AC (65.3% [59.3%-70.6%]), whereas 3-year DSS was the least favorable for small cell carcinoma (41.6% [31.3%-51.6%]) and sarcoma (45.4% [35.1%-55.1%]). In the multivariable analysis, independent predictors of DSS were age, marital status, grade, T-stage, N-stage, and variant histology. With respect to UCC, there was an increased risk of disease-specific death associated with all variants except AC. Sarcoma and spindle cell carcinoma were associated with the highest risk of death. With the exception of AC, the most common variant bladder cancer histologies are all independently associated with worse DSS relative to UCC in patients undergoing radical cystectomy.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability

PubMed Central

Dilworth, David; Gudavicius, Geoff; Xu, Xiaoxue; Boyce, Andrew K J; O’Sullivan, Connor; Serpa, Jason J; Bilenky, Misha; Petrochenko, Evgeniy V; Borchers, Christoph H; Hirst, Martin; Swayne, Leigh Anne; Howard, Perry; Nelson, Christopher J

2018-01-01

Abstract FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25–DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases. PMID:29361176

Slk19p of Saccharomyces cerevisiae Regulates Anaphase Spindle Dynamics Through Two Independent Mechanisms

PubMed Central

Havens, Kyle A.; Gardner, Melissa K.; Kamieniecki, Rebecca J.; Dresser, Michael E.; Dawson, Dean S.

2010-01-01

Slk19p is a member of the Cdc-14 early anaphase release (FEAR) pathway, a signaling network that is responsible for activation of the cell-cycle regulator Cdc14p in Saccharomyces cerevisiae. Disruption of the FEAR pathway results in defects in anaphase, including alterations in the assembly and behavior of the anaphase spindle. Many phenotypes of slk19Δ mutants are consistent with a loss of FEAR signaling, but other phenotypes suggest that Slk19p may have FEAR-independent roles in modulating the behavior of microtubules in anaphase. Here, a series of SLK19 in-frame deletion mutations were used to test whether Slk19p has distinct roles in anaphase that can be ascribed to specific regions of the protein. Separation-of-function alleles were identified that are defective for either FEAR signaling or aspects of anaphase spindle function. The data suggest that in early anaphase one region of Slk19p is essential for FEAR signaling, while later in anaphase another region is critical for maintaining the coordination between spindle elongation and the growth of interpolar microtubules. PMID:20923975

Combination Chemotherapy With or Without Temsirolimus in Treating Patients With Intermediate Risk Rhabdomyosarcoma

ClinicalTrials.gov

2018-06-27

Alveolar Rhabdomyosarcoma; Botryoid-Type Embryonal Rhabdomyosarcoma; Embryonal Rhabdomyosarcoma; Rhabdomyosarcoma; Sclerosing Rhabdomyosarcoma; Spindle Cell Rhabdomyosarcoma; Untreated Childhood Rhabdomyosarcoma

Microtubule Depolymerization as a Driver for Chromosome Motion

NASA Astrophysics Data System (ADS)

McIntosh, Richard

2014-03-01

Microtubules (MTs) are rigid polymers of the protein, tubulin, which function as intracellular struts. They are also tracks along which motor enzymes can run, carrying cargo to specific cellular locations. Most MTs are dynamic; they assemble and disassemble rapidly, particularly during cell division when the cell forms the ``mitotic spindle,'' a machine that organizes the duplicated chromosomes into a planar disk, then pulls the duplicate copies apart, moving them to opposite ends of the cell. This process is necessary for the daughter cells to have a full complement of DNA. The mitotic spindle is a labile framework that exerts several kinds of forces on the chromosomes to move them in well organized ways. It contains many motor enzymes that contribute to spindle formation, but genetic evidence shows that the motors that attach to chromosomes and might contribute to chromosome motion are dispensable for normal mitosis. Apparently MT dynamics can also serve as a motor and is an important source of force for chromosome motion. We have studied this process and find that MTs can be coupled to a load by specific spindle proteins so that MT depolymerization can exert substantial force. With the yeast protein, Dam1, a single MT can generate 30 pN, about 5-fold more than is generated by a motor enzyme like kinesin or myosin. The resulting motions are processive, so a depolymerizing MT can carry its load for many micrometers. However, Dam1 is found only in fungi. We have therefore sought other proteins that can serve as analogous couplers. Several MT-dependent motor enzymes can do the job in ways that do not require ATP, their normal source of energy. Some non-motor MT-associated proteins will also work, e.g., the kinetochore proteins NDC80 and CENP-F. Data will be presented that show the strengths and weaknesses of each coupler, allowing some generalization about how the mitotic machinery works. Supported by NIH GM033787.

Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos.

PubMed

Wakayama, Sayaka; Cibelli, Jose B; Wakayama, Teruhiko

2003-01-01

Cloning methods are now well described and becoming routine. Yet the frequency at which live cloned offspring are produced (as a percentage of starting one-cell embryos) remains below 5% irrespective of nucleus donor species or cell type. In considering the cause(s) of this universally low efficiency, features common to all cloning protocols are strong candidates. One such shared feature is enucleation; the donor nucleus is inserted into an enucleated cytoplast (ooplast). However, it is not known whether a nucleus-free metaphase II oocyte is developmentally impaired other than by virtue of lacking chromosomes, or if in nuclear transfer protocols, enucleation removes factors necessary to reprogram the incoming nucleus. We have here investigated the role of enucleation in nuclear transfer. Three hours after the injection of cumulus cell nuclei into non-enucleated oocytes, 65% contained two distinct metaphase spindles, with the remainder exhibiting a single spindle in which oocyte-derived and nucleus donor chromosomes were mixed. However, staining only one hour after donor nucleus insertion revealed that most had two discrete spindles. In the absence of staining, the donor nucleus spindle was not visible. This provided a straightforward way to identify and select the oocyte-derived metaphase chromosomes 1 h after donor nucleus microinjection, and 34-41% cloned embryo developed to the morulla-blastocyst stage following Sr(2+)-induced activation. Of these, two (1% of starting one-cell embryos) developed to term, an efficiency which is comparable to that obtained for controls (6 clone; 1-2%) in which enucleation preceded nuclear transfer. In conclusion, the timing of the removal of oocyte chromosomes before or after injection of somatic nucleus had no effect on cloned embryo development. These findings argue that neither oocyte chromosome depletion per se, nor the potential removal of "reprogramming" factors during enucleation explain the low efficiency of nuclear transfer cloning.

Trypanosome outer kinetochore proteins suggest conservation of chromosome segregation machinery across eukaryotes

PubMed Central

D’Archivio, Simon

2017-01-01

Kinetochores are multiprotein complexes that couple eukaryotic chromosomes to the mitotic spindle to ensure proper segregation. The model for kinetochore assembly is conserved between humans and yeast, and homologues of several components are widely distributed in eukaryotes, but key components are absent in some lineages. The recent discovery in a lineage of protozoa called kinetoplastids of unconventional kinetochores with no apparent homology to model organisms suggests that more than one system for eukaryotic chromosome segregation may exist. In this study, we report a new family of proteins distantly related to outer kinetochore proteins Ndc80 and Nuf2. The family member in kinetoplastids, KKT-interacting protein 1 (KKIP1), associates with the kinetochore, and its depletion causes severe defects in karyokinesis, loss of individual chromosomes, and gross defects in spindle assembly or stability. Immunopurification of KKIP1 from stabilized kinetochores identifies six further components, which form part of a trypanosome outer kinetochore complex. These findings suggest that kinetochores in organisms such as kinetoplastids are built from a divergent, but not ancestrally distinct, set of components and that Ndc80/Nuf2-like proteins are universal in eukaryotic division. PMID:28034897

Centromeres and kinetochores of Brassicaceae.

PubMed

Lermontova, Inna; Sandmann, Michael; Demidov, Dmitri

2014-06-01

The centromere-the primary constriction of monocentric chromosomes-is essential for correct segregation of chromosomes during mitosis and meiosis. Centromeric DNA varies between different organisms in sequence composition and extension. The main components of centromeric and pericentromeric DNA of Brassicaceae species are centromeric satellite repeats. Centromeric DNA initiates assembly of the kinetochore, the large protein complex where the spindle fibers attach during nuclear division to pull sister chromatids apart. Kinetochore assembly is initiated by incorporation of the centromeric histone H3 cenH3 into centromeric nucleosomes. The spindle assembly checkpoint acts during mitosis and meiosis at centromeres and maintains genome stability by preventing chromosome segregation before all kinetochores are correctly attached to microtubules. The function of the spindle assembly checkpoint in plants is still poorly understood. Here, we review recent advances of studies on structure and functional importance of centromeric DNA of Brassicaceae, assembly and function of cenH3 in Arabidopsis thaliana and characterization of core SAC proteins of A. thaliana in comparison with non-plant homologues.

Finite Element Analysis of Flexural Vibrations in Hard Disk Drive Spindle Systems

NASA Astrophysics Data System (ADS)

LIM, SEUNGCHUL

2000-06-01

This paper is concerned with the flexural vibration analysis of the hard disk drive (HDD) spindle system by means of the finite element method. In contrast to previous research, every system component is here analytically modelled taking into account its structural flexibility and also the centrifugal effect particularly on the disk. To prove the effectiveness and accuracy of the formulated models, commercial HDD systems with two and three identical disks are selected as examples. Then their major natural modes are computed with only a small number of element meshes as the shaft rotational speed is varied, and subsequently compared with the existing numerical results obtained using other methods and newly acquired experimental ones. Based on such a series of studies, the proposed method can be concluded as a very promising tool for the design of HDDs and various other high-performance computer disk drives such as floppy disk drives, CD ROM drives, and their variations having spindle mechanisms similar to those of HDDs.

Neural mechanisms of mental schema: a triplet of delta, low beta/spindle and ripple oscillations.

PubMed

Ohki, Takefumi; Takei, Yuichi

2018-02-06

Schemas are higher-level knowledge structures that integrate and organise lower-level representations. As internal templates, schemas are formed according to how events are perceived, interpreted and remembered. Although these higher-level units are assumed to play a fundamental role in our daily life from an early age, the neuronal basis and mechanisms of schema formation and use remain largely unknown. It is important to elucidate how the brain constructs and maintains these higher-level units. In order to examine the possible neural underpinnings of schema, we recapitulate previous work and discuss their findings related to schemas as the brain template. We specifically focused on low beta/spindle oscillations, which are assumed to be the key components of schemas, and propose that the brain template is implemented with a triplet of neural oscillations, that is delta, low beta/spindle and ripple oscillations. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

PubMed Central

Dou, Zhen; Liu, Xing; Wang, Wenwen; Zhu, Tongge; Wang, Xinghui; Xu, Leilei; Abrieu, Ariane; Fu, Chuanhai; Hill, Donald L.; Yao, Xuebiao

2015-01-01

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression. PMID:26240331

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment.

PubMed

Dou, Zhen; Liu, Xing; Wang, Wenwen; Zhu, Tongge; Wang, Xinghui; Xu, Leilei; Abrieu, Ariane; Fu, Chuanhai; Hill, Donald L; Yao, Xuebiao

2015-08-18

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C-MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.

The conserved Wdr8-hMsd1/SSX2IP complex localises to the centrosome and ensures proper spindle length and orientation

PubMed Central

Hori, Akiko; Morand, Agathe; Ikebe, Chiho; Frith, David; Snijders, Ambrosius P.; Toda, Takashi

2015-01-01

The centrosome plays a pivotal role in a wide range of cellular processes and its dysfunction is causally linked to many human diseases including cancer and developmental and neurological disorders. This organelle contains more than one hundred components, and yet many of them remain uncharacterised. Here we identified a novel centrosome protein Wdr8, based upon the structural conservation of the fission yeast counterpart. We showed that Wdr8 constitutively localises to the centrosome and super resolution microscopy uncovered that this protein is enriched at the proximal end of the mother centriole. Furthermore, we identified hMsd1/SSX2IP, a conserved spindle anchoring protein, as one of Wdr8 interactors by mass spectrometry. Wdr8 formed a complex and partially colocalised with hMsd1/SSX2IP. Intriguingly, knockdown of Wdr8 or hMsd1/SSX2IP displayed very similar mitotic defects, in which spindle microtubules became shortened and misoriented. Indeed, Wdr8 depletion resulted in the reduced recruitment of hMsd1/SSX2IP to the mitotic centrosome, though the converse is not true. Together, we propose that the conserved Wdr8-hMsd1/SSX2IP complex plays a critical role in controlling proper spindle length and orientation. PMID:26545777

53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.

PubMed

Fong, Chii Shyang; Mazo, Gregory; Das, Tuhin; Goodman, Joshua; Kim, Minhee; O'Rourke, Brian P; Izquierdo, Denisse; Tsou, Meng-Fu Bryan

2016-07-02

Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency.

Perivascular epithelioid cell tumor of the descending colon mimicking a gastrointestinal stromal tumor: a case report.

PubMed

Iwamoto, Ryuta; Kataoka, Tatsuki R; Furuhata, Ayako; Ono, Kazuo; Hirota, Seiichi; Kawada, Kenji; Sakai, Yoshiharu; Haga, Hironori

2016-11-14

We present a case of perivascular epithelioid cell tumor (PEComa), which clinically and histologically mimics a gastrointestinal stromal tumor (GIST). A 42-year-old woman was found to have a mass in the left flank during her annual medical checkup. Computed tomography examination revealed a submucosal tumor of the descending colon. Surgeons and radiologists suspected that the lesion was a GIST, and left hemicolectomy was performed without biopsy. Microscopic examination showed that the lesion was composed of spindle and epithelioid cells, which were immunohistochemically negative for c-kit and positive for platelet-derived growth factor receptor (PDGFR) α. Initial diagnosis of PDGFRα-positive GIST was made. However, gene analysis did not reveal mutations in PDGFRα. Additional immunohistochemistry showed that tumor cells were positive for human melanin black 45 (HMB45), melanA, and the myogenic marker calponin. A final diagnosis of PEComa was made. PEComa should be included in the differential diagnosis of PDGFRα-positive spindle cell tumors in the wall of the gastrointestinal tract.

Urinary bladder urothelial carcinoma with expression of KIT and PDGFRA and showing diverse differentiations into plasmacytoid, clear cell, acantholytic, nested, and spindle variants, and into adenocarcinoma, signet-ring cell carcinoma, small cell carcinoma, large cell carcinoma, and pleomorphic carcinoma.

PubMed

Terada, Tadashi

2013-01-01

Various tumors can arise in the urinary bladder (UB); most common is urothelial carcinoma (UC). UC of the UB have many variants. Other types of carcinomas such as adenocarcinoma (AC) and small cell carcinoma (SmCC) can occur in UB carcinomas. Expression of KIT and PDGFRA has not been reported. A 66-year-old man admitted to our hospital because of hematuria. Cystoscopy revealed papillary invasive tumor and a transurethral bladder tumorectomy (TUR-BT) was performed. The TUR-BT showed UC, AC, SmCC, large cell carcinoma (LCC), and pleomorphic carcinoma (PC). The UC component showed plasmacytoid, spindle, nested, clear cell, acantholytic variants. The AC element showed tubular adenocarcinoma and signet-ring cell carcinoma (Sig). Immunohistochemically, all of these subtypes were positive for cytokeratin (CK) AE1/3, CK CAM5.2, CK34BE12, CK5, CK6, CK7, CK8, CK18, CK19, CK20, EMA, CEA, p63, CA19-9, p53 (positive 45%), MUC1, NSE, NCAM, KIT, PDGFRA, and Ki-67 (87%). They were negative for vimentin, chromogranin, synaptophysin, S100 protein, CD34, CD14, α-smooth muscle actin, CD31, caldesmon, CD138, CD45, κ-chain, λ-chain, MUC2, MUC5AC and MUC6. Mucin histochemistry revealed mucins in AC element including Sig. A molecular genetic analysis using PCR-direct sequencing method identified no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes. The carcinoma was highly aggressive and invaded into muscular layer. The nuclear grade was very high, and there were numerous lymphovascular permeations were seen. The surface showed carcinoma in situ involving von-Brunn's nests. This case shows that carcinoma of UB can show diverse differentiations into numerous histological types and variants, and can express KIT and PDGFRA. The both genes showed no mutations in the present case.

CENP-A regulates chromosome segregation during the first meiosis of mouse oocytes.

PubMed

Li, Li; Qi, Shu-Tao; Sun, Qing-Yuan; Chen, Shi-Ling

2017-06-01

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A (CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Piezoelectric self-sensing actuator for active vibration control of motorized spindle based on adaptive signal separation

NASA Astrophysics Data System (ADS)

He, Ye; Chen, Xiaoan; Liu, Zhi; Qin, Yi

2018-06-01

The motorized spindle is the core component of CNC machine tools, and the vibration of it reduces the machining precision and service life of the machine tools. Owing to the fast response, large output force, and displacement of the piezoelectric stack, it is often used as the actuator in the active vibration control of the spindle. A piezoelectric self-sensing actuator (SSA) can reduce the cost of the active vibration control system and simplify the structure by eliminating the use of a sensor, because a SSA can have both actuating and sensing functions at the same time. The signal separation method of a SSA based on a bridge circuit is widely applied because of its simple principle and easy implementation. However, it is difficult to maintain dynamic balance of the circuit. Prior research has used adaptive algorithm to balance of the bridge circuit on the flexible beam dynamically, but those algorithms need no correlation between sensing and control voltage, which limit the applications of SSA in the vibration control of the rotor-bearing system. Here, the electromechanical coupling model of the piezoelectric stack is established, followed by establishment of the dynamic model of the spindle system. Next, a new adaptive signal separation method based on the bridge circuit is proposed, which can separate relative small sensing voltage from related mixed voltage adaptively. The experimental results show that when the self-sensing signal obtained from the proposed method is used as a displacement signal, the vibration of the motorized spindle can be suppressed effectively through a linear quadratic Gaussian (LQG) algorithm.

TD-60 links RalA GTPase function to the CPC in mitosis

PubMed Central

Papini, Diana; Langemeyer, Lars; Abad, Maria A.; Kerr, Alastair; Samejima, Itaru; Eyers, Patrick A.; Jeyaprakash, A. Arockia; Higgins, Jonathan M. G.; Barr, Francis A.; Earnshaw, William C.

2015-01-01

TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore–microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies. PMID:26158537

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore

PubMed Central

Marquardt, Joseph R.; Perkins, Jennifer L.; Beuoy, Kyle J.; Fisk, Harold A.

2016-01-01

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus. PMID:27339139

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore.

PubMed

Marquardt, Joseph R; Perkins, Jennifer L; Beuoy, Kyle J; Fisk, Harold A

2016-07-12

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus.

Functional Importance of the Anaphase-Promoting Complex-Cdh1-Mediated Degradation of TMAP/CKAP2 in Regulation of Spindle Function and Cytokinesis▿ †

PubMed Central

Hong, Kyung Uk; Park, Young Soo; Seong, Yeon-Sun; Kang, Dongmin; Bae, Chang-Dae; Park, Joobae

2007-01-01

Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G1/S and peaks at G2/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis. PMID:17339342

Functional importance of the anaphase-promoting complex-Cdh1-mediated degradation of TMAP/CKAP2 in regulation of spindle function and cytokinesis.

PubMed

Hong, Kyung Uk; Park, Young Soo; Seong, Yeon-Sun; Kang, Dongmin; Bae, Chang-Dae; Park, Joobae

2007-05-01

Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G(1)/S and peaks at G(2)/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis.

Duplication and Nuclear Envelope Insertion of the Yeast Microtubule Organizing Centre, the Spindle Pole Body.

PubMed

Rüthnick, Diana; Schiebel, Elmar

2018-05-10

The main microtubule organizing centre in the unicellular model organisms Saccharomyces cerevisiae and Schizosaccharomyces pompe is the spindle pole body (SPB). The SPB is a multilayer structure, which duplicates exactly once per cell cycle. Unlike higher eukaryotic cells, both yeast model organisms undergo mitosis without breakdown of the nuclear envelope (NE), a so-called closed mitosis. Therefore, in order to simultaneously nucleate nuclear and cytoplasmic MTs, it is vital to embed the SPB into the NE at least during mitosis, similarly to the nuclear pore complex (NPC). This review aims to embrace the current knowledge of the SPB duplication cycle with special emphasis on the critical step of the insertion of the new SPB into the NE.

Characterization of Topographically Specific Sleep Spindles in Mice

PubMed Central

Kim, Dongwook; Hwang, Eunjin; Lee, Mina; Sung, Hokun; Choi, Jee Hyun

2015-01-01

Study Objective: Sleep spindles in humans have been classified as slow anterior and fast posterior spindles; recent findings indicate that their profiles differ according to pharmacology, pathology, and function. However, little is known about the generation mechanisms within the thalamocortical system for different types of spindles. In this study, we aim to investigate the electrophysiological behaviors of the topographically distinctive spindles within the thalamocortical system by applying high-density EEG and simultaneous thalamic LFP recordings in mice. Design: 32-channel extracranial EEG and 2-channel thalamic LFP were recorded simultaneously in freely behaving mice to acquire spindles during spontaneous sleep. Subjects: Hybrid F1 male mice of C57BL/6J and 129S4/svJae. Measurements and Results: Spindle events in each channel were detected by spindle detection algorithm, and then a cluster analysis was applied to classify the topographically distinctive spindles. All sleep spindles were successfully classified into 3 groups: anterior, posterior, and global spindles. Each spindle type showed distinct thalamocortical activity patterns regarding the extent of similarity, phase synchrony, and time lags between cortical and thalamic areas during spindle oscillation. We also found that sleep slow waves were likely to associate with all types of sleep spindles, but also that the ongoing cortical decruitment/recruitment dynamics before the onset of spindles and their relationship with spindle generation were also variable, depending on the spindle types. Conclusion: Topographically specific sleep spindles show distinctive thalamocortical network behaviors. Citation: Kim D, Hwang E, Lee M, Sung H, Choi JH. Characterization of topographically specific sleep spindles in mice. SLEEP 2015;38(1):85–96. PMID:25325451

Automated frequency analysis of synchronous and diffuse sleep spindles.

PubMed

Huupponen, Eero; Saastamoinen, Antti; Niemi, Jukka; Virkkala, Jussi; Hasan, Joel; Värri, Alpo; Himanen, Sari-Leena

2005-01-01

Sleep spindles have different properties in different localizations in the cortex. First main objective was to develop an amplitude-independent multi-channel spindle detection method. Secondly the method was applied to study the anteroposterior frequency differences of pure synchronous (visible bilaterally, either frontopolarly or centrally) and diffuse (visible bilaterally both frontopolarly and centrally) sleep spindles. A previously presented spindle detector based on the fuzzy reasoning principle and a level detector were combined to form a multi-channel spindle detector. The spindle detector had a 76.17% true positive rate and 0.93% false-positive rate. Pure central spindles were faster and pure frontal spindles were slower than diffuse spindles measured simultaneously from both locations. The study of frequency relations of spindles might give new information about thalamocortical sleep spindle generating mechanisms. Copyright (c) 2005 S. Karger AG, Basel.

Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

PubMed Central

Quemin, Emmanuelle R. J.; Pietilä, Maija K.; Oksanen, Hanna M.; Forterre, Patrick; Rijpstra, W. Irene C.; Schouten, Stefan; Bamford, Dennis H.; Prangishvili, David

2015-01-01

ABSTRACT Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions. PMID:26355093

Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans

PubMed Central

Crowder, Marina E.; Flynn, Jonathan R.; McNally, Karen P.; Cortes, Daniel B.; Price, Kari L.; Kuehnert, Paul A.; Panzica, Michelle T.; Andaya, Armann; Leary, Julie A.; McNally, Francis J.

2015-01-01

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin. PMID:26133383

Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system

PubMed Central

1981-01-01

I microinjected calcium ions into echinoderm eggs during mitosis to determine the calcium sensitivity of microtubules (Mts) in vivo. Spindle birefringence (BR), a measure of the number of aligned Mts in the spindle, is locally, rapidly, and reversibly abolished by small volumes of microinjected CaCl2 (1 mM). Rapid return of BR is followed by anaphase, and subsequent divisions are normal. Similar doses of MgCl2, BaCl2, KCl, NaCl, pH buffers, distilled water, or vegetable oil have no effect on spindle BR, whereas large doses of such agents sometimes cause slow, uniform loss in BR over the course of a minute or more. Of the ions tested, only Sr++ causes effects comparable to Ca++. Ca-EGTA buffers, containing greater than micromolar free Ca++, abolishes BR in a manner similar to millimolar concentrations of injected CaCl2. Caffeine, a potent uncoupler of the Ca++-pump/ATPase of sarcoplasmic reticulum, causes a local, transient depression in spindle BR in the injected region. Finally, injection of potassium oxalate results in the formation of small, highly BR crystals, presumably CA- oxalate, in Triton-sensitive compartments in the cytoplasm. Taken together, these findings demonstrate that spindle Mts are sensitive to levels of free Ca++ in the physiological range, provide evidence for the existence of a strong cytoplasmic Ca++-sequestering system, and support the notion that Mt assembly and disassembly in local regions of the spindle may be orchestrated by local changes in the cytoplasmic free Ca++ concentration during mitosis. An appendix offers the design of a new chamber for immobilizing echinoderm eggs for injection, a new method for determining the volume of the injected solution, and a description of the microinjection technique, which was designed, but never fully described, by Hiramoto (Y. Hiramoto, Exp. Cell. Res., 1962, 27:416-426.). PMID:7194345

Mps1 as a link between centrosomes and genomic instability.

PubMed

Kasbek, Christopher; Yang, Ching-Hui; Fisk, Harold A

2009-10-01

Centrosomes are microtubule-organizing centers that must be precisely duplicated before mitosis. Centrosomes regulate mitotic spindle assembly, and the presence of excess centrosomes leads to the production of aberrant mitotic spindles which generate chromosome segregation errors. Many human tumors possess excess centrosomes that lead to the production of abnormal spindles in situ. In some tumors, these extra centrosomes appear before aneuploidy, suggesting that defects in centrosome duplication might promote genomic instability and tumorigenesis. The Mps1 protein kinase is required for centrosome duplication, and preventing the proteasome-dependent degradation of Mps1 at centrosomes increases its local concentration and causes the production of excess centrosomes during a prolonged S-phase. Here, we show that Mps1 degradation is misregulated in two tumor-derived cell lines, and that the failure to appropriately degrade Mps1 correlates with the ability of these cells to produce extra centrosomes during a prolonged S-phase. In the 21NT breast-tumor derived cell line, a mutant Mps1 protein that is normally constitutively degraded can accumulate at centrosomes and perturb centrosome duplication, suggesting that these cells have a defect in the mechanisms that target Mps1 to the proteasome. In contrast, the U2OS osteosarcoma cell line expresses a nondegradable form of Mps1, which we show causes the dose-dependent over duplication of centrioles even at very low levels of expression. Our data demonstrate that defects in Mps1 degradation can occur through multiple mechanisms, and suggest that Mps1 may provide a link between the control of centrosome duplication and genomic instability. (c) 2009 Wiley-Liss, Inc.

A new theory of the origin of cancer: quantum coherent entanglement, centrioles, mitosis, and differentiation.

PubMed

Hameroff, Stuart R

2004-11-01

Malignant cells are characterized by abnormal segregation of chromosomes during mitosis ("aneuploidy"), generally considered a result of malignancy originating in genetic mutations. However, recent evidence supports a century-old concept that maldistribution of chromosomes (and resultant genomic instability) due to abnormalities in mitosis itself is the primary cause of malignancy rather than a mere byproduct. In normal mitosis chromosomes replicate into sister chromatids which are then precisely separated and transported into mirror-like sets by structural protein assemblies called mitotic spindles and centrioles, both composed of microtubules. The elegant yet poorly understood ballet-like movements and geometric organization occurring in mitosis have suggested guidance by some type of organizing field, however neither electromagnetic nor chemical gradient fields have been demonstrated or shown to be sufficient. It is proposed here that normal mirror-like mitosis is organized by quantum coherence and quantum entanglement among microtubule-based centrioles and mitotic spindles which ensure precise, complementary duplication of daughter cell genomes and recognition of daughter cell boundaries. Evidence and theory supporting organized quantum states in cytoplasm/nucleoplasm (and quantum optical properties of centrioles in particular) at physiological temperature are presented. Impairment of quantum coherence and/or entanglement among microtubule-based mitotic spindles and centrioles can result in abnormal distribution of chromosomes, abnormal differentiation and uncontrolled growth, and account for all aspects of malignancy. New approaches to cancer therapy and stem cell production are suggested via non-thermal laser-mediated effects aimed at quantum optical states of centrioles.

P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation

PubMed Central

Lee, Kyunghee; Kenny, Alison E.

2010-01-01

Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by ∼40% in nontransformed human RPE-1, ∼80% in PtK2 (rat kangaroo), and ∼25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends. PMID:20462950

Truncation of POC1A associated with short stature and extreme insulin resistance.

PubMed

Chen, Jian-Hua; Segni, Maria; Payne, Felicity; Huang-Doran, Isabel; Sleigh, Alison; Adams, Claire; Savage, David B; O'Rahilly, Stephen; Semple, Robert K; Barroso, Inês

2015-10-01

We describe a female proband with primordial dwarfism, skeletal dysplasia, facial dysmorphism, extreme dyslipidaemic insulin resistance and fatty liver associated with a novel homozygous frameshift mutation in POC1A, predicted to affect two of the three protein products of the gene. POC1A encodes a protein associated with centrioles throughout the cell cycle and implicated in both mitotic spindle and primary ciliary function. Three homozygous mutations affecting all isoforms of POC1A have recently been implicated in a similar syndrome of primordial dwarfism, although no detailed metabolic phenotypes were described. Primary cells from the proband we describe exhibited increased centrosome amplification and multipolar spindle formation during mitosis, but showed normal DNA content, arguing against mitotic skipping, cleavage failure or cell fusion. Despite evidence of increased DNA damage in cells with supernumerary centrosomes, no aneuploidy was detected. Extensive centrosome clustering both at mitotic spindles and in primary cilia mitigated the consequences of centrosome amplification, and primary ciliary formation was normal. Although further metabolic studies of patients with POC1A mutations are warranted, we suggest that POC1A may be added to ALMS1 and PCNT as examples of centrosomal or pericentriolar proteins whose dysfunction leads to extreme dyslipidaemic insulin resistance. Further investigation of links between these molecular defects and adipose tissue dysfunction is likely to yield insights into mechanisms of adipose tissue maintenance and regeneration that are critical to metabolic health. © 2015 Society for Endocrinology.