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.

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.

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.

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

Zhang Bo; Huang Bo; School of Public Health, University of South China, Hengyang, Hunan 421001

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe,more » such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3{sigma} and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: > 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. > Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. > The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. > The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.« less

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.

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.

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.

Anti-Hu Antibody Associated Paraneoplastic Cerebellar Degeneration in Head and Neck Cancer.

PubMed

Huemer, Florian; Melchardt, Thomas; Tränkenschuh, Wolfgang; Neureiter, Daniel; Moser, Gerhard; Magnes, Teresa; Weiss, Lukas; Schlattau, Alexander; Hufnagl, Clemens; Ricken, Gerda; Höftberger, Romana; Greil, Richard; Egle, Alexander

2015-12-22

Paraneoplastic syndromes are most frequently associated with small cell lung carcinoma, hematologic and gynecologic malignancies while reports in head and neck cancer are rare. We present the case of a 60-year old female patient who developed paraneoplastic cerebellar degeneration upon locoregional recurrence of a poorly differentiated spindle cell carcinoma of the nasal cavity and paranasal sinus. The neurological symptoms, especially ataxia, stabilized after resection of tumor recurrence and concomitant chemoradiotherapy whereas anti-Hu-antibodies remained positive. Despite the unfavorable prognosis of paraneoplastic neurological disorders associated with onconeural antibodies, the patient achieved long-standing stabilization of neurological symptoms. We report the first patient with anti-Hu antibodies and paraneoplastic cerebellar degeneration associated with a spindle cell carcinoma of the head and neck. We recommend that evaluation of neurological symptoms in patients with this tumor entity should also include paraneoplastic syndromes as differential diagnoses and suggest early extensive screening for onconeural antibodies.

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

Well-differentiated liposarcoma of the tongue.

PubMed

Nunes, F D; Loducca, S V L; de Oliveira, E M F; de Araújo, V C

2002-01-01

Intraoral liposarcomas are rare, with most reported cases being of the myxoid histological type. We present a well-differentiated liposarcoma of the tongue, in a 65-year-old man. The tumour presented lipoblasts in various stages of differentiation, lipocytes in different sizes and shapes, mesenchymal and signet-ring cells. Lipoma, spindle-cell lipoma, myxoma, hibernoma, angiolipoma, fibrolipoma, pseudosarcomatous faciitis and malignant hysticytoma were considered in the diagnosis process. The patient was treated surgically and so far is free of disease.

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.

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.

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

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.

The Spindle Cell Neoplasms of the Oral Cavity.

PubMed

Shamim, Thorakkal

2015-01-01

Spindle cell neoplasms are defined as neoplasms that consist of spindle-shaped cells in the histopathology. Spindle cell neoplasms can affect the oral cavity. In the oral cavity, the origin of the spindle cell neoplasms may be traced to epithelial, mesenchymal and odontogenic components. This article aims to review the spindle cell neoplasms of the oral cavity with emphasis on histopathology.

The Spindle Cell Neoplasms of the Oral Cavity

PubMed Central

Shamim, Thorakkal

2015-01-01

Spindle cell neoplasms are defined as neoplasms that consist of spindle-shaped cells in the histopathology. Spindle cell neoplasms can affect the oral cavity. In the oral cavity, the origin of the spindle cell neoplasms may be traced to epithelial, mesenchymal and odontogenic components. This article aims to review the spindle cell neoplasms of the oral cavity with emphasis on histopathology. PMID:26351482

[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.

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.

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.

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

Gliosarcomas arising from the pineal gland region: uncommon localization and rare tumors.

PubMed

Sugita, Yasuo; Terasaki, Mizuhiko; Tanigawa, Ken; Ohshima, Koichi; Morioka, Motohiro; Higaki, Koichi; Nakagawa, Setsuko; Shimokawa, Shoko; Nakashima, Susumu

2016-02-01

Gliosarcomas are a variant of glioblastomas and present a biphasic pattern, with coexisting glial and mesenchymal components. In this study, two unusual cases are presented. Case 1 is a 52-year-old woman with a headache and memory disturbance for a month. Case 2 is an 18-year-old man with a headache lasting two weeks. In both cases, an MRI revealed enhancing T1-low to iso, T2-iso to high intensity lesions in the pineal gland region. Histologically, in case 1, the tumor showed spindle cell proliferation with disorganized fascicles and cellular pleomorphism. Tumor cells variously exhibited oncocytic transformation. Immunohistochemically, most of the spindle tumor cells were positive for myoglobin and desmin. Some of the tumor cells were positive for GFAP and S-100 protein. On the other hand, all tumor cells were positive for CD133, Musashi1, and SOX-2 which are the markers of neural stem cells. In case 2, the tumor showed monotonous proliferation of short spindle cells with disorganized fascicles and cellular atypism. The morphological distinction between glial and mesenchymal components was not apparent. Immunohistochemically, most of the spindle tumor cells were positive for desmin. Glial tumor cells that were dispersed within the sarcoma as single cells were positive for GFAP. In addition, all tumor cells were positive for CD133, Musashi1 and SOX-2. Based on these microscopic appearances, and immunohistochemical findings, these cases were diagnosed as gliosarcomas arising from the pineal gland region. These results also indicated that pluripotential cancer stem cells differentiated into glial and muscle cell lines at the time of tumor growth. In a survey of previous publications on gliosarcoma arising from the pineal gland, these cases are the second and third reports found in English scientific writings. © 2015 Japanese Society of Neuropathology.

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.

[Soft tissue sarcomas: the role of histology and molecular pathology for differential diagnosis].

PubMed

Poremba, C

2006-01-01

Soft tissue sarcomas include a wide spectrum of different entities. The so-called small round blue cell tumors and spindle cell tumors are difficult to classify based solely on conventional histology. To identify different subtypes of tumors special histochemical and immunohistochemical techniques are necessary. Analysis of protein expression by immunohistochemistry provides a helpful tool to investigate the histogenesis of tumors. A basic spectrum of antibodies should be included to study these tumors: Desmin and myogenin (or MyoD1) for skeletal differentiation; S-100, NSE, CD56, and synaptophysin for neural/neuroendocrine differentiation; CD3, CD20, and CD79 alpha for malignant lymphomas; CD34, sm-actin, and beta-catenin for spindle cell tumors; additional antigens, e. g. Ki-67 and p 53, for estimation of proliferation and tumor suppressor gene malfunctions. Nevertheless, the molecular analysis of soft tissue sarcomas is necessary for demonstration of specific translocations or gene defects to specify and proof a diagnosis. For this purpose, RT-PCR for RNA expression analysis of gene fusion transcripts and multi-color FISH for analysis of chromosomal rearrangements are used. Further investigations, using DNA microrrays may help to subclassify such tumors, with respect to prognosis or prediction of therapeutic response.

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.

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

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.

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.

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.

CD34-reactive fibrous papule of the nose.

PubMed

Shea, C R; Salob, S; Reed, J A; Lugo, J; McNutt, N S

1996-08-01

In human skin, the CD34 antigen is expressed on endothelium, periadnexal cells, and a population of reticular dermal interstitial cells. CD34 expression is characteristic of dermatofibrosarcoma protuberans and several other neoplasms, but not of typical fibrous papules of the nose. We describe a 16-year-old white girl with a slowly growing papule on the nose. Histopathology showed a dermal tumor with a superficial component of branched, thin-walled blood vessels and a deeper component of benign-appearing, spindle-shaped cells. These cells uniformly and strongly expressed CD34, but not factor XIIIa or markers of melanocytic, neural, muscular, vascular, or histiocytic differentiation. We consider this lesion a CD34-reactive fibrous papule. This benign tumor must be clearly distinguished from dermatofibrosarcoma protuberans, which also is composed of bundles of CD34-reactive spindle-shaped cells in most cases but has locally aggressive behavior.

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.

Tumor Cell Plasticity in Uveal Melanoma

PubMed Central

Folberg, Robert; Arbieva, Zarema; Moses, Jonas; Hayee, Amin; Sandal, Tone; Kadkol, ShriHari; Lin, Amy Y.; Valyi-Nagy, Klara; Setty, Suman; Leach, Lu; Chévez-Barrios, Patricia; Larsen, Peter; Majumdar, Dibyen; Pe’er, Jacob; Maniotis, Andrew J.

2006-01-01

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment. PMID:17003493

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.

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.

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

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.

Connections between cadherin-catenin proteins, spindle misorientation, and cancer

PubMed Central

Shahbazi, Marta N; Perez-Moreno, Mirna

2015-01-01

Cadherin-catenin mediated adhesion is an important determinant of tissue architecture in multicellular organisms. Cancer progression and maintenance is frequently associated with loss of their expression or functional activity, which not only leads to decreased cell-cell adhesion, but also to enhanced tumor cell proliferation and loss of differentiated characteristics. This review is focused on the emerging implications of cadherin-catenin proteins in the regulation of polarized divisions through their connections with the centrosomes, cytoskeleton, tissue tension and signaling pathways; and illustrates how alterations in cadherin-catenin levels or functional activity may render cells susceptible to transformation through the loss of their proliferation-differentiation balance. PMID:26451345

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.

MUC4, a novel immunohistochemical marker identified by gene expression profiling, differentiates pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

PubMed

Amatya, Vishwa Jeet; Kushitani, Kei; Mawas, Amany Sayed; Miyata, Yoshihiro; Okada, Morihito; Kishimoto, Takumi; Inai, Kouki; Takeshima, Yukio

2017-05-01

Sarcomatoid mesothelioma, a histological subtype of malignant pleural mesothelioma, is a very aggressive tumor with a poor prognosis. Histological diagnosis of sarcomatoid mesothelioma largely depends on the histomorphological feature of spindled tumor cells with immunohistochemical reactivity to cytokeratins. Diagnosis also requires clinico-radiological and/or macroscopic evidence of an extrapulmonary location to differentiate it from lung sarcomatoid carcinoma. Although there are promising immunohistochemical antibody panels to differentiate mesothelioma from lung carcinoma, a consensus on the immunohistochemical markers that distinguish sarcomatoid mesothelioma from lung sarcomatoid carcinoma has not been reached and requires further study. We performed whole gene expression analysis of formalin-fixed paraffin-embedded tissue from sarcomatoid mesothelioma and lung sarcomatoid carcinoma and observed significant differences in the expression of MUC4 and other genes between sarcomatoid mesothelioma and lung sarcomatoid carcinoma. Immunohistochemistry demonstrated that MUC4 was expressed in the spindled tumor cells of lung sarcomatoid carcinoma (21/29, 72%) but was not expressed in any sarcomatoid mesothelioma (0/31, 0%). To differentiate sarcomatoid mesothelioma from lung sarcomatoid carcinoma, negative MUC4 expression showed 100% sensitivity and 72% specificity and accuracy rate of 87%, which is higher than immunohistochemical markers such as calretinin, D2-40 and Claudin-4. Therefore, we recommend to include MUC4 as a novel and useful negative immunohistochemical marker for differentiating sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

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.

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.

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.

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.

Regulation of spindle integrity and mitotic fidelity by BCCIP

PubMed Central

Huhn, S C; Liu, J; Ye, C; Lu, H; Jiang, X; Feng, X; Ganesan, S; White, E; Shen, Z

2017-01-01

Centrosomes together with the mitotic spindle ensure the faithful distribution of chromosomes between daughter cells, and spindle orientation is a major determinant of cell fate during tissue regeneration. Spindle defects are not only an impetus of chromosome instability but are also a cause of developmental disorders involving defective asymmetric cell division. In this work, we demonstrate BCCIP, especially BCCIPα, as a previously unidentified component of the mitotic spindle pole and the centrosome. We demonstrate that BCCIP localizes proximal to the mother centriole and participates in microtubule organization and then redistributes to the spindle pole to ensure faithful spindle architecture. We find that BCCIP depletion leads to morphological defects, disoriented mitotic spindles, chromosome congression defects and delayed mitotic progression. Our study identifies BCCIP as a novel factor critical for microtubule regulation and explicates a mechanism utilized by BCCIP in tumor suppression. PMID:28394342

Cutaneous Clear Cell Sarcoma: a clinicopathologic, immunohistochemical and molecular analysis of 12 cases emphasizing its distinction from dermal melanoma

PubMed Central

Hantschke, Markus; Mentzel, Thomas; Rütten, Arno; Palmedo, Gabriele; Calonje, Eduardo; Lazar, Alexander J.; Kutzner, Heinz

2010-01-01

Clear cell sarcoma (CCS) of tendons and aponeuroses / malignant melanoma of soft parts is a rare tumor and in the majority of cases presents a characteristic reciprocal translocation t(12;22)(q13;q12) that results in fusion of the EWS and ATF1 genes. Although the melanocytic differentiation of CCS is indisputable, its precise lineage remains unclear. Typically, the slowly growing tumor affects the extremities of adolescents or young adults, especially around the ankle and foot. CCS is classically regarded as a deep soft tissue tumor associated with tendons or aponeuroses. This traditional view is put into perspective by the description of primary CCS of the gastrointestinal tract, which may have a variant fusion gene EWSR1-CREB1. We describe 12 cases of cutaneous CCS and discuss the differential diagnoses. These 12 cases share an identical immunohistochemical profile with malignant melanoma (MM) and thus can easily be confused with a dermal variant of spindle cell MM or metastasis of MM. The patients' ages ranged from 6 to 74 years (median: 25 y), and there was a female predominance (10 females, 2 males). Most tumors (n = 9) were located on the extremities, 2 tumors arose on the back, and 1 on the abdomen. The mean tumor size was 0,97 cm (range, 0,4 to 1,7 cm). Six cases showed invasion of the subcutis, the other 6 cases were entirely dermal. Tumor necrosis was evident in 2 cases, melanin pigment in 2 cases, and ulceration in 1 tumor. All cases showed uniform nests and fascicles of pale spindled or slightly epitheloid cells with finely granular eosinophilic or clear cytoplasm. There was fair pleomorphism with plump spindled nuclei and significantly prominent nucleoli. Multinucleated wreath-like tumor giant cells were observed in two-thirds of cases, but were usually present only focally. The dense cellular aggregates were encased by delicate fibrous septa. The stroma showed a sclerotic reticulated pattern. Partly, the nests of spindle cells bordered the epidermis, prima vista mimicking junctional nests of melanocytes. The specific translocation pattern was confirmed in all cases by fluorescence in situ hybridization. Local recurrences and metastases developed in 2 and 3 patients, respectively, and 1 patient was dead of the disease. PMID:20087159

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.

Inhibition of Bcl-xL sensitizes cells to mitotic blockers, but not mitotic drivers

PubMed Central

Bennett, Ailsa; Sloss, Olivia; Topham, Caroline; Nelson, Louisa; Tighe, Anthony

2016-01-01

Cell fate in response to an aberrant mitosis is governed by two competing networks: the spindle assembly checkpoint (SAC) and the intrinsic apoptosis pathway. The mechanistic interplay between these two networks is obscured by functional redundancy and the ability of cells to die either in mitosis or in the subsequent interphase. By coupling time-lapse microscopy with selective pharmacological agents, we systematically probe pro-survival Bcl-xL in response to various mitotic perturbations. Concentration matrices show that BH3-mimetic-mediated inhibition of Bcl-xL synergises with perturbations that induce an SAC-mediated mitotic block, including drugs that dampen microtubule dynamics, and inhibitors targeting kinesins and kinases required for spindle assembly. By contrast, Bcl-xL inhibition does not synergize with drugs which drive cells through an aberrant mitosis by overriding the SAC. This differential effect, which is explained by compensatory Mcl-1 function, provides opportunities for patient stratification and combination treatments in the context of cancer chemotherapy. PMID:27512141

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.

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.

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

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

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

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.

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.

Mechanical design principles of a mitotic spindle.

PubMed

Ward, Jonathan J; Roque, Hélio; Antony, Claude; Nédélec, François

2014-12-18

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This 'pushing' mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindle's fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length.

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.

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.

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.

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.

Robust control of mitotic spindle orientation in the developing epidermis

PubMed Central

Poulson, Nicholas D.

2010-01-01

Progenitor cells must balance self-amplification and production of differentiated progeny during development and homeostasis. In the epidermis, progenitors divide symmetrically to increase surface area and asymmetrically to promote stratification. In this study, we show that individual epidermal cells can undergo both types of division, and therefore, the balance is provided by the sum of individual cells’ choices. In addition, we define two control points for determining a cell’s mode of division. First is the expression of the mouse Inscuteable gene, which is sufficient to drive asymmetric cell division (ACD). However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, we show that p63, a transcriptional regulator of stratification, does not control either of these processes. These data have uncovered two important regulatory points controlling ACD in the epidermis and allow a framework for analysis of how external cues control this important choice. PMID:21098114

Embryonal rhabdomyosarcoma in an immature Baird's tapir (Tapirus bairdii).

PubMed

Bonar, Christopher J; Lewandowski, Albert H; Skowronek, Anthony J

2007-03-01

An immature Baird's tapir (Tapirus bairdii) with a history of seizure-like episodes developed signs of respiratory disease. The initial clinical diagnosis was pneumonia, and antibiotic therapy was started. The animal failed to improve after 14 days of therapy and developed unilateral, bloody nasal discharge. Endoscopic examination and radiography revealed a soft tissue mass in the nasopharynx depressing the soft palate. The tapir died 32 days after initial presentation. Histologic examination of the mass demonstrated a mesenchymal tumor composed of spindle cells with elongate nuclei forming densely packed fascicles. The neoplastic spindle cells showed prominent cross-striations. Immunohistochemistry revealed the cells to be positive for desmin and myoglobin, but negative for smooth muscle actin, confirming diagnosis of rhabdomyosarcoma. Embryonal rhabdomyosarcoma is the most common nasopharyngeal soft tissue tumor of humans, and it has been reported infrequently in dogs, horses, and pigs. Neoplasia should be a differential diagnosis in cases of unilateral nasal discharge and inspiratory stridor, even in young animals.

Malignant perivascular epithelioid cell tumor of the retroperitoneum

PubMed Central

Wu, Ji-Hua; Zhou, Jin-Lian; Cui, Yan; Jing, Qing-Ping; Shang, Le; Zhang, Jian-Zhong

2013-01-01

Perivascular epithelioid cell tumors (PEComas) are a rare type of mesenchymal neoplasms characterized by a proliferation of perivascular cells with an epithelioid phenotype and expression of myo-melanocytic markers. The majority of PEComas seem to be benign and usually their prognosis is good. Malignant cases are extremely rare, exhibiting a malignant course with local recurrences and distant metastases. We herein report a case of a malignant PEComa arising in the retroperitoneum. The patient was a 55-year-old woman experiencing abdominal discomfort for approximately one month. Ultrasound and computer tomography (CT) scans of the abdomen revealed a solid mass arising from the retroperitoneum. Microscopically, the tumor was composed of epithelioid cells mixed with spindled cells. The nucleus had significant atypia, and the mitoses were obvious. The focal intravascular tumor embolus was visible. Immunohistochemically, the epithelioid tumor cells were positive for HMB45 and Melan-A, and the spindled tumor celLs were positive for SMA and desmin. Seven months after a surgical resection, an ultrasound revealed liver metastases. In conclusion, the malignant PEComas of the retroperitoneum is a very rare neoplasm with unique morphological and immunohistochemical characteristics. It should be differentiated from other epithelioid cell tumors of the retroperitoneum. PMID:24133607

Malignant perivascular epithelioid cell tumor of the retroperitoneum.

PubMed

Wu, Ji-Hua; Zhou, Jin-Lian; Cui, Yan; Jing, Qing-Ping; Shang, Le; Zhang, Jian-Zhong

2013-01-01

Perivascular epithelioid cell tumors (PEComas) are a rare type of mesenchymal neoplasms characterized by a proliferation of perivascular cells with an epithelioid phenotype and expression of myo-melanocytic markers. The majority of PEComas seem to be benign and usually their prognosis is good. Malignant cases are extremely rare, exhibiting a malignant course with local recurrences and distant metastases. We herein report a case of a malignant PEComa arising in the retroperitoneum. The patient was a 55-year-old woman experiencing abdominal discomfort for approximately one month. Ultrasound and computer tomography (CT) scans of the abdomen revealed a solid mass arising from the retroperitoneum. Microscopically, the tumor was composed of epithelioid cells mixed with spindled cells. The nucleus had significant atypia, and the mitoses were obvious. The focal intravascular tumor embolus was visible. Immunohistochemically, the epithelioid tumor cells were positive for HMB45 and Melan-A, and the spindled tumor celLs were positive for SMA and desmin. Seven months after a surgical resection, an ultrasound revealed liver metastases. In conclusion, the malignant PEComas of the retroperitoneum is a very rare neoplasm with unique morphological and immunohistochemical characteristics. It should be differentiated from other epithelioid cell tumors of the retroperitoneum.

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

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.

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.

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

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.

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.

Numerous eosinophilic globules (skeinoid fibers) in a duodenal stromal tumor: an exceptional case showing smooth muscle differentiation.

PubMed

Matsukuma, S; Doi, M; Suzuki, M; Ikegawa, K; Sato, K; Kuwabara, N

1997-11-01

A unique case of duodenal stromal tumor in a 51-year-old man is reported. The tumor histologically showed spindle cell proliferation and numerous eosinophilic globules. Most globules were composed of tangled 45 nm thick fibrils, which were ultrastructurally identical to 'skeinoid fibers'. The presence of glycogen granules in the tumor cells and the immunoreactivity for alpha-smooth muscle actin suggested smooth muscle differentiation. Focal ultrastructural findings also supported the smooth muscle nature of this tumor. There were no immunohistochemical and ultrastructural features indicating neural differentiation. In previous studies, the presence of such 'skeinoid fibers' was suggested to be a histological marker for neural differentiation in gastrointestinal stromal tumor. However, the findings in the present case suggest that numerous 'skeinoid fibers' can be identified in duodenal stromal tumor with smooth muscle differentiation, although this condition may be rare.

Chromokinesin Kid and kinetochore kinesin CENP-E differentially support chromosome congression without end-on attachment to microtubules.

PubMed

Iemura, Kenji; Tanaka, Kozo

2015-03-06

Chromosome congression is the alignment of chromosomes at the spindle equator, and is a prerequisite for faithful chromosome segregation. Recent data suggest that before kinetochores attach to the end of microtubules (end-on attachment), chromosomes can move along microtubules towards the spindle equator through attachment of kinetochores to the lateral surface of microtubules (lateral attachment). Here we address this mechanism, focusing on the contribution of two mitotic motors, Kid and CENP-E. In cells depleted of Hec1, which is essential for end-on attachment, chromosomes show partial and transient congression. This transient congression is further perturbed by co-depletion of Kid, suggesting its role in chromosome congression. In comparison, CENP-E suppresses chromosome congression, probably by tethering kinetochores to short, unstable microtubules, and works in congression only when microtubules are stabilized. Our results may reflect the differential contributions of Kid and CENP-E in chromosome congression in physiological conditions where stabilized microtubules are becoming increased.

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

Model of myosin recruitment to the cell equator for cytokinesis: feedback mechanisms and dynamical regimes

NASA Astrophysics Data System (ADS)

Veksler, Alexander; Vavylonis, Dimitrios

2011-03-01

The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During animal cell cytokinesis, cortical myosin filaments (MF) disassemble at the flanking regions and concentrate in the equator. This recruitment depends on myosin motor activity and the Rho proteins that regulate MF assembly and disassembly. Central spindle and astral microtubules help establish a spatial pattern of differential Rho activity. We propose a reaction-diffusion model for the dynamics of MF recruitment to the equatorial region. In the model, the central spindle and mechanical stress promote self-reinforcing MF assembly. Negative feedback is introduced by MF-induced recruitment of inhibitor myosin phosphatase. Our model yields various dynamical regimes and explains both the recruitment of MF to the cleavage furrow and the observed damped MF oscillations in the flanking regions, as well as steady MF assembly. Space and time parameters of MF oscillations are calculated. We predict oscillatory relaxation of cortical MF upon removal of locally-applied external stress.

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.

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

Mechanical design principles of a mitotic spindle

PubMed Central

Ward, Jonathan J; Roque, Hélio; Antony, Claude; Nédélec, François

2014-01-01

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This ‘pushing’ mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindle's fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length. DOI: http://dx.doi.org/10.7554/eLife.03398.001 PMID:25521247

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.

Recognition of extremophilic archaeal viruses by eukaryotic cells: a promising nanoplatform from the third domain of life

PubMed Central

Uldahl, Kristine Buch; Wu, Linping; Hall, Arnaldur; Papathanasiou, Pavlos; Peng, Xu; Moghimi, Seyed Moein

2016-01-01

Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration of novel medical nanoplatforms. Here, we selected two archaeal viruses Sulfolobus monocaudavirus 1 (SMV1) and Sulfolobus spindle shaped virus 2 (SSV2) owing to their unique spindle shape, hyperthermostable and acid-resistant nature and studied their interaction with mammalian cells. Accordingly, we followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path for selective cell targeting. On internalization, both viruses localize to the lysosomal compartments. Neither SMV1, nor SSV2 induced any detrimental effect on cell morphology, plasma membrane and mitochondrial functionality. This is the first study demonstrating recognition of archaeal viruses by eukaryotic cells which provides good basis for future exploration of archaeal viruses in bioengineering and development of multifunctional vectors. PMID:27892499

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

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

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.

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.

Morphological and immunohistochemical diversity of endometrial stromal sarcoma in rats.

PubMed

Kumabe, Shino; Sato, Junko; Tomonari, Yuki; Takahashi, Miwa; Inoue, Kaoru; Yoshida, Midori; Doi, Takuya; Wako, Yumi; Tsuchitani, Minoru

2018-04-01

To clarify the histopathological characteristics of rat endometrial stromal sarcoma (ESS), we morphologically reviewed 12 malignant uterine tumors protruding into the lumen in previous rat carcinogenicity studies. The 12 cases were classified into the following 6 types based on their morphological features: spindle cell and collagen rich type, pleomorphic/spindle cell and compact type, decidual alteration type, histiocytic and multinucleated giant cell mixture type, Antoni A-type schwannoma type, and Antoni B-type schwannoma type. Immunohistochemically, tumor cells in all cases exhibited focal or diffuse positive reactions for vimentin, and 11 of the 12 cases were positive for S-100. Interestingly, 9 cases were positive for desmin or αSMA, indicating tumor cells expressing smooth muscle properties. Both Antoni A- and B-type schwannoma types showed low reactions for both muscle markers. Positive results for estrogen receptor α in the 11 cases suggested that they were derived from endometrial stromal cells. On the basis of their immunohistochemical profiles, they were considered to be derived from endometrial stromal cells while they showed morphological variation. The detection of a basement membrane surrounding tumor cells might not be a definitive indicator for differential diagnosis of ESS from malignant schwannoma. In conclusion, ESS could exhibit wide morphological and immunohistochemical variation including features of schwannoma or smooth muscle tumor.

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

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

PubMed Central

Mossel, Eric C.; Wang, Jieru; Jeffers, Scott; Edeen, Karen E.; Wang, Shuanglin; Cosgrove, Gregory P.; Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Pearson, Leonard D.; Holmes, Kathryn V.; Mason, Robert J.

2008-01-01

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 hours and peaked at approximately 105 pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection. PMID:18022664

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

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.

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.

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

Balloon cell nevus of the iris.

PubMed

Morcos, Mohib W; Odashiro, Alexandre; Bazin, Richard; Pereira, Patricia Rusa; O'Meara, Aisling; Burnier, Miguel N

2014-12-01

Balloon cell nevus is a rare histopathological lesion characterized by a predominance of large, vesicular and clear cells, called balloon cells. There is only 1 case of balloon cell nevus of the iris reported in the literature. A 55 year-old man presented a pigmented elevated lesion in the right iris since the age of 12 years old. The lesion had been growing for the past 2 years and excision was performed. Histopathological examination showed a balloon cell nevus composed of clear and vacuolated cells without atypia. A typical spindle cell nevus of the iris was also observed. The differential diagnosis included xanthomatous lesions, brown adipocyte or other adipocytic lesions, clear cell hidradenoma, metastatic clear cell carcinoma of the kidney and clear cell sarcoma. The tumor was positive for Melan A, S100 protein and HMB45. Balloon cell nevus of the iris is rare but should be considered in the differential diagnosis of melanocytic lesions of the iris. Copyright © 2014 Elsevier GmbH. All rights reserved.

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.

Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning

PubMed Central

Manatschal, Cristina; Farcas, Ana-Maria; Degen, Miriam Steiner; Bayer, Mathias; Kumar, Anil; Landgraf, Christiane; Volkmer, Rudolf; Barral, Yves; Steinmetz, Michel O.

2016-01-01

The Kar9 pathway promotes nuclear fusion during mating and spindle alignment during metaphase in budding yeast. How Kar9 supports the different outcome of these two divergent processes is an open question. Here, we show that three sites in the C-terminal disordered domain of Kar9 mediate tight Kar9 interaction with the C-terminal dimerization domain of Bim1 (EB1 orthologue). Site1 and Site2 contain SxIP motifs; however, Site3 defines a novel type of EB1-binding site. Whereas Site2 and Site3 mediate Kar9 recruitment to microtubule tips, nuclear movement, and karyogamy, only Site2 functions in spindle positioning during metaphase. Site1 in turn plays an inhibitory role during mating. Additionally, the Kar9-Bim1 complex is involved in microtubule-independent activities during mating. Together, our data reveal how multiple and partially redundant EB1-binding sites provide a microtubule-associated protein with the means to modulate its biochemical properties to promote different molecular processes during cell proliferation and differentiation. PMID:27682587

Systems cell biology of the mitotic spindle.

PubMed

Saleem, Ramsey A; Aitchison, John D

2010-01-11

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog.

PubMed

Pérez, J; Bautista, M J; Rollón, E; de Lara, F C; Carrasco, L; Martin de las Mulas, J

1996-07-01

The immunohistochemical expression of muscle actin has been studied in 45 canine hemangiopericytomas (CHP) using a monoclonal antibody (HHF35) and formalin-fixed, paraffin-embedded specimens. The distribution of vimentin, desmin, cytokeratins, lysozyme, factor VIII-related antigen, S-100 protein, and glial fibrillary acidic protein was studied both in CHP and in some canine soft-tissue neoplasms (seven fibrosarcomas, seven benign schwannomas, seven benign fibrous histiocytomas, and six leiomyosarcomas) used as controls for differential diagnosis. All CHP and control tumors expressed vimentin. Twenty-three CHP expressed muscle actin, whereas all control tumors analyzed were muscle actin-negative, with the exception of leiomyosarcomas. Among muscle actin- and vimentin-positive CHP, one case could be reclassified as leiomyosarcoma because it was desmin-positive, two cases expressed lysozyme, and nine cases expressed S-100 protein. Among muscle actin-negative and vimentin-positive CHP, seven expressed S-100 protein. In addition, S-100 protein was detected in five schwannomas. All CHP and control tumors analyzed were negative for cytokeratins, factor VIII-related antigen, and glial fibrillary acidic protein. Our results support the hypothesis of a pericytic origin of CHP, and suggest that muscle actin, desmin, vimentin, and lysozyme could be useful for the differential diagnosis of canine spindle cell tumors, but not all these neoplasms can be identified with these tumor tissue markers.

A neuronal morphologic type unique to humans and great apes

PubMed Central

Nimchinsky, Esther A.; Gilissen, Emmanuel; Allman, John M.; Perl, Daniel P.; Erwin, Joseph M.; Hof, Patrick R.

1999-01-01

We report the existence and distribution of an unusual type of projection neuron, a large, spindle-shaped cell, in layer Vb of the anterior cingulate cortex of pongids and hominids. These spindle cells were not observed in any other primate species or any other mammalian taxa, and their volume was correlated with brain volume residuals, a measure of encephalization in higher primates. These observations are of particular interest when considering primate neocortical evolution, as they reveal possible adaptive changes and functional modifications over the last 15–20 million years in the anterior cingulate cortex, a region that plays a major role in the regulation of many aspects of autonomic function and of certain cognitive processes. That in humans these unique neurons have been shown previously to be severely affected in the degenerative process of Alzheimer’s disease suggests that some of the differential neuronal susceptibility that occurs in the human brain in the course of age-related dementing illnesses may have appeared only recently during primate evolution. PMID:10220455

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

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.

Effect of chitosan conduit under a dynamic culture on the proliferation and neural differentiation of human exfoliated deciduous teeth stem cells.

PubMed

Su, Wen-Ta; Shih, Yi-An; Ko, Chih-Sheng

2016-06-01

Ex vivo engineering of artificial nerve conduit is a suitable alternative clinical treatment for nerve injuries. Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. These cells, when cultured in six-well plates, exhibited a spindle fibroblastic morphology, whereas those under a dynamic culture aggregated into neurosphere-like clusters in the chitosan conduit. In this study, we confirmed that SHEDs efficiently express the neural stem cell marker nestin, the early neural cell marker β-III-tubulin, the late neural marker neuron-specific enolase and the glial cell markers glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). The three-dimensional chitosan conduit and dynamic culture system generated fluid shear stress and enhanced nutrient transfer, promoting the differentiation of SHEDs to neural cells. In particular, the gene expressions of GFAP and CNPase increased by 28- and 53-fold, respectively. This study provides evidence for the dynamic culture of SHEDs during ex vivo neural differentiation and demonstrates its potential for cell therapy in neurological diseases. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.

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

Cytologically atypical anal sac adenocarcinoma in a dog.

PubMed

Sakai, Hiroki; Murakami, Mami; Mishima, Hiroyuki; Hoshino, Yuki; Mori, Takashi; Maruo, Kohji; Yanai, Tokuma

2012-06-01

A 10-year-old intact female Shetland Sheepdog with tenesmus had a subcutaneous mass at the left ventral aspect of the anus. On cytologic examination, 2 types of cells were observed. Most of the cells were oval to polygonal and had elliptical or elongate nuclei and a moderate amount of pale to basophilic cytoplasm. The remaining cells had round to oval nuclei and pale to basophilic cytoplasm. Cells of both types were loosely adhered to each other and were arranged in rosette-like structures. Both neoplastic cell types had fine homogenous chromatin and either a small indistinct nucleolus or no visible nucleolus. Mild anisokaryosis and anisocytosis were observed. Histologically, the mass consists of glandular structures formed by cuboidal cells admixed with bundles of spindle cells. Eosinophilic PAS- and Alcian blue-positive secretory material was found in the center of some glandular structures. Both neoplastic cell types had positive staining with paradoxical concanavalin A and expressed cytokeratin, but not vimentin, S-100, α-smooth muscle actin, or desmin. Based on location and histologic and immunohistochemical features, the final diagnosis was adenocarcinoma of the apocrine gland of the anal sac, which should be included as a cytologic differential diagnosis when spindle cells and typical epithelial cells are observed in masses in the region of the anal sac of dogs. © 2012 American Society for Veterinary Clinical Pathology.

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.

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

Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins

PubMed Central

Roychowdhury, Sukla; Rasenick, Mark. M

2009-01-01

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. G proteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation. In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between G protein subunits (α and βγ) and microtubules, and the molecular details emerging from these studies suggest that α and βγ subunits of G proteins interact with tubulin/microtubules to regulate assembly/dynamics of microtubules, providing a novel mechanism for hormone or neurotransmitter induced rapid remodeling of cytoskeleton, regulation of mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation where structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission. PMID:18754776

Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

PubMed

Jiao, Fei; Wang, Juan; Dong, Zhao-Lun; Wu, Min-Juan; Zhao, Ting-Bao; Li, Dan-Dan; Wang, Xin

2012-08-01

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

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

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

Search, capture and signal: games microtubules and centrosomes play.

PubMed

Schuyler, S C; Pellman, D

2001-01-01

Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.

Differential sensitivity of Glioma stem cells to Aurora kinase A inhibitors: implications for stem cell mitosis and centrosome dynamics.

PubMed

Mannino, Mariella; Gomez-Roman, Natividad; Hochegger, Helfrid; Chalmers, Anthony J

2014-07-01

Glioma stem-cell-like cells are considered to be responsible for treatment resistance and tumour recurrence following chemo-radiation in glioblastoma patients, but specific targets by which to kill the cancer stem cell population remain elusive. A characteristic feature of stem cells is their ability to undergo both symmetric and asymmetric cell divisions. In this study we have analysed specific features of glioma stem cell mitosis. We found that glioma stem cells appear to be highly prone to undergo aberrant cell division and polyploidization. Moreover, we discovered a pronounced change in the dynamic of mitotic centrosome maturation in these cells. Accordingly, glioma stem cell survival appeared to be strongly dependent on Aurora A activity. Unlike differentiated cells, glioma stem cells responded to moderate Aurora A inhibition with spindle defects, polyploidization and a dramatic increase in cellular senescence, and were selectively sensitive to Aurora A and Plk1 inhibitor treatment. Our study proposes inhibition of centrosomal kinases as a novel strategy to selectively target glioma stem cells. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

PubMed Central

Dewey, Evan B.; Johnston, Christopher A.

2017-01-01

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. PMID:28747439

Human Mesenchymal Stem Cells Derived From Limb Bud Can Differentiate into All Three Embryonic Germ Layers Lineages

PubMed Central

Jiao, Fei; Wang, Juan; Dong, Zhao-lun; Wu, Min-juan; Zhao, Ting-bao; Li, Dan-dan

2012-01-01

Abstract Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them “human limb bud–derived mesenchymal stem cells” (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro. PMID:22775353

Collision of Ductal Carcinoma In Situ of Anogenital Mammary-like Glands and Vulvar Sarcomatoid Squamous Cell Carcinoma.

PubMed

Tran, Tien A N; Deavers, Michael T; Carlson, J Andrew; Malpica, Anais

2015-09-01

A spectrum of invasive adenocarcinomas presumably arising from the anogenital mammary-like glands of the vulva has been reported. Even rarer are the cases of pure ductal carcinoma in situ that originated from these unique glandular structures. Herein, we report an 81-yr-old woman presented with an invasive well-differentiated squamous cell carcinoma of the vulva. Unexpectedly, the underlying dermis demonstrated a cystically dilated structure that displayed a layer of malignant squamous cells in the periphery, and a second centrally located population of neoplastic cells exhibiting glandular differentiation. In addition, a spindle and pleomorphic malignant cell population consistent with a sarcomatoid carcinoma was identified around the cystic structure. Scattered benign anogenital mammary-like glands were present in the adjacent dermis. The histologic and immunohistochemical findings were consistent with those of vulvar squamous cell carcinoma that has undergone sarcomatoid transformation after spreading in a pagetoid fashion into an underlying focus of ductal carcinoma in situ of anogenital mammary-like gland origin.

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

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

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

Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

2014-11-01

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Lossmore » of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.« less

Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

PubMed

Chen, Y K; Huang, Anderson H C; Chan, Anthony W S; Lin, L M

2016-06-01

Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

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

Epidermal development, growth control, and homeostasis in the face of centrosome amplification.

PubMed

Kulukian, Anita; Holland, Andrew J; Vitre, Benjamin; Naik, Shruti; Cleveland, Don W; Fuchs, Elaine

2015-11-17

As nucleators of the mitotic spindle and primary cilium, centrosomes play crucial roles in equal segregation of DNA content to daughter cells, coordination of growth and differentiation, and transduction of homeostatic cues. Whereas the majority of mammalian cells carry no more than two centrosomes per cell, exceptions to this rule apply in certain specialized tissues and in select disease states, including cancer. Centrosome amplification, or the condition of having more than two centrosomes per cell, has been suggested to contribute to instability of chromosomes, imbalance in asymmetric divisions, and reorganization of tissue architecture; however, the degree to which these conditions are a direct cause of or simply a consequence of human disease is poorly understood. Here we addressed this issue by generating a mouse model inducing centrosome amplification in a naturally proliferative epithelial tissue by elevating Polo-like kinase 4 (Plk4) expression in the skin epidermis. By altering centrosome numbers, we observed multiciliated cells, spindle orientation errors, and chromosome segregation defects within developing epidermis. None of these defects was sufficient to impart a proliferative advantage within the tissue, however. Rather, impaired mitoses led to p53-mediated cell death and contributed to defective growth and stratification. Despite these abnormalities, mice remained viable and healthy, although epidermal cells with centrosome amplification were still appreciable. Moreover, these abnormalities were insufficient to disrupt homeostasis and initiate or enhance tumorigenesis, underscoring the powerful surveillance mechanisms in the skin.

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.

The cytology of a thyroid granular cell tumor.

PubMed

Chang, Shu-Mei; Wei, Chang-Kuo; Tseng, Chih-En

2009-01-01

Granular cell tumor (GCT) of the thyroid is rare. Before this report, only four cases of thyroid GCT have been reported, none of which presented a cytopathological examination. In this paper, we report the fine needle aspiration cytology and pathological analysis of a thyroid GCT from a 12-year-old girl who presented with a painless neck mass. The tumor cells were single, in syncytial clusters, or pseudofollicles, contained small round, oval, or spindle nuclei, indistinct nucleoli, and a large amount of grayish, granular fragile cytoplasm. The background contained granular debris and naked nuclei. A differential diagnosis of thyroid GCT with more frequent thyroid lesions containing cytoplasmic granules, including Hurthle cells, macrophages, follicular cells, and cells of black thyroid syndrome, was also performed.

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.

Differentiation of pigmented Spitz nevi and Reed nevi by integration of dermatopathologic and dermatoscopic findings

PubMed Central

Bär, Michael; Tschandl, Philipp; Kittler, Harald

2012-01-01

Background: It is unclear whether pigmented Spitz and Reed nevi are distinct morphologic entities or part of the spectrum of Spitz nevi. Methods: In a retrospective observational study we analyzed dermatopathologic slides of 22 cases with clinical and dermatoscopic features indicative of pigmented Spitz or Reed nevus in a blinded fashion according to predefined criteria and subsequently correlated dermatopathologic with clinical and dermatoscopic findings. Results: We differentiated pigmented Spitz and Reed nevus dermatopathologically by their capacity of melanin production and a vertical versus horizontal growth pattern. Based on histopathology 20 nevi (91%) could be reliably diagnosed as Reed nevus (68%, n=15) or as pigmented Spitz nevus (23%, n=5). In two cases (9%, n=2) it was not possible to make a clear distinction from a dermatopathologic point of view. Dermatopathologic-dermatoscopic correlation showed that Reed nevi were characterized by a dermatoscopic pattern of peripheral radial lines or pseudopods (fascicular growth pattern), whereas pigmented Spitz nevi were typified by a pattern consisting of clods (nested growth pattern). “Spitz cells” (large epithelioid melanocytes) were more commonly found in Spitz nevi (100%, n = 5) but were also present in Reed nevi (n=6, 40%). Spindle cells were found in both types of nevi. Conclusions: Pigmented Spitz and Reed nevi can be reliably distinguished based on their dermatopathologic and dermatoscopic patterns. The specific dermatopathologic patterns of pigmented Spitz and Reed nevi correspond well to their dermatoscopic patterns. The presence of “Spitz cells” or spindle cells should not be regarded as the decisive criterion to differentiate between these two entities. PMID:24765545

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

Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy.

PubMed

Mukherjee, Subhas; Brat, Daniel J

2017-01-01

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

Mixed endometrial stromal and smooth muscle tumor: report of a case with focal anaplasia and early postoperative lung metastasis.

PubMed

Shintaku, Masayuki; Hashimoto, Hiromi

2013-04-01

A rare case of a mixed endometrial stromal and smooth muscle tumor arising in the uterus of a 74-year-old woman is reported. The patient underwent hysterectomy for an enlarging uterine mass, and a large intramural tumor, showing marked central hyaline necrosis with calcification, was found. The tumor consisted of an admixture of a low-grade endometrial stromal sarcoma (ESS) and a fascicular proliferation of spindle cells suggesting smooth muscle differentiation, and a characteristic 'star-burst' appearance was found. In the ESS region, there were a few small foci of anaplasia where large polygonal cells with atypical nuclei and abundant eosinophilic cytoplasm proliferated, and the proliferative activity was locally increased in these foci. A small metastatic nodule appeared in the lung nine months after the hysterectomy, and the resected metastatic lesion showed features of anaplastic spindle cell sarcoma which was immunoreactive for CD10 but not for smooth muscle markers. Mixed endometrial stromal and smooth muscle tumors should be regarded as malignant neoplasms with the potential for hematogenous metastasis, particularly when they contain foci of cellular anaplasia. © 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

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.

A Comparative Study to Evaluate Myogenic Differentiation Potential of Human Chorion versus Umbilical Cord Blood-derived Mesenchymal Stem Cells.

PubMed

Bana, Nikoo; Sanooghi, Davood; Soleimani, Mansoureh; Hayati Roodbari, Nasim; Alavi Moghaddam, Sepideh; Joghataei, Mohammad Taghi; Sayahpour, Forough Azam; Faghihi, Faezeh

2017-08-01

Musculodegenerative diseases threaten the life of many patients in the world. Since drug administration is not efficient in regeneration of damaged tissues, stem cell therapy is considered as a good strategy to restore the lost cells. Since the efficiency of myogenic differentiation potential of human Chorion- derived Mesenchymal Stem Cells (C-MSCs) has not been addressed so far; we set out to evaluate myogenic differentiation property of these cells in comparison with Umbilical Cord Blood- derived Mesenchymal Stem Cells (UCB-MSCs) in the presence of 5-azacytidine. To do that, neonate placenta Umbilical Cord Blood were transferred to the lab. After characterization of the isolated cells using flowcytometry and multilineage differentiation capacity, the obtained Mesenchymal Stem Cells were cultured in DMEM/F12 supplemented with 2% FBS and 10μM of 5-azacytidine to induce myogenic differentiation. Real-time PCR and immunocytochemistry were used to assess the myogenic properties of the cells. Our data showed that C-MSCs and UCB-MSCs were spindle shape in morphology. They were positive for CD90, CD73 and CD44 antigens, and negative for hematopoietic markers. They also differentiated into osteoblast and adipoblast lineages. Real-time PCR results showed that the cells could express MyoD, desmin and α-MHC at the end of the first week (P<0.05). No significant upregulation was detected in the expression of GATA-4 in both groups. Immunocytochemical staining revealed the expression of Desmin, cTnT and α-MHC. Results showed that these cells are potent to differentiate into myoblast- like cells. An upregulation in the expression of some myogenic markers (desmin, α- MHC) was observed in C-MSCs in comparison with UCB-MSCs. Copyright © 2017. Published by Elsevier Ltd.

Hemangiopericytoma in a young dog: Evaluation of histopathological and immunohistochemical features.

PubMed

Namazi, Fatemeh; Abbaszadeh Hasiri, Mohammad; Oryan, Ahmad; Moshiri, Ali

2014-01-01

In the present study, we describe a subcutaneous mass between the left flank and hip in a 2-year-old male Great Dane dog. Histopathologically, cells appeared to be spindle shaped around a central capillary together with a fingerprint pattern. Immunohistochemical analysis presented that the neoplastic cells expressed vimentin, but did not stain for S-100 protein. On the basis of histopathology and immunohistochemical findings, the present tumor was diagnosed as canine hemangiopericytoma. Hemangiopericytoma could be considered in differential diagnosis list of any mass in the skin (even in young dogs) and must be identified histopathologically.

Hemangiopericytoma in a young dog: Evaluation of histopathological and immunohistochemical features

PubMed Central

Namazi, Fatemeh; Abbaszadeh Hasiri, Mohammad; Oryan, Ahmad; Moshiri, Ali

2014-01-01

In the present study, we describe a subcutaneous mass between the left flank and hip in a 2-year-old male Great Dane dog. Histopathologically, cells appeared to be spindle shaped around a central capillary together with a fingerprint pattern. Immunohistochemical analysis presented that the neoplastic cells expressed vimentin, but did not stain for S-100 protein. On the basis of histopathology and immunohistochemical findings, the present tumor was diagnosed as canine hemangiopericytoma. Hemangiopericytoma could be considered in differential diagnosis list of any mass in the skin (even in young dogs) and must be identified histopathologically. PMID:25568712

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.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

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

Sung, Min Sun; Biosystems and Bioengineering Program, University of Science and Technology; Mun, Ji-Young

2013-07-19

Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineeredmore » the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.« less

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

PubMed

Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

2017-02-01

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

P21-activated kinase 4 (PAK4) is required for metaphase spindle positioning and anchoring.

PubMed

Bompard, G; Rabeharivelo, G; Cau, J; Abrieu, A; Delsert, C; Morin, N

2013-02-14

The oncogenic kinase PAK4 was recently found to be involved in the regulation of the G1 phase and the G2/M transition of the cell cycle. We have also identified that PAK4 regulates Ran GTPase activity during mitosis. Here, we show that after entering mitosis, PAK4-depleted cells maintain a prolonged metaphase-like state. In these cells, chromosome congression to the metaphase plate occurs with normal kinetics but is followed by an extended period during which membrane blebbing and spindle rotation are observed. These bipolar PAK4-depleted metaphase-like spindles have a defective astral microtubule (MT) network and are not centered in the cell but are in close contact with the cell cortex. As the metaphase-like state persists, centrosome fragmentation occurs, chromosomes scatter from the metaphase plate and move toward the spindle poles with an active spindle assembly checkpoint, a phenotype that is reminiscent of cohesion fatigue. PAK4 also regulates the acto-myosin cytoskeleton and we report that PAK4 depletion results in the induction of cortical membrane blebbing during prometaphase arrest. However, we show that membrane blebs, which are strongly enriched in phospho-cofilin, are not responsible for the poor anchoring of the spindle. As PAK4 depletion interferes with the localization of components of the dynein/dynactin complexes at the kinetochores and on the astral MTs, we propose that loss of PAK4 could induce a change in the activities of motor proteins.

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

HMB-45 negative angiomyolipoma of the orbit: a case report and review of the literature.

PubMed

Lin, Che-Yu; Tsai, Chieh-Chih; Kau, Hui-Chuan; Yu, Wei-Kuang; Kao, Shu-Ching; Liu, Catherine Jui-Ling

2016-01-11

Angiomyolipoma is a benign mesenchymal tumor composed of variable amounts of smooth muscle, adipose tissue and thick-walled blood vessels, and usually named PEComas (perivascular epithelioid cell tumors). PEComas share overlapping histopathological features with epithelioid cells along a perivascular distribution and characteristic immunohistochemistry with coexpression of myoid and melanocytic markers (HMB-45 /or Melan-A). We report the first case of primary orbital angiomyolipoma with negative melanocytic marker. An 80-year-old Asian woman had a 2-year history of progressive swelling in the left upper eyelid. External examination revealed 3 cm of relative proptosis of the left eye and a palpable mass in the left superonasal orbit. Computed tomographic scan demonstrated a circumscribed, heterogeneous orbital mass. Excision biopsy was done and the histological finding demonstrated the orbital mass was composed of mature adipocytes, intermingled with spindle or oval-shaped cells, and accompanied by thick-walled blood vessels. Immunohistochemically, tumor cells were positive for CD34 and HHF-35, but negative for cytokeratin, HMB-45 and Melan-A. The diagnosis of angiomyolipoma was made. No recurrence was noted at 2-year follow-up. In our case, the HMB-45 negativity may be explained by the rarity of the epithelioid cells, and the HMB-45 positivity is often weaker or absent in spindle cells. Angiomyolipoma, although rare, should be added to the differential diagnosis of space-occupying orbital lesion.

Perivascular epithelioid cell tumor (PEComa) of gynecologic origin: a clinicopathological study of three cases.

PubMed

Ye, H Y; Chen, J G; Luo, D L; Jiang, Z M; Chen, Z H

2012-01-01

Perivascular epithelioid cell tumors (PEComas), occasionally associated with the tuberous sclerosis complex, are characterized by varying amounts of spindle and epithelioid cells with clear to eosinophilic cytoplasm that display immunoreactivity for melanocytic markers, most frequently HMB-45. Perivascular epithelioid cell tumor of gynecologic origin is very rare, and there have been only a few reported cases. This study describes the clinical, histological, and immunohistochemical features and prognoses of three cases of gynecologic origin. Two of the three tumors were confined to the uterus and one to the vagina. None of the patients had tuberous sclerosis complex. Immunohistochemistry indicated that all three cases expressed at least one melanocytic marker, and HMB45 was a positive marker for all of them. These markers can be found in both epithelial cells and spindle cells. Except for MiTF, which was located in the nucleus, all the other antibodies were located in the cytoplasm. The three cases have been followed up for 26, 22, and three months, respectively, with disease-free survival in all cases. We conclude that PEComas of gynecologic origin have morphological and immunohistochemical features of the PEComa family, which are rare and should be included in the differential diagnosis with other tumors. Until more cases of this rare tumor are evaluated with longer follow-up, firm criteria for malignancy remain uncertain.

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.

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.

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.

Expression of microphthalmia transcription factor, S100 protein, and HMB-45 in malignant melanoma and pigmented nevi.

PubMed

Xia, Jianxin; Wang, Yanlong; Li, Fuqiu; Wang, Jinfeng; Mu, Yan; Mei, Xianglin; Li, Xue; Zhu, Wenjing; Jin, Xianhua; Yu, Kai

2016-09-01

Malignant melanoma (MM) is a type of malignant tumor, which originates from neural crest melanocytes. MM progresses rapidly and results in a high mortality rate. The present study aims to investigate the expression of microphthalmia transcription factor (MITF), the S100 protein, and HMB-45 in MM and pigmented nevi. A total of 32 MM samples (including three skin metastasis, three lymph node metastasis and two spindle cell MM samples), two Spitz nevus samples, four pigmented nevus samples and two blue nevus samples were collected. The expression levels of S100 protein, HMB-45, and MITF were observed via immunostaining. The S100 protein exhibited high positive rates in MM and pigment disorders (96.7 and 100%, respectively), but with low specificity. The S100 protein was also expressed in fibroblasts, myoepithelial cells, histocytes and Langerhans cells in normal skin samples. HMB-45 had high specificity. Its positive expression was only confined to MM cells and junctional nevus cells. Furthermore, HMB-45 was not expressed in melanocytes in the normal tissue samples around the tumor or in the benign intradermal nevus cells. MITF exhibited high specificity and high sensitivity. It was expressed in the nuclei of melanocytes, MM cells and nevus cells. It was observed to be strongly expressed in metastatic MM and spindle cell MMs. Thus, MITF may present as a specific immunomarker for the diagnosis and differential diagnosis of MM.

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.

Small cell type neuroendocrine carcinoma colliding with squamous cell carcinoma at esophagus

PubMed Central

Yang, Luoluo; Sun, Xun; Zou, Yabin; Meng, Xiangwei

2014-01-01

Collision tumor is an extremely rare tumor which defined as the concrescence of two distinct primaries neoplasms. We report here a case of collision tumor at lower third esophagus composed of small cell type neuroendocrine carcinoma (NEC), which is an very rare, highly aggressive and poorly prognostic carcinoma and squamous cell carcinoma (SqCC). In our case, pathologically, the small cell carcinoma display the characteristic of small, round, ovoid or spindle-shaped tumor cells with scant cytoplasm, which colliding with a moderately differentiated squamous cell carcinoma. Immunohistochemical staining demonstrated positive activities for CD56, synaptophysin, 34βE12, CK 5/6, ki-67 (70%-80%), but negative for CD99, chromogranin A, and TTF-1. Accurate diagnosis was made base on these findings. PMID:24817981

The Differential Roles of Budding Yeast Tem1p, Cdc15p, and Bub2p Protein Dynamics in Mitotic ExitD⃞V⃞

PubMed Central

Molk, Jeffrey N.; Schuyler, Scott C.; Liu, Jenny Y.; Evans, James G.; Salmon, E. D.; Pellman, David; Bloom, Kerry

2004-01-01

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade. PMID:14718561

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

Temporal Dynamics of Parvalbumin-Expressing Axo-axonic and Basket Cells in the Rat Medial Prefrontal Cortex In Vivo

PubMed Central

Hartwich, Katja; Borhegyi, Zsolt; Somogyi, Peter; Klausberger, Thomas

2015-01-01

Axo-axonic interneurons, innervating exclusively axon initial segments, and parvalbumin-expressing basket interneurons, targeting somata, dendrites, and spines of pyramidal cells, have been proposed to control neuronal activity in prefrontal circuits. We recorded the spike-timing of identified neurons in the prelimbic cortex of anesthetized rats, and show that axo-axonic cells increase their firing during tail pinch-induced brain state-activation. In addition, axo-axonic cells differ from other GABAergic parvalbumin-expressing cells in their spike timing during DOWN- to UP-state transitions of slow oscillations and in their coupling to gamma and spindle oscillations. The distinct firing dynamics and synaptic targets of axo-axonic and other parvalbumin-expressing cells provide differential contributions to the temporal organization of prefrontal networks. PMID:23152631

A fetal human heart cardiac-inducing RNA (CIR) promotes the differentiation of stem cells into cardiomyocytes.

PubMed

Kochegarov, Andrei; Moses-Arms, Ashley; Lemanski, Larry F

2015-08-01

A specific human fetal heart RNA has been discovered, which has the ability to induce myocardial cell formation from mouse embryonic and human-induced pluripotent stem cells in culture. In this study, commercially obtained RNA from human fetal heart was cloned, sequenced, and synthesized using standard laboratory approaches. Molecular analyses of the specific fetal cardiac-inducing RNA (CIR), revealed that it is a fragment of N-sulfoglucosaminesulfohydrolase and the caspase recruitment domain family member 14 precursor. Stem cells transfected with CIRs often form into spindle-shaped cells characteristic of cardiomyocytes,and express the cardiac-specific contractile protein marker, troponin-T, in addition to tropomyosin and α-actinin as detected by immunohistochemical staining. Expression of these contractile proteins showed organization into sarcomeric myofibrils characteristic of striated cardiac muscle cells. Computer analyses of the RNA secondary structures of the active CIR show significant similarities to a RNA from salamander or myofibril-inducing RNA (MIR), which also promotes non-muscle cells to differentiate into cardiac muscle. Thus, these two RNAs, salamander MIR and the newly discovered human-cloned CIR reported here, appear to have evolutionarily conserved secondary structures suggesting that both play major roles in vertebrate heart development and, particularly, in the differentiation of cardiomyocytes from non-muscle cells during development.

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

Intermediate filament proteins and actin isoforms as markers for soft-tissue tumor differentiation and origin. III. Hemangiopericytomas and glomus tumors.

PubMed Central

Schürch, W.; Skalli, O.; Lagacé, R.; Seemayer, T. A.; Gabbiani, G.

1990-01-01

Intermediate filament proteins and actin isoforms of a series of 12 malignant hemangiopericytomas and five glomus tumors were examined by light microscopy, transmission electron microscopy, two-dimensional gel electrophoresis (2D-GE), and by immunohistochemistry, the latter using monoclonal or affinity-purified polyclonal antibodies to desmin, vimentin, cytokeratins, alpha-smooth muscle, and alpha-sarcomeric actins. By light microscopy, all hemangiopericytomas disclosed a predominant vascular pattern with scant storiform, myxoid and spindle cell areas, and with variable degrees of perivascular fibrosis. By ultrastructure, smooth muscle differentiation was observed in each hemangiopericytoma. Immunohistochemically, neoplastic cells of hemangiopericytomas expressed vimentin as the sole intermediate filament protein and lacked alpha-smooth muscle or alpha-sarcomeric actins. 2D-GE revealed only beta and gamma actins, in proportions typical for fibroblastic tissues. Glomus tumors revealed vimentin and alpha-smooth muscle actin within glomus cells by immunohistochemical techniques and disclosed ultrastructurally distinct smooth muscle differentiation. Therefore hemangiopericytomas represent a distinct soft-tissue neoplasm with uniform morphologic, immunohistochemical, and biochemical features most likely related to glomus tumors, the former representing an aggressive and potentially malignant neoplasm of vascular smooth muscle cells and the latter a well-differentiated neoplasm of vascular smooth muscle cells. Because malignant hemangiopericytomas disclose smooth muscle differentiation by ultrastructure, but do not express alpha-smooth muscle actin, as normal pericytes and glomus cells, it is suggested that these neoplasms represent highly vascularized smooth muscle neoplasms, ie, poorly differentiated leiomyosarcomas derived from vascular smooth muscle cells or their equivalent, the pericytes, which have lost alpha-smooth muscle actin as a differentiation marker that is similar to many conventional poorly differentiated leiomyosarcomas. Images Figure 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2158236

Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity.

PubMed

Dewey, Evan B; Johnston, Christopher A

2017-09-15

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial-mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation. © 2017 Dewey and Johnston. 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).

Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin.

PubMed

Little, D; Said, J W; Siegel, R J; Fealy, M; Fishbein, M C

1986-06-01

Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.

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

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).

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.

PEComa: morphology and genetics of a complex tumor family.

PubMed

Thway, Khin; Fisher, Cyril

2015-10-01

Perivascular epithelioid cell tumors, or PEComas, are mesenchymal neoplasms composed of histologically and immunohistochemically distinctive epithelioid or spindle cells, which are immunoreactive for both smooth muscle and melanocytic markers. The cells in PEComas are typically arranged around blood vessels and appear to form the vessel wall, often infiltrating the smooth muscle of small- to medium-sized vessels. Periluminal cells are usually epithelioid and the more peripheral cells are spindle shaped. The cells have small, round to oval nuclei, sometimes with focal nuclear atypia, and clear to eosinophilic cytoplasm, and no counterpart normal cell has been identified. The PEComa "family" now includes angiomyolipoma, pulmonary clear cell "sugar" tumor and lymphangioleiomyomatosis, primary extrapulmonary sugar tumor, clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres, abdominopelvic sarcoma of perivascular epithelioid cells, and other tumors with similar features at various sites that are simply termed PEComa. Some PEComas occur in patients with tuberous sclerosis complex and share the genetic abnormalities. There is a behavioral spectrum from benign to frankly malignant, and histologic criteria have been proposed for assessing malignant potential. The differential diagnosis can include carcinomas, smooth muscle tumors, other clear cell neoplasms, and adipocytic tumors. PEComas constitute a genetically diverse group that includes neoplasms harboring TFE3 gene rearrangements and those with TSC2 mutations, indicating alternative tumorigenic pathways. Recent advances in therapy of malignant PEComas relate to increased knowledge of specific genetic changes and their effects on metabolic pathways that are susceptible to specific interventions. We review PEComas, emphasizing the diagnostic spectrum and recent immunohistochemical and genetic findings. Copyright © 2015 Elsevier Inc. All rights reserved.

Correlation of histological grade of dedifferentiation with clinical outcome in 55 patients with dedifferentiated liposarcomas.

PubMed

Dantey, Kossivi; Schoedel, Karen; Yergiyev, Oleksandr; Bartlett, David; Rao, Uma N M

2017-08-01

In this study the histologic grade of dedifferentiated liposarcomas was correlated with outcome in surgically resected specimens in 55 patients over a 19-year period at the University of Pittsburgh Medical Center. The tumors were located in the retroperitoneum (N=35); the extremities and thigh (N=16), and the remainder involved the spermatic cord and head and neck. Most tumors were large (mean=21 cm.) Follow-up was available in all 55 patients (median=36 months). Forty-one tumors classified as high-grade dedifferentiated liposarcoma (HG-DDLPS) had mitotically active pleomorphic and spindle cells and foci of necrosis. They included tumors with foci of smooth muscle differentiation (N=12), osteosarcoma (N=4), and myxoid areas (N=9). Fourteen tumors classified as low-grade dedifferentiated liposarcoma (LG-DDLPS) displayed a predominantly bland, monomorphic, spindle cell population with few mitoses and scant necrosis. The Kaplan-Meier method and log-rank test were used for statistical analysis. All tumors had unequivocal foci of well-differentiated liposarcoma (WDLPS). Fluorescence in situ hybridization (FISH) detected amplification of MDM2 in 29 cases. Twenty of 41 patients (49%) with HG-DDLPS died of tumor, and two patients died with LG-DDLPS (14%). The overall survival of patients with LG-DDLPS was significantly longer (P=.02). The median survival was 113 months for the LG-DDLPS and 48 months for the HG-DDLPS. Metastases (N=4) occurred only in the high-grade tumors and were independent of the type of heterologous differentiation. Patients with HG-DDLPS were at a greater risk of earlier death. Distinction between the two groups is important for patient selection for possible adjuvant therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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

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

[Clinicopathological features of uterine neoplasms with perivascular epithelioid cell differentiation].

PubMed

Lu, Hai-zhen; Zhang, Hong-tu; Liu, Xiu-yun; Xue, Xin-hua; Xie, Yong-qiang; Liu, Shang-mei; Su, Qin

2009-03-01

To study the neoplasm with perivascular epithelioid cell differentiation (PEComa) with respect to their morphologic, immunohistochemical and clinical phenotypes. Three PEComas were included in this study, one located at the left uterine horn, and two presented as a mass in the uterine corpus. The tumors were examined by histopathology and immunohistochemistry. The lesions were composed of spindle, blunt epithelioid cells, with foci of, or scattered, cells showing adipose differentiation in two cases. The myomelanocytic differentiation was demonstrated, proving the diagnosis as PEComa. Mild nuclear atypia and focal necrosis was observed in one lesion, and the rest two showed malignant morphologic phenotypes including moderate nuclear atypia and coagulative necrosis. The mitotic and Ki67-labelling indices ranged from 0.5/10 HPF to 14/10 HPF and 0.6% to 7.0%, respectively. All of the three patients remain alive. Malignant nature of the two lesions with worrisome morphology was confirmed by occurrence of metastases after hysterectomy. PEComa is a rare tumor, occurring preferentially in the uterus. It is regarded as a tumor with uncertain malignant potential, but a minority of them shows malignant clinical behaviors. Some pathologic parameters including large tumor size, sheet-like necrosis, marked nuclear atypia, elevated mitotic index (> or = 10/10 HPF), aberrant mitotic figure and vascular invasion may help to establish a diagnosis of malignant PEComa.

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

Genetic Instability of Breast Cancer Cells Induced by Aberrant Expression of hMpS1

DTIC Science & Technology

2004-09-01

and function of the mitotic spindle is dependent on the MpS1 protein kinase. The components of mitotic spindle checkpoints were first identified in...yeast and their homologs of higher organisms were then identified and characterized. These include Bubl, Bub2, Bub3, Madl, Mad2, Mad3 and MpS1 . MpS 1...spindle checkpoint and centrosome duplication (1-6). Although several recent reports demonstrated that vertebrate MpS1 proteins regulate the spindle

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.

Histologic differentiation of desmoplastic melanoma from cicatrices.

PubMed

Kaneishi, N K; Cockerell, C J

1998-04-01

Desmoplastic malignant melanoma (DMM) is a rare variant of melanoma that can be very difficult to diagnose correctly both clinically and histologically. The problem is compounded by the fact that many lesions persist at previous biopsy or excision sites so that scar tissue is often present admixed with or adjacent to the spindle cell neoplasm which may exhibit fibroblastic differentiation itself. In order to assess this problem, we compared and contrasted the histologic features of six DMM with 15 examples of cicatrices from various sources. Mature scars were readily differentiated from DMM by light microscopy. In contrast, immature scar and DMM had many features in common including hypercellularity, nodular lymphoid infiltrates, myxoid stroma, and atypical nuclei. The presence of a melanocytic proliferation within the epidermis above the dermal component, neurotropism, and S-100 and/or HMB-45 positivity of neoplastic cells were the only features that permitted reliable differentiation between the two. Clinical correlation and review of previous biopsy specimens are crucial in preventing a delayed diagnosis of DMM. Re-excision is advised in all questionable cases.

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...

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.

Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

PubMed Central

Tamada, Masako; Zallen, Jennifer A.

2015-01-01

Summary Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand, Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation. PMID:26506305

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

Comparison of Osteogenic and Chondrogenic Differentiation Ability of Buccal Fat Pad Derived Mesenchymal Stem Cells and Gingival Derived Cells.

PubMed

Ghaderi, Hamid; Razmkhah, Mahboobeh; Kiany, Farin; Chenari, Nooshafarin; Haghshenas, Mohammad Reza; Ghaderi, Abbas

2018-06-01

One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells (MSCs) with higher differentiation ability. In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs (BFP-MSCs) with gingival derived cells (GDCs) were compared. BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I (COLL), BGLA and bone morphogenetic protein 2 (BMP2) using qRT-PCR. Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls. Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries.

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.

Differentiation and characteristics of the enhanced green fluorescent protein gene transgenic goat neural stem cells cultured in attached and non-attached plates.

PubMed

Zheng, Yue-Mao; Dang, Yong-Hui; Qiu, Shuang; Qi, Ying-Pei; Xu, Yong-Ping; Sai, Wu-Jia-Fu

2011-08-01

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of embryonic day 98 fetal goat, (ii) to determine if these stem cells have the capability of multipotent differentiation following transfection with a reporter gene, EGFP (enhanced green fluorescent protein) and (iii) to study the characteristics of the stem cells cultured in attached and non-attached plates. NSCs were isolated from embryonic day 98 fetal goat brain, transfected with EGFP gene using lipofection, and subcultured in attached and non-attached plates respectively. The transgenic stem cells were induced to differentiate into osteogenic and endothelial cells in vitro respectively. Markers associated with undifferentiated NSCs and their differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that stem cells could be isolated from embryonic day 98 fetal goat brain, and EGFP gene could be transfected into the cells. The transgenic NSCs were capable of self-renewal, a defining property of stem cells, and were grown as free-floating neurospheres in non-attached plates. When the neurospheres were transferred and cultured in attached plates, cells migrate from the neurospheres and are grown as spindle cells. The stem cells were grown as quasi-circular cells when the single stem cells were cultured in attached plates. Both the NSCs cultured in non-attached and attached plates could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4), Nanog, Sox2 [SRY (sex-determining region Y)-box 2] and Nestin, while following differentiation cells expressed markers for osteogenic cells (Osteocalcin+ and Osteonectin+) and endothelium (CD34+ and eNOS+). The results demonstrated that the goat EGFP gene transgenic NSCs have the capability of multipotent differentiation, which means that the transgenic NSCs may be useful in cell transplantation studies in future.

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.

Intranasal pericytic tumors (glomus tumor and sinonasal hemangiopericytoma-like tumor): report of two cases with review of the literature.

PubMed

Li, Xiao-Qiu; Hisaoka, Masanori; Morio, Takashi; Hashimoto, Hiroshi

2003-05-01

An intranasal glomus tumor and a sinonasal hemangiopericytoma-like tumor are reported. Both patients were elderly women suffering from nasal bleeding, and presented with a polypoid mass arising in the nasal septum. Microscopically, the glomus tumor displayed a proliferation of uniform rounded or cuboidal epithelioid cells arranged in sheets and interrupted by a rich vasculature with a characteristic configuration mimicking the normal glomus bodies, while the sinonasal hemangiopericytoma-like tumor featured a perivascular proliferation of spindle- to oval-shaped cells that were arranged in short fascicles. Both tumors shared immunohistochemical features supporting their myoid differentiation by the expression of vimentin, alpha-smooth muscle actin and muscle-specific actin, albeit with no immunoreaction to desmin. Both the intranasal glomus tumor and sinonasal hemangiopericytoma-like tumor are characterized by a perivascular growth pattern and myoid differentiation, having a close relation to the 'perivascular myomas', which was recently designated.

SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study

PubMed Central

Laga, Alvaro C.; Zhan, Qian; Weishaupt, Carsten; Ma, Jie; Frank, Markus H.; Murphy, George F.

2012-01-01

SOX2 is an embryonic neural crest stem-cell transcription factor recently shown to be expressed in human melanoma and to correlate with experimental tumor growth. SOX2 binds to an enhancer region of the gene that encodes for nestin, also a neural progenitor cell biomarker. To define further the potential relationship between SOX2 and nestin, we examined co-expression patterns in 135 melanomas and 37 melanocytic nevi. Immunohistochemical staining in 27 melanoma tissue sections showed an association between SOX2 positivity, spindle cell shape and a peripheral nestin distribution pattern. In contrast, SOX2-negative cells were predominantly epithelioid, and exhibited a cytoplasmic pattern for nestin. In tissue microarrays, co-expression correlated with tumor progression, with only 11% of nevi co-expressing SOX2 and nestin in contrast to 65% of metastatic melanomas, and preliminarily, with clinical outcome. Human melanoma lines that differentially expressed constitutive SOX2 revealed a positive correlation between SOX2 and nestin expression. Experimental melanomas grown from these respective cell lines in murine subcutis and dermis of xenografted human skin maintained the association between SOX2-positivity, spindle cell shape, and peripheral nestin distribution. Moreover, the cytoplasmic pattern of nestin distribution was observed in xenografts generated from SOX2-knockdown A2058 melanoma cells, in contrast to the periperhal nestin pattern seen in tumors grown from A2058 control cells transfected with non-target shRNA. In aggregate, these data further support a biologically significant linkage between SOX2 and nestin expression in human melanoma. PMID:21410764

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.

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.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

PubMed

Lin, Han-Tso; Chiou, Shih-Hwa; Kao, Chung-Lan; Shyr, Yi-Ming; Hsu, Chien-Jen; Tarng, Yih-Wen; Ho, Larry L-T; Kwok, Ching-Fai; Ku, Hung-Hai

2006-07-28

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa

PubMed Central

Webber, Beau R; Osborn, Mark J; McElroy, Amber N; Twaroski, Kirk; Lonetree, Cara-lin; DeFeo, Anthony P; Xia, Lily; Eide, Cindy; Lees, Christopher J; McElmurry, Ron T; Riddle, Megan J; Kim, Chong Jai; Patel, Dharmeshkumar D; Blazar, Bruce R; Tolar, Jakub

2016-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB. PMID:28250968

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.

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

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

Isolation, Characterization and Growth Kinetic Comparison of Bone Marrow and Adipose Tissue Mesenchymal Stem Cells of Guinea Pig.

PubMed

Aliborzi, Ghaem; Vahdati, Akbar; Mehrabani, Davood; Hosseini, Seyed Ebrahim; Tamadon, Amin

2016-05-30

Mesenchymal stem cells (MSCs) from different sources have different characteristics. Moreover, MSCs are not isolated and characterized in Guinea pig for animal model of cell therapy. was the isolating of bone marrow MSCs (BM-MSCs) and adipose tissue MSCs (AT-MSCs) from Guinea pig and assessing their characteristics. In this study, bone marrow and adipose tissue were collected from three Guinea pigs and cultured and expanded through eight passages. BM-MSCs and AT-MSCs at passages 2, 5 and 8 were seeded in 24-well plates in triplicate. Cells were counted from each well 1~7 days after seeding to determine population doubling time (PDT) and cell growth curves. Cells of passage 3 were cultured in osteogenic and adipogenic differentiation media. BM-MSCs and AT-MSCs attached to the culture flask and displayed spindle-shaped morphology. Proliferation rate of AT-MSCs in the analyzed passages was more than BM-MSCs. The increase in the PDT of MSCs occurs with the increase in the number of passages. Moreover, after culture of BM-MSCs and AT-MSCs in differentiation media, the cells differentiated toward osteoblasts and adipocytes as verified by Alizarin Red staining and Oil Red O staining, respectively. BM-MSCs and AT-MSCs of Guinea pig could be valuable source of multipotent stem cells for use in experimental and preclinical studies in animal models.

Synergistic effect between bioactive glass foam and a perfusion bioreactor on osteogenic differentiation of human adipose stem cells.

PubMed

Silva, A R P; Paula, A C C; Martins, T M M; Goes, A M; Pereria, M M

2014-03-01

Tissue engineering is a multidisciplinary science that combines a structural scaffold and cells to form a construct able to promote regeneration of injured tissue. Bioactive glass foam produced by sol-gel is an osteoinductive material with a network of interconnected macropores necessary for cell colonization. The use of human adipose-derived stem cell (hASC) presents advantages as the potential for a large number of cells, rapid expansion in vitro and the capability of differentiating into osteoblasts. The use of a bioreactor in three-dimensional cell culture enables greater efficiency for cell nutrition and application of mechanical forces, important modulators of bone physiology. The hASC seeded in a bioactive glass scaffold and cultured in osteogenic Leibovitz L-15 medium in a bioreactor with a flow rate of 0.1 mL min(-1) demonstrated a significant increase in cell proliferation and viability and alkaline phosphatase (ALP) activity peak after 14 days. The immunofluorescence assay revealed an expression of osteopontin, osteocalcin and type I collagen from 7 to 21 days after culture. The cells changed from a spindle shape to a cuboidal morphology characteristic of osteoblasts. The polymerase chain reaction assay confirmed that osteopontin, osteocalcin, and ALP genes were expressed. These results indicate that hASCs differentiated into an osteogenic phenotype when cultured in bioactive glass scaffold, osteogenic Leibovitz L-15 medium and a perfusion bioreactor. Therefore, these results highlight the synergism between a bioactive glass scaffold and the effect of perfusion on cells and indicate the differentiation into an osteogenic phenotype. Copyright © 2013 Wiley Periodicals, Inc.

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.

Welcoming the new WHO classification of pituitary tumors 2017: revolution in TTF-1-positive posterior pituitary tumors.

PubMed

Shibuya, Makoto

2018-04-01

The fourth edition of the World Health Organization classification of endocrine tumors (EN-WHO2017) was released in 2017. In this new edition, changes in the classification of non-neuroendocrine tumors are proposed particularly in tumors arising in the posterior pituitary. These tumors are a distinct group of low-grade neoplasms of the sellar region that express thyroid transcription factor-1, and include pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, and sellar ependymoma. This short review focuses on the classification of posterior pituitary tumors newly proposed in EN-WHO2017, and controversies in their pathological differential diagnosis are discussed based on recent cases.

[Incidence of anaplastic tumor in structure of other histologic forms of the thyroid gland cancer].

PubMed

Vinnik, Iu A; Gorbenko, V N; Vas'ko, A R; Kikhtenko, E V; Gargin, V V

2014-01-01

The degrees of invasiveness, proliferative activity, morphofunctional activity of nuclei in the thyroidal gland tumors were studied, while analyzing material, obtained in 1343 patients, suffering thyroidal gland cancer (THGC) and operated on in 2000-2013 yrs. Morphological point quantity of malignancy (as a criterion of the tumor progression grade) and mitotic activity in cellular population were determined in various kinds of THGC. Undifferentiated (anaplastic carcinoma) type of THGC is the most malignant one. There were determined a spindle-like, giant-cell and squamous-cell forms of undifferentiated THGC. The presence of sites of differentiated cancer in 33% of histological preparations witnesses the interrelationship with the earlier existed pathological process.

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

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.

Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes.

PubMed

Johnson, Mary Kathrine; Cooksey, Amanda M; Wise, Dwayne A

2008-11-01

CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented. (c) 2008 Wiley-Liss, Inc.

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

Loss of centrioles causes chromosomal instability in vertebrate somatic cells.

PubMed

Sir, Joo-Hee; Pütz, Monika; Daly, Owen; Morrison, Ciaran G; Dunning, Mark; Kilmartin, John V; Gergely, Fanni

2013-12-09

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.

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

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.

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

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.

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

"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.

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.

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.

A T-Type Capacitive Sensor Capable of Measuring 5-DOF Error Motions of Precision Spindles

PubMed Central

Xiang, Kui; Qiu, Rongbo; Mei, Deqing; Chen, Zichen

2017-01-01

The precision spindle is a core component of high-precision machine tools, and the accurate measurement of its error motions is important for improving its rotation accuracy as well as the work performance of the machine. This paper presents a T-type capacitive sensor (T-type CS) with an integrated structure. The proposed sensor can measure the 5-degree-of-freedom (5-DOF) error motions of a spindle in-situ and simultaneously by integrating electrode groups in the cylindrical bore of the stator and the outer end face of its flange, respectively. Simulation analysis and experimental results show that the sensing electrode groups with differential measurement configuration have near-linear output for the different types of rotor displacements. What’s more, the additional capacitance generated by fringe effects has been reduced about 90% with the sensing electrode groups fabricated based on flexible printed circuit board (FPCB) and related processing technologies. The improved signal processing circuit has also been increased one times in the measuring performance and makes the measured differential output capacitance up to 93% of the theoretical values. PMID:28846631

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

Myoepithelial carcinoma of the parotid gland: A case of adequate fine-needle aspiration cytology specimens rendering a conclusive diagnosis possible.

PubMed

Guo, Xin; Watanabe, Jiro; Ariyasu, Sanae; Sasaguri, Yasuyuki; Kurose, Nozomu; Fukushima, Kei; Yamada, Sohsuke

2018-01-01

An 80-year-old male presented with a history of a hard right parotid mass that had gradually increased in size, with subsequent facial paralysis. A fine-needle aspiration biopsy was performed. The cytologic specimens contained a substantial number of sheet-like clusters or small groups of a mixture of plasmacytoid, oval to spindled, or large epithelioid cells having hyperchromatic pleomorphic nuclei, abundant cytoplasm with occasional inclusion body-like materials, and prominent nucleoli, in a relatively clear background. We first interpreted it as a carcinoma, suggestive of myoepithelial differentiation. Radical parotidectomy was performed, and a gross examination of the neoplasm revealed a non-capsulated and ill-defined tumor lesion, with a grayish or yellowish cut surface, associated with fat invasion. On a microscopic examination, the tumor was predominantly composed of the solid proliferation of atypical cells including a mixture of oval to spindled, plasmacytoid, or epithelioid cells, often arranged in a trabecular and reticular growth pattern with patchy eosinophilic hyalinized stroma. Immunohistochemistry showed that the carcinoma cells were specifically positive for p63, cytokeratins, and vimentin. Finally, electron microscopy demonstrated that their phenotype was consistent with a myoepithelial origin containing many bundles of variably thin actin filaments. Therefore, we finally made a diagnosis of myoepithelial carcinoma, defined as the malignant counterpart of benign myoepithelioma. We should be aware that owing to its characteristic cytological features, cytopathologists may be able to make a correct diagnosis of myoepithelial carcinoma, based on multiple and adequate samplings.

Influence of in vitro biomimicked stem cell 'niche' for regulation of proliferation and differentiation of human bone marrow-derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment.

PubMed

Kim, Jae Hyung; Shin, Sang-Hyun; Li, Tian Zhu; Suh, Hwal

2016-01-01

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) undergo senescence-related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5-azacytidine was not associated with the phenomenon, but the serum-starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM-MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle-shape morphology, increased in proliferation rate by two-fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.

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.

Differentiated intraepithelial neoplasia of the vulva.

PubMed

Mulvany, Nicholas J; Allen, David G

2008-01-01

We present the clinical and pathological findings of 6 women with intraepithelial neoplasia of differentiated or simplex type (DVIN). The mean age was 68 years (range 55-82). One lesion was still in situ, whereas 5 were associated with squamous carcinoma, 4 of well-differentiated keratinizing type and 1 of poorly differentiated spindle-cell type. The invasive depth of the squamous carcinomas ranged from 0.6 to 8 mm and the surgical margins of all of the resection specimens were uninvolved by neoplastic cells. In contrast, DVIN involved the surgical margins in 5 specimens while the remaining specimen had normal surgical margins. In all 6 vulvar specimens, DVIN showed intense immunoreactivity for Ki-67 in the basal and parabasal cells while only 4 specimens showed reactivity for p53. In 5 surgical specimens with DVIN the number of CD1a cells was increased but little if any immunoreactivity could be found amongst the corresponding invasive neoplastic cells. Four squamous carcinomas also showed diffuse p53 reactivity. There was little difference in the pattern of Ki-67 expression between DVIN and squamous carcinoma. For a number of reasons, DVIN present diagnostic difficulty and considerable interobserver variation also exists. Our study suggests that Ki-67 and p16 are useful for distinguishing DVIN and classical VIN 3, whereas p53 and CD1a are useful for distinguishing DVIN and invasive squamous carcinoma. Furthermore, p53 appears to have higher specificity than sensitivity for distinguishing DVIN from normal squamous epithelium.

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

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.

Fine-needle aspiration cytology of hemangiopericytoma: A report of five cases.

PubMed

Chhieng, D; Cohen, J M; Waisman, J; Fernandez, G; Cangiarella, J

1999-08-25

Hemangiopericytoma (HPC) is a relatively rare neoplasm, accounting for approximately 2.5% of all soft tissue tumors. Its histopathology has been well documented but to the authors' knowledge reports regarding its fine-needle aspiration (FNA) cytology rarely are encountered. In the current study the authors report the cytologic findings in FNA specimens from nine confirmed cases of HPC and attempt to correlate the cytologic features with the biologic outcomes. FNA was performed with or without radiologic guidance. Corresponding sections of tissue were reviewed in conjunction with the cytologic preparations. Nine FNAs were performed in 5 patients (3 men and 2 women) with an age range of 38-77 years (mean, 56 years). Two lesions were primary soft tissue lesions arising in the lower extremities; seven were recurrent or metastatic lesions from bone (one lesion), kidney (one lesion), pelvic fossa (one lesion), lower extremities (two lesions), trunk (one lesion), and breast (one lesion). All aspirates were cellular and were comprised of single and tightly packed clusters of oval to spindle-shaped cells aggregated around branched capillaries. Basement membrane material was observed in 6 cases (67%). The nuclei were uniform and oval, with finely granular chromatin and inconspicuous nucleoli in all cases except one. No mitotic figures or areas of necrosis were identified. A correct diagnosis of HPC was made on one primary lesion and all recurrent or metastatic lesions. HPCs show a spindle cell pattern in cytologic preparations and must be distinguished from more common spindle cell lesions. The presence of branched capillaries and abundant basement membrane material supports a diagnosis of HPC. Immunohistochemistry and electron microscopy performed on FNA samples may be helpful in the differential diagnosis. FNA is a useful and accurate tool with which to confirm recurrent or metastatic HPC; however, prediction of the biologic behavior of HPC based on cytologic features is not feasible. Cancer (Cancer Cytopathol) Copyright 1999 American Cancer Society.

Orofacial haemangiopericytoma--a rare case.

PubMed

Shetty, S R; Chatra, L; Shenai, K P

2007-10-01

Haemangiopericytoma is an uncommon vascular tumour that originates from small pericapillary spindle-shaped cells. Clinical diagnosis of such a lesion is an extremely challenging task because there are very few detailed reports of such lesions and one would hardly think of a diagnosis such as haemangiopericytoma while examining a lesion. We report here a case of haemangiopericytoma arising from the infratemporal fossa involving the orbit, the lateral wall of the nasal cavity, the maxillary sinus, the maxillary alveolar process and the ethmoidal sinus. The clinical features, radiological findings and differential diagnoses to be considered, and the available treatment options, are discussed in this case report.

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

Striker Suitability Challenges in a Complex Threat Environment

DTIC Science & Technology

2008-04-23

áåÑçêãÉÇ=ÅÜ~åÖÉ=======- 163 - = = large number of wheel spindles developed fatigue cracks and had to be replaced early. Drive shafts are also failing... spindles developing fatigue cracks – drive shafts breaking – prescribed tire pressure is 80 PSI, however, with slat armor/sandbags – must maintain...drive shafts , differentials – Impairs off-road ops, larger footprint • Though not designed primarily for the urban fight (MOUT), Stryker is well-suited

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

Illusion caused by vibration of muscle spindles reveals an involvement of muscle spindle inputs in regulating isometric contraction of masseter muscles.

PubMed

Tsukiboshi, Taisuke; Sato, Hajime; Tanaka, Yuto; Saito, Mitsuru; Toyoda, Hiroki; Morimoto, Toshifumi; Türker, Kemal Sitki; Maeda, Yoshinobu; Kang, Youngnam

2012-11-01

Spindle Ia afferents may be differentially involved in voluntary isometric contraction, depending on the pattern of synaptic connections in spindle reflex pathways. We investigated how isometric contraction of masseter muscles is regulated through the activity of their muscle spindles that contain the largest number of intrafusal fibers among skeletal muscle spindles by examining the effects of vibration of muscle spindles on the voluntary isometric contraction. Subjects were instructed to hold the jaw at resting position by counteracting ramp loads applied on lower molar teeth. In response to the increasing-ramp load, the root mean square (RMS) of masseter EMG activity almost linearly increased under no vibration, while displaying a steep linear increase followed by a slower increase under vibration. The regression line of the relationship between the load and RMS was significantly steeper under vibration than under no vibration, suggesting that the subjects overestimated the ramp load and excessively counteracted it as reflected in the emergence of bite pressure. In response to the decreasing-ramp load applied following the increasing one, the RMS hardly decreased under vibration unlike under no vibration, leading to a generation of bite pressure even after the offset of the negative-ramp load until the vibration was ceased. Thus the subjects overestimated the increasing rate of the load while underestimating the decreasing rate of the load, due to the vibration-induced illusion of jaw opening. These observations suggest that spindle Ia/II inputs play crucial roles both in estimating the load and in controlling the isometric contraction of masseter muscles in the jaw-closed position.

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

Scribble Modulates the MAPK/Fra1 Pathway to Disrupt Luminal and Ductal Integrity and Suppress Tumour Formation in the Mammary Gland

PubMed Central

Godde, Nathan J.; Sheridan, Julie M.; Smith, Lorey K.; Pearson, Helen B.; Britt, Kara L.; Galea, Ryan C.; Yates, Laura L.; Visvader, Jane E.; Humbert, Patrick O.

2014-01-01

Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression. PMID:24852022

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.

Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach.

PubMed

Innocenti, Paolo; Woodward, Hannah L; Solanki, Savade; Naud, Sébastien; Westwood, Isaac M; Cronin, Nora; Hayes, Angela; Roberts, Jennie; Henley, Alan T; Baker, Ross; Faisal, Amir; Mak, Grace Wing-Yan; Box, Gary; Valenti, Melanie; De Haven Brandon, Alexis; O'Fee, Lisa; Saville, Harry; Schmitt, Jessica; Matijssen, Berry; Burke, Rosemary; van Montfort, Rob L M; Raynaud, Florence I; Eccles, Suzanne A; Linardopoulos, Spiros; Blagg, Julian; Hoelder, Swen

2016-04-28

Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model.

Decursin inhibits vasculogenesis in early tumor progression by suppression of endothelial progenitor cell differentiation and function.

PubMed

Jung, Seok Yun; Choi, Jin Hwa; Kwon, Sang-Mo; Masuda, Haruchika; Asahara, Takayuki; Lee, You-Mie

2012-05-01

Endothelial progenitor cells (EPCs) contribute to the tumor vasculature during tumor progression. Decursin isolated from the herb Angelica gigas is known to possess potent anti-inflammatory activities. Recently, we reported that decursin is a novel candidate for an angiogenesis inhibitor [Jung et al., 2009]. In this study, we investigated whether decursin regulates EPC differentiation and function to inhibit tumor vasculogenesis. We isolated AC133+ cells from human cord blood and decursin significantly decreased the number of EPC colony forming units of human cord blood-derived AC133+ cells that produce functional EPC progenies. Decursin dose-dependently decreased the cell number of EPC committing cells as demonstrated by EPC expansion studies. Decursin inhibited EPC differentiation from progenitor cells into spindle-shaped EPC colonies. Additionally, decursin inhibited proliferation and migration of early EPCs isolated from mouse bone marrow. Furthermore, decursin suppressed expression of angiopoietin-2, angiopoietin receptor Tie-2, Flk-1 (vascular endothelial growth factor receptor-2), and endothelial nitric oxide synthase in mouse BM derived EPCs in a dose-dependent manner. Decursin suppressed tube formation ability of EPCs in collaboration with HUVEC. Decursin (4 mg/kg) inhibited tumor-induced mobilization of circulating EPCs (CD34 + /VEGFR-2+ cells) from bone marrow and early incorporation of Dil-Ac-LDL-labeled or green fluorescent protein (GFP)+ EPCs into neovessels of xenograft Lewis lung carcinoma tumors in wild-type- or bone-marrow-transplanted mice. Accordingly, decursin attenuated EPC-derived endothelial cells in neovessels of Lewis lung carcinoma tumor masses grown in mice. Together, decursin likely affects EPC differentiation and function, thereby inhibiting tumor vasculogenesis in early tumorigenesis. Copyright © 2012 Wiley Periodicals, Inc.

Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes.

PubMed

Eichenlaub-Ritter, Ursula; Staubach, Nora; Trapphoff, Tom

2010-12-01

It has been known for more than half a century that the risk of conceiving a child with trisomy increases with advanced maternal age. However, the origin of the high susceptibility to nondisjunction of whole chromosomes and precocious separation of sister chromatids, leading to aneuploidy in aged oocytes and embryos derived from them, cannot be traced back to a single disturbance and mechanism. Instead, analysis of recombination patterns of meiotic chromosomes of spread oocytes from embryonal ovary, and of origins and exchange patterns of extra chromosomes in trisomies, as well as morphological and molecular studies of oocytes and somatic cells from young and aged females, show chromosome-specific risk patterns and cellular aberrations related to the chronological age of the female. In addition, analysis of the function of meiotic- and cell-cycle-regulating genes in oogenesis, and the study of the spindle and chromosomal status of maturing oocytes, suggest that several events contribute synergistically to errors in chromosome segregation in aged oocytes in a chromosome-specific fashion. For instance, loss of cohesion may differentially predispose chromosomes with distal or pericentromeric chiasmata to nondisjunction. Studies on expression in young and aged oocytes from human or model organisms, like the mouse, indicate that the presence and functionality/activity of gene products involved in cell-cycle regulation, spindle formation and organelle integrity may be altered in aged oocytes, thus contributing to a high risk of error in chromosome segregation in meiosis I and II. Genes that are often altered in aged mouse oocytes include MCAK (mitotic-centromere-associated protein), a microtubule depolymerase, and AURKB (Aurora kinase B), a protein of the chromosomal passenger complex that has many targets and can also phosphorylate and regulate MCAK localization and activity. Therefore we explored the role of MCAK in maturing mouse oocytes by immunofluorescence, overexpression of a MCAK-EGFP (enhanced green fluorescent protein) fusion protein, knockdown of MCAK by RNAi (RNA interference) and inhibition of AURKB. The observations suggest that MCAK is involved in spindle regulation, chromosome congression and cell-cycle control, and that reductions in mRNA and protein in a context of permissive SAC (spindle assembly checkpoint) predispose to aneuploidy. Failure to recruit MCAK to centromeres and low expression patterns, as well as disturbances in regulation of enzyme localization and activity, e.g. due to alterations in activity of AURKB, may therefore contribute to maternal age-related rises in aneuploidy in mammalian oocytes.

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

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.

Spontaneous Differentiation of Human Mesenchymal Stem Cells on Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold.

PubMed

Sonomoto, Koshiro; Yamaoka, Kunihiro; Kaneko, Hiroaki; Yamagata, Kaoru; Sakata, Kei; Zhang, Xiangmei; Kondo, Masahiro; Zenke, Yukichi; Sabanai, Ken; Nakayamada, Shingo; Sakai, Akinori; Tanaka, Yoshiya

2016-01-01

Mesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints. MSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro. Healthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts. Our PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction.

[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.

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

PubMed

Jackson, Hartland W; Waterhouse, Paul; Sinha, Ankit; Kislinger, Thomas; Berman, Hal K; Khokha, Rama

2015-03-01

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

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

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

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.

Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.

PubMed

Sacristan, Carlos; Kops, Geert J P L

2015-01-01

Error-free chromosome segregation relies on stable connections between kinetochores and spindle microtubules. The spindle assembly checkpoint (SAC) monitors such connections and relays their absence to the cell cycle machinery to delay cell division. The molecular network at kinetochores that is responsible for microtubule binding is integrated with the core components of the SAC signaling system. Molecular-mechanistic understanding of how the SAC is coupled to the kinetochore-microtubule interface has advanced significantly in recent years. The latest insights not only provide a striking view of the dynamics and regulation of SAC signaling events at the outer kinetochore but also create a framework for understanding how that signaling may be terminated when kinetochores and microtubules connect. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Giant cell angiofibroma or localized periorbital lymphedema?

PubMed

Lynch, Michael C; Chung, Catherine G; Specht, Charles S; Wilkinson, Michael; Clarke, Loren E

2013-12-01

Giant cell angiofibroma represents a rare soft tissue neoplasm with a predilection for the orbit. We recently encountered a mass removed from the lower eyelid of a 56-year-old female that histopathologically resembled giant cell angiofibroma. The process consisted of haphazardly arranged CD34-positive spindled and multinucleated cells within an edematous, densely vascular stroma. However, the patient had recently undergone laryngectomy and radiotherapy for a laryngeal squamous cell carcinoma. A similar mass had arisen on the contralateral eyelid, and both had developed several months post-therapy. Lymphedema of the orbit can present as tumor-like nodules and in some cases may share histopathologic features purported to be characteristic of giant cell angiofibroma. A relationship between giant cell angiofibroma and lymphedema has not been established, but our case suggests there may be one. The potential overlap of these two conditions should be recognized, as should other entities that may enter the differential diagnosis. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

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

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

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

Fibrosarcoma of the jaws: two cases of primary tumors with intraosseous growth.

PubMed

Angiero, Francesca; Rizzuti, Tommaso; Crippa, Rolando; Stefani, Michele

2007-01-01

Fibrosarcoma (FS) is a malignant mesenchymal neoplasm of the fibroblasts that rarely affects the oral cavity. Two cases of primary FS of the jaws with intraosseous growth (2 men, aged 53 and 71 years) are described. Microscopically, in one case the tumor showed an intense proliferation of spindle-shaped cells, varying little in size and shape and arranged in parallel bands, partly crossing each other, with significant mitotic activity and nuclear pleomorphism; the second case was characterized by low cellularity comprising spindle-shaped cells, deposited in a variably fibrous and myxoid stroma. On immunohistochemistry, cells in both cases were strongly immunoreactive for MIB-1 and vimentin, focally positive for CD68, and negative for S-100 protein, pancytokeratin, HMB45, CD34, desmin, smooth muscle actin (SMA) and epithelial membrane antigen (EMA). Based on clinical, histological and immunohistochemical findings, the final diagnosis was FS in the first case, myxofibrosarcoma in the second. Treatment was radical surgery with mandibular reconstruction. After two years, the first patient displayed multiple metastases and died during the third year after the initial diagnosis; the second patient was still alive and doing well five years after treatment. We discuss the differential diagnosis versus other forms of sarcoma, examining the morphological appearance that is frequently very similar, the immunohistochemical expression of MIB-1, vimentin, S-100, CD-34, CD68, EMA, as well as conventional clinicopathological features that may help to distinguish FS from other sarcomas.

[Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

PubMed

Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

2017-01-01

Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

Diagnostic utility of aP2/FABP4 expression in soft tissue tumours.

PubMed

Kashima, T G; Turley, H; Dongre, A; Pezzella, F; Athanasou, N A

2013-04-01

Adipocyte P2 (aP2), also known as fatty acid-binding protein 4 (FABP4), is a fatty acid-binding protein found in the cytoplasm of cells of adipocyte differentiation. In this study, we examined a large number of soft tissue tumours with a commercial polyclonal anti-aP2/FABP4 antibody and a newly developed mouse monoclonal antibody raised against this protein to determine the diagnostic utility of aP2/FABP4 as a marker of tumours of adipose differentiation. A mouse monoclonal antibody, clone 175d, was raised against a mixture of synthetic peptides corresponding to the amino acid sequence of residues 10-28 and 121-132 of the human aP2/FABP4 protein. Antigen expression with polyclonal and monoclonal antibodies was immunohistochemically determined in paraffin sections of normal adipose tissue and a wide range of benign and malignant primary soft tissue tumours (n = 200). aP2/FABP4 was expressed around the cytoplasmic lipid vacuole in white and brown fat cells in benign lipomas and hibernomas. Immature fat cells and lipoblasts in spindle cell/pleomorphic lipoma, atypical lipomatous tumour/well-differentiated liposarcoma, myxoid/round cell liposarcoma and pleomorphic liposarcoma also reacted strongly for aP2/FABP4. No specific staining was seen in non-adipose benign and malignant mesenchymal and non-mesenchymal tumours. aP2/FABP4 is expressed by mature and immature fat cells and is a marker of tumours of adipose differentiation. Immunophenotypic aP2/FABP4 expression is useful in identifying lipoblasts, and immunohistochemistry with polyclonal/monoclonal anti-aP2/FABP4 antibodies should be useful in distinguishing liposarcoma from other malignancies, particularly round cell, myxoid and pleomorphic soft tissue sarcomas.

Embryonic development of the axial column in the little skate, Leucoraja erinacea.

PubMed

Criswell, Katharine E; Coates, Michael I; Gillis, J Andrew

2017-03-01

The morphological patterns and molecular mechanisms of vertebral column development are well understood in bony fishes (osteichthyans). However, vertebral column morphology in elasmobranch chondrichthyans (e.g., sharks and skates) differs from that of osteichthyans, and its development has not been extensively studied. Here, we characterize vertebral development in an elasmobranch fish, the little skate, Leucoraja erinacea, using microCT, paraffin histology, and whole-mount skeletal preparations. Vertebral development begins with the condensation of mesenchyme, first around the notochord, and subsequently around the neural tube and caudal artery and vein. Mesenchyme surrounding the notochord differentiates into a continuous sheath of spindle-shaped cells, which forms the precursor to the mineralized areolar calcification of the centrum. Mesenchyme around the neural tube and caudal artery/vein becomes united by a population of mesenchymal cells that condenses lateral to the sheath of spindle-shaped cells, with this mesenchymal complex eventually differentiating into the hyaline cartilage of the future neural arches, hemal arches, and outer centrum. The initially continuous layers of areolar tissue and outer hyaline cartilage eventually subdivide into discrete centra and arches, with the notochord constricted in the center of each vertebra by a late-forming "inner layer" of hyaline cartilage, and by a ring of areolar calcification located medial to the outer vertebral cartilage. The vertebrae of elasmobranchs are distinct among vertebrates, both in terms of their composition (i.e., with centra consisting of up to three tissues layers-an inner cartilage layer, a calcified areolar ring, and an outer layer of hyaline cartilage), and their mode of development (i.e., the subdivision of arch and outer centrum cartilage from an initially continuous layer of hyaline cartilage). Given the evident variation in patterns of vertebral construction, broad taxon sampling, and comparative developmental analyses are required to understand the diversity of mechanisms at work in the developing axial skeleton of vertebrates. J. Morphol. 278:300-320, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

PubMed

Yong, Kwon Joong; Milenic, Diane E; Baidoo, Kwamena E; Brechbiel, Martin W

2014-01-01

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

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.

Fault tolerant linear actuator

DOEpatents

Tesar, Delbert

2004-09-14

In varying embodiments, the fault tolerant linear actuator of the present invention is a new and improved linear actuator with fault tolerance and positional control that may incorporate velocity summing, force summing, or a combination of the two. In one embodiment, the invention offers a velocity summing arrangement with a differential gear between two prime movers driving a cage, which then drives a linear spindle screw transmission. Other embodiments feature two prime movers driving separate linear spindle screw transmissions, one internal and one external, in a totally concentric and compact integrated module.

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

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.

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.

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.

Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe.

PubMed

Yukawa, Masashi; Yamada, Yusuke; Yamauchi, Tomoaki; Toda, Takashi

2018-01-04

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by kinesin-5, which generates outward force. This outward force is balanced by antagonistic inward force elicited by kinesin-14 and/or dynein. In fission yeast, two kinesin-14 proteins, Pkl1 and Klp2, play an opposing role against the kinesin-5 motor protein Cut7. However, how the two kinesin-14 proteins coordinate individual activities remains elusive. Here, we show that although deletion of either pkl1 or klp2 rescues temperature-sensitive cut7 mutants, deletion of only pkl1 can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, whereas Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two kinesin-14 proteins. Interestingly, human kinesin-14 (KIFC1 or HSET) can replace either Pkl1 or Klp2. Moreover, overproduction of HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism whereby two different Kinesin-14 motor proteins exert their antagonistic roles against kinesin-5 in a spatially distinct manner. © 2018. Published by The Company of Biologists Ltd.

Aspiration biopsy of mammary analogue secretory carcinoma of accessory parotid gland: another diagnostic dilemma in matrix-containing tumors of the salivary glands.

PubMed

Levine, Pascale; Fried, Karen; Krevitt, Lane D; Wang, Beverly; Wenig, Bruce M

2014-01-01

Mammary analogue secretory carcinoma (MASC) is a newly described rare salivary gland tumor, which shares morphologic features with acinic cell carcinoma, low-grade cystadenocarcinoma, and secretory carcinoma of the breast. This is the first reported case of MASC of an accessory parotid gland detected by aspiration biopsy with radiologic and histologic correlation in a 34-year-old patient. Sonographically-guided aspiration biopsy showed cytologic features mimicking those of low-grade mucoepidermoid carcinoma, including sheets of bland epithelial cells, dissociated histiocytoid cells with intracytoplasmic mucinous material, and spindle cells lying in a web-like matrix. Histologic sections showed a circumscribed tumor with microcystic spaces lined by bland uniform epithelial cells and containing secretory material. The tumor cells expressed mammaglobin and BRST-2. The cytologic features, differential diagnosis, and pitfalls are discussed. The pathologic stage was pT1N0. The patient showed no evidence of disease at 1 year follow-up. Copyright © 2012 Wiley Periodicals, Inc.

Squamous cell carcinoma of the lung with highly proliferating fibromatosis-like stroma: a rare phenomenon.

PubMed

Tajima, Shogo; Takanashi, Yusuke; Koda, Kenji

2015-01-01

Few cases of carcinoma with exuberant stromal proliferation have been documented, apart from scirrhous carcinoma. To the best of our knowledge, previous cases of carcinoma exhibiting exuberant stromal proliferation have exclusively been reported in the thyroid gland, specifically as papillary carcinoma. The exuberant stromal proliferation has been recognized to be similar to either fibromatosis or nodular fasciitis. Herein, we report a case of a 74-year-old Japanese man whose tumor in the upper lobe of his right lung displayed highly proliferating stroma with dispersed, poorly differentiated squamous cell carcinoma nests. The stromal spindle cells (fibroblasts/myofibroblasts) had similar molecular profiles to those typically observed in fibromatosis rather than nodular fasciitis, resulting in the designation of "fibromatosis-like" stroma. The presence of carcinoma cells, along with stromal cells, expressing TGF-β in this case likely fostered continuous stromal proliferation, presumably in conjunction with the unique microenvironment in which the carcinoma cells were present.

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

Plane of vertebral movement eliciting muscle lengthening history in the low back influences the decrease in muscle spindle responsiveness of the cat

PubMed Central

Ge, Weiqing; Cao, Dong-Yuan; Long, Cynthia R.

2011-01-01

Proprioceptive feedback is thought to play a significant role in controlling both lumbopelvic and intervertebral orientations. In the lumbar spine, a vertebra's positional history along the dorsal-ventral axis has been shown to alter the position, movement, and velocity sensitivity of muscle spindles in the multifidus and longissimus muscles. These effects appear due to muscle history. Because spinal motion segments have up to 6 degrees of freedom for movement, we were interested in whether the axis along which the history is applied differentially affects paraspinal muscle spindles. We tested the null hypothesis that the loading axis, which creates a vertebra's positional history, has no effect on a lumbar muscle spindle's subsequent response to vertebral position or movement. Identical displacements were applied along three orthogonal axes directly at the L6 spinous process using a feedback motor system under displacement control. Single-unit nerve activity was recorded from 60 muscle spindle afferents in teased filaments from L6 dorsal rootlets innervating intact longissimus or multifidus muscles of deeply anesthetized cats. Muscle lengthening histories along the caudal-cranial and dorsal-ventral axis, compared with the left-right axis, produced significantly greater reductions in spindle responses to vertebral position and movement. The spinal anatomy suggested that the effect of a lengthening history is greatest when that history had occurred along an axis lying within the anatomical plane of the facet joint. Speculation is made that the interaction between normal spinal mechanics and the inherent thixotropic property of muscle spindles poses a challenge for feedback and feedforward motor control of the lumbar spine. PMID:21960662

Evaluation of an anal sac adenocarcinoma tumor in a Spitz dog.

PubMed

Javanbakht, Javad; Tavassoli, Abbas; Sabbagh, Atefeh; Hassan, Mehdy Aghamohammmad; Samakkhah, Shohreh Alian; Shafiee, Radmehr; Lakzian, Ali; Ghalee, Vahideh Rahmani; Gharebagh, Sonia Shoja

2013-01-01

A 9-year-old emasculated male Spitz with tenesmus and constipation had a subcutaneous mass at the left ventral aspect of the anus with history of polyuria and polydipsia. A complete blood cell count, serum biochemistry panel, and urinalysis (cystocentesis sample) were evaluated. Abnormalities in the serum biochemistry panel included a mildly elevated serum cholesterol concentration (7.28 mmol/L; reference interval, 2.70-5.94 mmol/L), increased serum alkaline phosphatase activity (184 U/L; reference interval, 9-90 U/L), alanine transaminase (122 U/L; reference interval, 5-60 U/L) activity and aspartate aminotransferase (80 U/L; reference interval, 5-55 U/L) activity, severe increased total calcium concentration (16.3 mg/dL; reference interval, 8.2-12.4 mg/dL or 9.3-11.4 mg/dL), and decreased total calcium concentration (3.4 mg/dL, reference interval, 2.5-5.6mg/dL). Furthermore, testing revealed an increased intact parathyroid hormone concentration (38.6 pmol/L; reference interval, 3-17 pmol/L). On cytologic and histopathologic examinations, various types of cells were observed. Most of the cells were oval to polygonal and had elliptical or elongate nuclei and a moderate amount of pale to basophilic cytoplasm. The remaining cells had round to oval nuclei and pale to basophilic cytoplasm. Cells of both types were loosely adhered to each other and were arranged in rosette-like structures. Both neoplastic cell types had fine homogenous chromatin and either a small indistinct nucleolus or no visible nucleolus. Mild anisokaryosis and anisocytosis were observed. Histologically, the mass consists of glandular structures formed by cuboidal cells admixed with bundles of spindle cells. Based on location and histologic features, the final diagnosis was adenocarcinoma of the apocrine gland of the anal sac, which should be included as a cytologic differential diagnosis when spindle cells and typical epithelial cells are observed in masses in the region of the anal sac of dogs.

GTPase Ran strongly accumulates at the kinetochores of somatic chromosomes in the spermatogonial mitoses of Acricotopus lucidus (Diptera, Chironomidae).

PubMed

Staiber, Wolfgang

2014-07-01

Unequal chromosome segregation and spindle formation occurs in the last gonial mitosis in the germ line of the chironomid Acricotopus lucidus. During this differential mitosis, all germ line-limited chromosomes (=Ks) migrate undivided to only one pole of the cell, while the somatic chromosomes (=Ss) first remain in the metaphase plane, and with the arrival of the Ks at the pole, they then separate equally. The evolutionarily conserved GTPase Ran plays a crucial role in many cellular processes. This includes the regulation of microtubule nucleation and stabilisation at kinetochores and of spindle assembly during mitosis, which is promoted by a RanGTP concentration gradient that forms around the mitotic chromosomes (Kalab et al. in Science 295:2452-2456, 2002, Nature 440:697-701, 2006). In the present study, a strong accumulation of Ran was detected by immunofluorescence at the kinetochores of the Ss in normal gonial and differential gonial mitoses of males of A. lucidus. In contrast, no Ran accumulation was observed at the kinetochores of the Ss in the metaphases of brain ganglia mitoses or of aberrant spermatocytes or in metaphases I and II of spermatocyte meiotic divisions. Likewise, there was no accumulation at the kinetochores of Drosophila melanogaster mitotic chromosomes from larval brains. The specific accumulation of Ran at the kinetochores of the Ss in differential gonial mitoses of A. lucidus strongly suggests that Ran is involved in a mechanism acting in this exceptional mitosis, which retains the Ss at the metaphase plane and prevents a premature separation and unequal segregation of the Ss during monopolar migration of the Ks.

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.

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

PubMed

Wagoner, Michael D; Bohrer, Laura R; Aldrich, Benjamin T; Greiner, Mark A; Mullins, Robert F; Worthington, Kristan S; Tucker, Budd A; Wiley, Luke A

2018-05-08

The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4 , SOX2 , KLF4 and c-MYC Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3 , SOX9 , TFAP2A , SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1 , COL8A1 , COL8A2 , AQP1 and CDH2 and the proteins ZO-1, N-Cad, AQP-1 and Na + /K + ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics. © 2018. Published by The Company of Biologists Ltd.

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

Mesenchymal stromal cells (MSCs) induce ex vivo proliferation and erythroid commitment of cord blood haematopoietic stem cells (CB-CD34+ cells)

PubMed Central

Perucca, Simone; Di Palma, Andrea; Piccaluga, Pier Paolo; Gemelli, Claudia; Zoratti, Elisa; Bassi, Giulio; Giacopuzzi, Edoardo; Lojacono, Andrea; Borsani, Giuseppe; Tagliafico, Enrico; Scupoli, Maria Teresa; Bernardi, Simona; Zanaglio, Camilla; Cattina, Federica; Cancelli, Valeria; Malagola, Michele; Krampera, Mauro; Marini, Mirella; Almici, Camillo; Ferrari, Sergio; Russo, Domenico

2017-01-01

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis. PMID:28231331

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.

Meningioma-like tumor of the skin. An ultrastructural and immunohistochemical study.

PubMed

Barr, R J; Yi, E S; Jensen, J L; Wuerker, R B; Liao, S Y

1993-08-01

Three unusual cutaneous tumors are described along with ultrastructural and immunohistochemical studies. All lesions were asymptomatic red-brown papulonodules. Light microscopic examination revealed a whorled configuration of spindle-shaped cells, some concentrically arranged around blood vessels. Immunohistochemical panels exhibited positive staining only with antibody to vimentin and negative staining with antibodies against S-100 protein, muscle markers, cytokeratin, epithelial membrane antigen, Leu 7, type IV collagen, and factor XIIIa, ruling out obvious nevomelanocytic, nerve sheath, meningothelial, smooth muscle, and perithelial differentiation. Electron microscopic examination demonstrated cells producing poorly formed collagen fibrils, sparse collagen fibers, and possessing occasional ill-defined intercellular junctions between their elongated cell processes. This rare tumor is considered to be either an immature fibrohistiocytic or possibly a nerve sheath neoplasm with striking similarities to so-called canine hemangiopericytoma. Because the prominent whorled pattern was reminiscent of meningioma, the lesion was referred to as meningioma-like tumor of the skin.

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

Diagnostic Exercise: Circling and Behavioral Changes in a Cat.

PubMed

Faller, K; Leach, J; Gutierrez-Quintana, R; Finck, M; Hammond, G; Penderis, J; Marchesi, F

2015-07-01

A 4-year old spayed male domestic shorthair cat was presented with a history of circling and behavioral changes. Neurologic examination showed mild proprioceptive deficits. The lesion was localized in the forebrain, and magnetic resonance imaging revealed the presence of a large midline intracranial mass extending from the frontal lobe to the tentorial region of the brain. Euthanasia was elected due to poor prognosis. Histopathologic evaluation confirmed the presence of a mass composed by sheets and aggregates of large round/polygonal cells and multinucleate cells associated with deposits of cholesterol clefts, scattered hemorrhages and hemosiderin-laden macrophages. Immunohistochemistry showed that the round/polygonal cells and multinucleate cells were strongly positive for major histocompatibility complex class II antigen, variably positive for CD18, and occasionally positive for S100. Subsets of spindle cells showing variable expression of vimentin, S100, and neuron-specific enolase were also present. The final diagnosis was cholesterol granuloma. Differential diagnosis with meningioma is discussed. © The Author(s) 2014.

[Clinicopathologic characteristics of hemangiopericytoma/solitary fibrous tumor with giant cells].

PubMed

Wang, Hai-yan; Fan, Qin-he; Gong, Qi-xing; Wang, Zheng

2009-03-01

To study the pathological characteristics, diagnosis and differential diagnoses of hemangiopericytoma-solitary fibrous tumor with giant cells. Pathological characteristics of seven cases of orbital and extraorbital hemangiopericytoma-solitary fibrous tumors with giant cells were evaluated by HE and immunohistochemistry (EnVision method). Two cases were located in the orbit, one of which had recurred. Five cases were located in the extraorbital regions. Histologically, the tumors were well-circumscribed and composed of non-atypical, round to spindle cells with collagen deposition in the stroma. The tumors had prominent vasculatures and in areas, pseudovascular spaces lined by multinucleated giant cells lining which were also present in the stroma. Immunohistochemically, both neoplastic cells and multinucleate giant cells expressed CD34. Seven patients underwent tumor excision and were well and without tumor recurrence upon the clinical follow-up. Hemangiopericytoma-solitary fibrous tumor with giant cells is an intermediate soft tissue tumor. It typically involves the orbital or extraorbital regions. Histologically, the tumor should be distinguished from giant cell fibroblastoma, pleomorphic hyalinzing angiectatic tumor of soft part and angiomatoid fibrous histiocytoma.

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.

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.

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

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

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

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.

Thrombopoietin-induced Polyploidization of Bone Marrow Megakaryocytes Is Due to a Unique Regulatory Mechanism in Late Mitosis

PubMed Central

Nagata, Yuka; Muro, Yoshinao; Todokoro, Kazuo

1997-01-01

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process. PMID:9334347

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.

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

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.

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

Dedifferentiated Liposarcoma Mimicking a Primary Colon Mass.

PubMed

Hollowoa, Blake; Lamps, Laura W; Mizell, Jason S; English, George W; Bridge, Julia A; Ram, Roopa; Gardner, Jerad M

2018-04-01

Dedifferentiated liposarcoma is typically a nonlipogenic high-grade sarcoma that arises from well-differentiated liposarcoma. It most commonly presents as a large mass in the retroperitoneum. Significant involvement of the gastrointestinal tract by dedifferentiated liposarcoma is uncommon. We present a unique case of dedifferentiated liposarcoma radiographically mimicking a primary colon mass with resulting intussusception; stranding of the adjacent adipose tissue was presumed to be a secondary reactive change. On histopathologic analysis of the hemicolectomy specimen, a high-grade sarcoma was seen growing through the colonic wall, and the majority of the surrounding pericolonic adipose tissue was actually composed of well-differentiated liposarcoma with characteristic fibrous bands rather than benign fat with reactive fibrosis. This case raises awareness that well-differentiated liposarcoma and dedifferentiated liposarcoma can rarely present as a primary intestinal mass mimicking colon cancer or other more common entities. When radiographic examination shows a perigastrointestinal or retroperitoneal fatty mass and/or stranding of the fat adjacent to a solid gastrointestinal mass, this unusual scenario should be considered in the radiologic differential diagnosis. Pathologists should keep dedifferentiated liposarcoma in the initial histologic differential diagnosis for any high-grade spindle cell tumor of the retroperitoneum or intra-abdominal visceral organs.

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

Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development

PubMed Central

Kohara, Yukihiro; Soeta, Satoshi; Izu, Yayoi; Amasaki, Hajime

2015-01-01

In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle cells in the osteonal cavities. These results indicate that Col VI provides a characteristic microenvironment in the osteonal cavity of the primary osteon, and that differentiation and proliferation of the osteoblast lineage occur in the Col VI-immunoreactive area. Interaction of Col VI and NG2 may be involved in the structural organization of the primary osteon by regulating osteoblast lineages. PMID:25943007

Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development.

PubMed

Kohara, Yukihiro; Soeta, Satoshi; Izu, Yayoi; Amasaki, Hajime

2015-05-01

In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle cells in the osteonal cavities. These results indicate that Col VI provides a characteristic microenvironment in the osteonal cavity of the primary osteon, and that differentiation and proliferation of the osteoblast lineage occur in the Col VI-immunoreactive area. Interaction of Col VI and NG2 may be involved in the structural organization of the primary osteon by regulating osteoblast lineages. © 2015 Anatomical Society.

Alternate-1 and Alternate-2 Orientations in Interchange (Reciprocal Translocation) Quadrivalents

PubMed Central

Rickards, Geoffrey K.

1983-01-01

The dynamic properties of the spindle, and published numerical data, argue that the differentiation of alternate-1 and alternate-2 orientations of an interchange quadrivalent is real and meaningful, contra Boussy (1982). PMID:17246129

Spindle epithelial tumor with thymus-like differentiation of the thyroid in a 70-year-old man.

PubMed

Lee, Sunhye; Kim, Yon Seon; Lee, Jeong Hyeon; Hwang, Sung Ho; Oh, Yu-Hwan; Ko, Byung Kyun; Ham, Soo-Youn

2018-06-01

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a very rare tumor of the thyroid gland mostly occurring in young patients. The imaging findings of SETTLE tumors are yet to be defined. However, they are usually described as well-defined heterogeneously enhanced masses on CT scan. The current case has the potential growth as compared with a 2009 chest radiography. We took into account the possibility of SETTLE in the case of a bulky mass in patients over 70 years old, particularly in the lower neck. Herein, we report a case of the oldest patient so far. The patient underwent a right lobectomy of the thyroid and mass excision. Follow-up CT scans after 6 months revealed no local recurrence. Surgery is the gold standard treatment for SETTLE. Chemotherapy and radiotherapy could be another possible option for patients with advanced stage SETTLE.

Comparison of the effects of BPA and BPAF on oocyte spindle assembly and polar body release in mice.

PubMed

Nakano, Kei; Nishio, Manami; Kobayashi, Norio; Hiradate, Yuuki; Hoshino, Yumi; Sato, Eimei; Tanemura, Kentaro

2016-04-01

Bisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 μg/ml) for 18 h. At concentrations of 50 and 100 μg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 μg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 μg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 μg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.

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).

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.

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

PubMed

Inoue, D; Santiago, P; Horne, W C; Baron, R

1997-10-03

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

[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.

Intra-abdominal primary monophasic synovial sarcoma with hemangiopericytoma-like areas.

PubMed

Rao, Lakshmi; Jaiprakash, Padmapriya; Palankar, Nagaraj; Gowda, Vinay

2013-01-01

We report a case of retroperitoneal intra-abdominal primary monophasic synovial sarcoma (SS) with hemangiopericytomatous (HPC) pattern in a 25-year-old male arising from the triangular ligament on the superior surface of liver encasing the inferior vena cava (IVC) and masquerading as a hepatic tumor. A large heterogeneously enhancing, well defined, lobulated, exophytic lesion was seen involving segment VIII of the liver with foci of calcification in the periphery. A biopsy, followed by total resection of the tumor, showed a spindle cell sarcoma with HPC pattern, which was consistent with monophasic SS on histology and immunohistochemistry. The unusual clinical presentation, radiology, pathology, and differential diagnosis will be discussed in detail.

A resected perivascular epithelioid cell tumor (PEComa) of the pancreas diagnosed using endoscopic ultrasound-guided fine-needle aspiration.

PubMed

Okuwaki, Kosuke; Kida, Mitsuhiro; Masutani, Hironori; Yamauchi, Hiroshi; Katagiri, Hiroyuki; Mikami, Tetuo; Miyazawa, Shiro; Iwai, Tomohisa; Takezawa, Miyoko; Imaizumi, Hiroshi; Koizumi, Wasaburo

2013-01-01

Primary perivascular epithelioid cell tumors (PEComas) of the pancreas are extremely rare. We herein report our experience with a patient who had a primary PEComa of the pancreas that was diagnosed by the preoperative histopathological examination of a biopsy specimen obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). The patient was a 43-year-old woman whose chief complaint was abdominal pain. Imaging studies revealed a pancreatic tumor. Gastrointestinal stromal tumor (GIST), solid pseudopapillary tumor and neuroendocrine tumor were considered in the differential diagnosis. A histopathological examination of a specimen of the tumor obtained using EUS-FNA showed spindle-shaped tumor cells with enlarged nuclei and eosinophilic cytoplasm. The tumor cells proliferated in a sheet-like fashion and stained positive for the melanoma-associated antigen HMB-45. A PEComa was thus diagnosed. If an adequate tumor specimen can be obtained using EUS-FNA, immunostaining may facilitate the diagnosis of extremely rare diseases and therefore assist in deciding the treatment policy.

Pancreatic perivascular epithelioid cell tumor: A case report with clinicopathological features and a literature review.

PubMed

Jiang, Hui; Ta, Na; Huang, Xiao-Yi; Zhang, Ming-Hua; Xu, Jing-Jing; Zheng, Kai-Lian; Jin, Gang; Zheng, Jian-Ming

2016-04-07

Perivascular epithelioid cell tumor (PEComa) of the pancreas is an unusual tumor deriving from mesenchyma. This paper described a case of pancreatic PEComa, which was initially suspected as neuroendocrine carcinoma by biopsy, and therefore surgical treatment was recommended due to undetermined diagnosis. Examination of the surgical specimen under a microscope showed that the tumor cell's morphology was epithelioid or spindle-shaped, and ranged in a nested pattern. Additionally, these cells had a large extent of acidophilic cytoplasm, no mitotic figures, and expressed HMB-45, melan-p, and smooth muscle actin immunohistochemically. Pathological examination indicated that PEComa originated from the pancreas, but symptoms related to tuberous sclerosis were absent. Since PEComa is extremely rare in the pancreas, it is likely to be ignored in differential diagnosis. In conclusion, our article highlighted the clinicopathological features of PEComa, and we conducted a literature review focusing on PEComa so as to deepen the understanding of this tumor type.

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.

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

Propylthiouracil, independent of its antithyroid effect, promotes vascular smooth muscle cells differentiation via PTEN induction.

PubMed

Chen, Wei-Jan; Pang, Jong-Hwei S; Lin, Kwang-Huei; Lee, Dany-Young; Hsu, Lung-An; Kuo, Chi-Tai

2010-01-01

Propylthiouracil (PTU), independent of its antithyroid effect, is recently found to have an antiatherosclerotic effect. The aim of this study is to determine the impact of PTU on phenotypic modulation of vascular smooth muscle cells (VSMCs), as phenotypic modulation may contribute to the growth of atherosclerotic lesions and neointimal formation after arterial injury. Propylthiouracil reduced neointimal formation in balloon-injured rat carotid arteries. In vitro, PTU may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of SMC differentiation marker contractile proteins, including calponin and smooth muscle (SM)-myosin heavy chain (SM-MHC). Transient transfection studies in VSMCs demonstrated that PTU induced the activity of SMC marker genes (calponin and SM-MHC) promoters, indicating that PTU up-regulates these genes expression predominantly at the transcriptional level. Furthermore, PTU enhanced the expression of PTEN and inhibition of PTEN by siRNA knockdown blocked PTU-induced activation of contractile proteins expression and promoter activity. In the rat carotid injury model, PTU reversed the down-regulation of contractile proteins and up-regulated PTEN in the neointima induced by balloon injury. Propylthiouracil promotes VSMC differentiation, at lest in part, via induction of the PTEN-mediated pathway. These findings suggest a possible mechanism by which PTU may contribute to its beneficial effects on atherogenesis and neointimal formation after arterial injury.

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.

A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on β-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro.

PubMed

An, Shaofeng; Gao, Yan; Huang, Xiangya; Ling, Junqi; Liu, Zhaohui; Xiao, Yin

2015-05-01

The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. β-tricalcium phosphate (β-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous β-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell-Counting kit-8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the β-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the β-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the β-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on β-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects on hPDLCs than the inorganic elements.

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.

Suspended animation in C. elegans requires the spindle checkpoint.

PubMed

Nystul, Todd G; Goldmark, Jesse P; Padilla, Pamela A; Roth, Mark B

2003-11-07

In response to environmental signals such as anoxia, many organisms enter a state of suspended animation, an extreme form of quiescence in which microscopically visible movement ceases. We have identified a gene, san-1, that is required for suspended animation in Caenorhabditis elegans embryos. We show that san-1 functions as a spindle checkpoint component in C. elegans. During anoxia-induced suspended animation, embryos lacking functional SAN-1 or a second spindle checkpoint component, MDF-2, failed to arrest the cell cycle, exhibited chromosome missegregation, and showed reduced viability. These data provide a model for how a dynamic biological process is arrested in suspended animation.

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.

The Justy mutant mouse strain produces a spontaneous murine model of salivary gland cancer with myoepithelial and basal cell differentiation

PubMed Central

Simons, Andrean L.; Lu, Ping; Gibson-Corley, Katherine N.; Robinson, Robert A.; Meyerholz, David K.; Colgan, John D.

2013-01-01

We previously identified a novel mutant mouse strain on the C3HeB/FeJ background named Justy. This strain bears a recessive mutation in the Gon4l gene that greatly reduces expression of the encoded protein, a nuclear factor implicated in transcriptional regulation. Here, we report that Justy mutant mice aged 6 months or older spontaneously developed carcinomas with myoepithelial and basaloid differentiation in salivary glands with an incidence of ~25%. Tumors developed proximate to submandibular glands and to a lesser extent in the sublingual and parotid glands. Histologically, tumors often had central cavitary lesions filled with necrotic debris that was lined by tumors cells and had spindle and epithelioid cell differentiation with lesser basaloid to clear cell features. Tumor tissue often had variable evidence of a high mitotic rate, pleomorphism and invasion into adjacent salivary glands. Neoplastic cells had diffuse immunoreactivity for pancytokeratin (AE1/AE3) and p63. While CK5/6 immunostaining was seen in the much of the tumor cells, it was often lacking in pleomorphic areas. Tumor cells lacked immunoreactivity for alpha-smooth muscle actin, S100, c-Kit and glial fibrillary acid protein. Additionally, tumors had immunoreactivity for phosphorylated and total epidermal growth factor receptor (EGFR), suggesting that EGFR signaling may participate in growth regulation of these tumors. These findings indicate that the salivary gland carcinomas occur spontaneously in Justy mice and that these tumors may offer a valuable model for study of EGFR regulation. Combined, our data suggest that Justy mice warrant further investigation for use as a mouse model for human salivary gland neoplasia. PMID:23608756

Distribution of TTX-sensitive voltage-gated sodium channels in primary sensory endings of mammalian muscle spindles.

PubMed

Carrasco, Dario I; Vincent, Jacob A; Cope, Timothy C

2017-04-01

Knowledge of the molecular mechanisms underlying signaling of mechanical stimuli by muscle spindles remains incomplete. In particular, the ionic conductances that sustain tonic firing during static muscle stretch are unknown. We hypothesized that tonic firing by spindle afferents depends on sodium persistent inward current (INaP) and tested for the necessary presence of the appropriate voltage-gated sodium (NaV) channels in primary sensory endings. The NaV 1.6 isoform was selected for both its capacity to produce INaP and for its presence in other mechanosensors that fire tonically. The present study shows that NaV 1.6 immunoreactivity (IR) is concentrated in heminodes, presumably where tonic firing is generated, and we were surprised to find NaV 1.6 IR strongly expressed also in the sensory terminals, where mechanotransduction occurs. This spatial pattern of NaV 1.6 IR distribution was consistent for three mammalian species (rat, cat, and mouse), as was tonic firing by primary spindle afferents. These findings meet some of the conditions needed to establish participation of INaP in tonic firing by primary sensory endings. The study was extended to two additional NaV isoforms, selected for their sensitivity to TTX, excluding TTX-resistant NaV channels, which alone are insufficient to support firing by primary spindle endings. Positive immunoreactivity was found for NaV 1.1 , predominantly in sensory terminals together with NaV 1.6 and for NaV 1.7 , mainly in preterminal axons. Differential distribution in primary sensory endings suggests specialized roles for these three NaV isoforms in the process of mechanosensory signaling by muscle spindles. NEW & NOTEWORTHY The molecular mechanisms underlying mechanosensory signaling responsible for proprioceptive functions are not completely elucidated. This study provides the first evidence that voltage-gated sodium channels (NaVs) are expressed in the spindle primary sensory ending, where NaVs are found at every site involved in transduction or encoding of muscle stretch. We propose that NaVs contribute to multiple steps in sensory signaling by muscle spindles as it does in other types of slowly adapting sensory neurons. Copyright © 2017 the American Physiological Society.

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

Kinetic Analysis of a Molecular Model of the Budding Yeast Cell Cycle

PubMed Central

Chen, Katherine C.; Csikasz-Nagy, Attila; Gyorffy, Bela; Val, John; Novak, Bela; Tyson, John J.

2000-01-01

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. PMID:10637314

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

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.

[Myofibroma/myofibromatosis: a clinicopathologic analysis of 9 cases].

PubMed

Fu, Y; Guan, W Y; Wu, H Y; Wu, H Y; Fan, Z W; Ye, Q; Meng, F Q

2018-01-08

Objective: To investigate the clinical and histological features, diagnosis and differential diagnosis of myofibroma/myofibromatosis. Methods: The clinical data and pathology features of nine cases of myofibroma/myofibromatosis were collected from August 2011 to November 2016 in Affiliated Drum Tower Hospital, Nanjing University Medical School and Children's Hospital of Nanjing Medical University. Immunohistochemistry(IHC), PDGFRB molecular analysis and ETV6-NTRK3 gene fusion were performed and relevant literature reviewed. Results: There were 7 males and 2 females, with age ranging from 3 days to 18 years (mean 5 years). The tumors were located in head and neck (eight cases) and trunk (one case). Clinically, the tumors presented as freely movable nodules. Microscopically, they appeared biphasic with alternating light- and dark-staining areas. The light-staining area consisted mainly of plump myoid spindle cells with eosinophilic cytoplasm arranged in nodules, short fascicles, or whorls.The dark-staining area was composed of round or polygonal cells with slightly hyperchromatic nuclei or small spindle cells arranged around a distinct hemangiopericytoma-like vascular pattern. IHC showed the tumor cells in the light-staining area were strongly positive for vimentin and SMA, while cells in dark-staining area were strongly positive for vimentin, and weakly for SMA. Tumor cells were negative for desmin, S-100 protein, h-Caldesmon, CD34 and STAT6. Analysis of PDGFRB mutations was performed in seven cases. Two cases showed 12 exon point mutation c. 1681 c>T(p.R561C), one case showed 14 exon point mutation c. 1998C>G (p.N666K). ETV6-NTRK3 gene fusion was not detected by fluorescence in situ hybridization in four patients under three years old. All cases were followed for 6 to 68 months, with two recurrences. Conclusions: Myofibroma/myofibromatosis is an uncommon benign myofibroblastic tumor of infancy and childhood. The tumor can appear biphasic, and may show PDGFRB point mutation which is of potential diagnostic value.

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

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

Development of collagen peptide-based biomaterials for tissue engineering applications

NASA Astrophysics Data System (ADS)

Hernandez Gordillo, Victor

The transition from in vitro to in vivo use of stem cells in regenerative medicine requires biomaterial scaffolds that can maintain stem cell viability and at the same time allow cell differentiation. We have previously reported the design of a collagen mimetic peptide (CMP) that assembles into a mesh-like three-dimensional (3D) structure upon the addition of metal ions and its potential for the culture of human cells. The addition of a chelating solution, such as EDTA, results in disassembly of the 3D structure, demonstrating the flexibility in the assembly/disassembly process. In the second chapter of this dissertation, we report the design of CMPs that can be functionalized with His-tagged cargoes within the 3D scaffold, via metal coordination. We show that the addition of GFP-His8 and human epidermal growth factor (hEGF-His6) has minimal effect in the assembly process. Additionally, we show that the bound hEGF-His6 can be released gradually in vitro for 5 days and induces cell proliferation in an EGF-dependent cell line. Furthermore, we functionalized the CMPs with the cell adhesion sequence (RGDS) to promote cell differentiation of two human non-tumorigenic cells lines, MCF10A and 3522-S1. In the third chapter, we evaluated the possibility of using the collagen mimetic-peptide-based (CMP) scaffolds for cell encapsulation and differentiation of human mesenchymal stem cells (hMSC). We show that hMSC encapsulated within the CMP scaffold are viable for up to 24 days post encapsulation. Moreover, hMSC at days 1, 4 and 8 days after encapsulation can be recovered from the scaffold and retain their stemness properties when analyzed for in vitro differentiation. We also demonstrate by real time PCR (RT-PCR) that genes important for osteogenic and chondrogenic differentiation are over-expressed in the absence of stimulating factors when the cells are encapsulated in the 3D scaffold at 8 and 24 days post encapsulation. Lastly, the incorporation of the cell adhesion sequence (RGDS) was shown to influence the scaffold-cell interaction. hMSCs within these RGDS-modified scaffold adopted spindle shape morphology and a complex cell organization at the outermost layer of the scaffold. In contrast, in the scaffold lacking the RGDS sequence hSMCs formed cell aggregates.

Study of irradiated bone. III. Scintigraphic and radiographic detection of radiation-induced osteosarcomas. [/sup 99m/Tc-pyrophosphate; rabbits

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

King, M.A.; Casarett, G.W.; Weber, D.A.

1980-05-01

Within 1 yr after localized irradiation of a hind limb with single (1756 rads) or fractionated (4650 rads in 3 wk) x-ray doses, radiation-induced osteosarcomas were observed in four of nine single-dose rabbits and two of 11 fractionated-dose rabbits. Tumors were observed in the proximal tibia in five cases and the distal femur in one case. In terms of production of osteoid or osseous tissue, three tumors were well differentiated, one slightly differentiated, and two (spindle-cell tumors) undifferentiated. This report summarizes the Tc-99m pyrophosphate (TcPPi) imaging and autoradiographic, radiographic, and histologic studies of these osteosarcomas. The four differentiated osteosarcomas weremore » detected 1 to 2.5 mo earlier by TcPPi imaging than by radiography, whereas the two undifferentiated tumors were suspected 2 wk or 3.5 mo earlier radiographically. Autoradiograms showed TcPPi localization in bone produced by differentiated osteosarcomas, and in regions of reactive bone resorption and formation peripheral to tumors. The results support a recommendation for combined radiographic and scintigraphic techniques for the early detection of osteosarcomas.« 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.

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

Evaluation of an anal sac adenocarcinoma tumor in a Spitz dog

PubMed Central

Javanbakht, Javad; Tavassoli, Abbas; Sabbagh, Atefeh; Hassan, Mehdy Aghamohammmad; Samakkhah, Shohreh Alian; Shafiee, Radmehr; Lakzian, Ali; Ghalee, Vahideh Rahmani; Gharebagh, Sonia Shoja

2013-01-01

A 9-year-old emasculated male Spitz with tenesmus and constipation had a subcutaneous mass at the left ventral aspect of the anus with history of polyuria and polydipsia. A complete blood cell count, serum biochemistry panel, and urinalysis (cystocentesis sample) were evaluated. Abnormalities in the serum biochemistry panel included a mildly elevated serum cholesterol concentration (7.28 mmol/L; reference interval, 2.70–5.94 mmol/L), increased serum alkaline phosphatase activity (184 U/L; reference interval, 9–90 U/L), alanine transaminase (122 U/L; reference interval, 5–60 U/L) activity and aspartate aminotransferase (80 U/L; reference interval, 5–55 U/L) activity, severe increased total calcium concentration (16.3 mg/dL; reference interval, 8.2–12.4 mg/dL or 9.3–11.4 mg/dL), and decreased total calcium concentration (3.4 mg/dL, reference interval, 2.5–5.6mg/dL). Furthermore, testing revealed an increased intact parathyroid hormone concentration (38.6 pmol/L; reference interval, 3–17 pmol/L). On cytologic and histopathologic examinations, various types of cells were observed. Most of the cells were oval to polygonal and had elliptical or elongate nuclei and a moderate amount of pale to basophilic cytoplasm. The remaining cells had round to oval nuclei and pale to basophilic cytoplasm. Cells of both types were loosely adhered to each other and were arranged in rosette-like structures. Both neoplastic cell types had fine homogenous chromatin and either a small indistinct nucleolus or no visible nucleolus. Mild anisokaryosis and anisocytosis were observed. Histologically, the mass consists of glandular structures formed by cuboidal cells admixed with bundles of spindle cells. Based on location and histologic features, the final diagnosis was adenocarcinoma of the apocrine gland of the anal sac, which should be included as a cytologic differential diagnosis when spindle cells and typical epithelial cells are observed in masses in the region of the anal sac of dogs. PMID:23570021

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.

Fine needle aspiration (FNA) of synovial sarcoma--a comparative histological-cytological study of 15 cases, including immunohistochemical, electron microscopic and cytogenetic examination and DNA-ploidy analysis.

PubMed

Akerman, M; Willén, H; Carlén, B; Mandahl, N; Mertens, F

1996-06-01

A retrospective study of 25 FNAs (11 aspirates from primary tumours and 14 from recurrencies and metastases) from 15 synovial sarcomas was performed. The cytological findings were correlated with the histopathology and the value of immunohistochemical and electron microscopic examination as well as DNA-ploidy and cytogenetic analysis for diagnosis were assessed. A reproducible cellular pattern with a reliable diagnosis of spindle cell sarcoma was possible provided that the aspirates were cell rich. However, a true biphasic pattern indicative of synovial sarcoma was only seen in one of the 25 specimens. Electron microscopic examination of the aspirates was a valuable adjunctive diagnostic method, whereas immunocytochemistry and DNA-ploidy analysis were not. Immunohistochemical, electron microscopic and cytogenetic analysis were all valuable ancillary methods when performed on surgical specimens. Malignant haemangiopericytoma and fibrosarcoma were the most important differential diagnoses in the FNA specimens.

Incidental synovial myxoma with extensive intermuscular infiltration in a dog.

PubMed

Izawa, Takeshi; Tanaka, Miyuu; Aoki, Mika; Ohashi, Fumihito; Yamate, Jyoji; Kuwamura, Mitsuru

2012-12-01

A 16-year-old male mixed-breed dog was euthanized due to progression of renal failure caused by renal adenocarcinoma in the left kidney. Apart from main symptomatic lesion, accumulation of transparent jelly-like fluid was observed between the right femoral muscles. Gross examination of the right hindlimb revealed multiple nodules in the articular surface and capsule of the stifle joints, which extended into the crural muscles. Histopathologically, the joint and intermuscular masses were characterized by variously-sized hypocellular nodules consisting of spindle to stellate cells suspended in an abundant myxoid matrix. There were cystic structures within the intermuscular masses, lined by synoviocyte-like cells. Based on the gross and histopathologic findings, the case was diagnosed as synovial myxoma with extensive intermuscular infiltration. Synovial myxoma should be considered in the differential diagnosis of dogs with myxomatous tumor between skeletal muscles, even in absence of joint or muscle symptoms.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

PubMed

Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki

2016-01-01

Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.

Fat-forming solitary fibrous tumor of the kidney: a case report and literature review

PubMed Central

Chen, Yanyang; Wang, Fen; Han, Anjia

2015-01-01

Fat-forming solitary fibrous tumor (SFT) is a rare soft tissue tumor. Herein, we reported a 30-year-old woman was found to have a solid mass measuring 60×45 mm in the right kidney on an abdominal computed tomography scan. The tumor was well-circumscribed and composed of cellular nodules with the classic SFT admixed with clusters and lobules of mature adipocytes. Immunohistochemistry staining showed that the tumor cells were diffusely and strongly positive for CD34 and Bcl-2, focally and weakly positive for CD99 and EMA. Mature adipocytes were positive for S-100 protein. Ki-67 expression was found in approximately 2% of tumor cells. However, tumor cells were negative for cytokeratin, S-100 protein, HMB-45, Melan-A, SMA, and CD117. We made the pathological diagnosis of fat-forming SFT of the right kidney. The differential diagnosis includes angiomyolipoma, liposarcoma, spindle cell lipoma, sarcomatoid renal cell carcinoma, synovial sarcoma, and gastrointestinal stromal tumor. The patient was alive and well without evidence of recurrence or metastasis at 19 months after tumor resection. PMID:26339447

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

Extreme Spindles and Leukoencephalopathy after Acute Lymphoblastic Leukemia Treatment: An Undescribed Association.

PubMed

Kanda, Paulo Afonso Medeiros; Kanda, Rafael Guimarães; Mei, Paulo Afonso; Cury, Ivan José

2015-12-01

We report a case of a child whose EEG demonstrated extreme spindles (ES) after acute lymphoblastic leukemia treatment. This finding has not been reported previously. In 1962, Gibbs and Gibbs described the ES EEG pattern due to its high amplitude (200 to 400 μV). ES are a rare spindle variant that is found in EEGs of 0.05% of normal children (average age, 3 years, with a range of 1 to 12 years), and are even rarer after 11 years. Moreover due to changes in the white matter of the frontal lobe, ES have been associated with such conditions as cerebral palsy and mental retardation, residual brain damage, undefined infections, infantile neuroaxonal dystrophy, Menkes' kinky-hair syndrome, congenital muscular dystrophy, hydrocephalus, porencephaly, epilepsy, progressive cerebellar degeneration, and mycoplasma encephalitis. Methotrexate has a notably toxic effect on the central nervous system, with leukoencephalopathy being the most common form. In our case, frontocentral ES were associated with hyperintense lesions in the white matter of the frontal lobe. Lesional deafferentation can be the substrate for an almost continuous ES, since both initiation and termination of spindle oscillations are thought to originate in thalamocortical neurons. Thus, we postulate that in some cases a partial functional cortical differentiation could generate ES.

[Retroperitoneal dedifferentiated liposarcoma with rhabdomyoblastic differentiation: a clinicopathological analysis].

PubMed

Liu, L; Wang, L H; Ren, Y B; Rao, X S; Yang, S M

2018-02-08

Objective: To investigate the clinicopathological features, differential diagnosis, treatment and prognosis of dedifferentiated liposarcoma with rhabdomyoblastic differentiation. Methods: Six cases of retroperitoneal dedifferentiated liposarcoma with rhabdomyoblastic features were collected from December 2014 to August 2017 at Peking University International Hospital. The clinical manifestations, histomorphology, immunophenotype, treatment and follow-up data were analyzed, and relevant literature reviewed. Results: The six patients included two males and four females, with age range of 47 to 66 years (mean 56 years). One case was primary and the five cases were recurred; four cases received radiotherapy and/or chemotherapy. The tumor diameters were 10 to 30 cm. Microscopically, the dedifferentiated areas were well demarcated from the well-differentiated areas, and resembled malignant fibrous histiocytoma, fibrosarcoma or solitary fibrous tumor with obvious mitotic figures or necrosis. Rhabdomyoblastic cells made up 10% to 30% of dedifferentiated area, and were scattered or focally distributed, being rounded, band-like or spindled, mostly with abundant eosinophilic cytoplasm. No striated structure was found, and the nucleis were rounded, oval or irregular shape with central or eccentric prominent nucleoli. Rare rhabdomyoblastic cells were lymphocytoid. The tumors encroached the muscular layer of intestinal wall in two cases and perirenal adipose tissue in one case. By immunohistochemical staining, the rhabdomyoblastic cells of all cases were all positive for desmin, myogenin, myoD1 and SMA; S-100 protein was expressed in one case (1/6). Well-differentiated area in two cases and dedifferentiated areas in all six cases were positive for MDM2, CDK4 and p16. After resection of the tumor and adjacent organs, one case recurred three months later, but there was no distant metastasis. Conclusions: Dedifferentiated liposarcoma with rhabdomyoblastic differentiation is a rare dedifferentiated liposarcoma. Pathological diagnosis is based on morphology, with supplementary immunohistochemical or molecular evaluation for further differential diagnosis. Multiple relapses may occur after surgical ablation plus adjuvant therapy.

Pancreatic perivascular epithelioid cell tumor: A case report with clinicopathological features and a literature review

PubMed Central

Jiang, Hui; Ta, Na; Huang, Xiao-Yi; Zhang, Ming-Hua; Xu, Jing-Jing; Zheng, Kai-Lian; Jin, Gang; Zheng, Jian-Ming

2016-01-01

Perivascular epithelioid cell tumor (PEComa) of the pancreas is an unusual tumor deriving from mesenchyma. This paper described a case of pancreatic PEComa, which was initially suspected as neuroendocrine carcinoma by biopsy, and therefore surgical treatment was recommended due to undetermined diagnosis. Examination of the surgical specimen under a microscope showed that the tumor cell’s morphology was epithelioid or spindle-shaped, and ranged in a nested pattern. Additionally, these cells had a large extent of acidophilic cytoplasm, no mitotic figures, and expressed HMB-45, melan-p, and smooth muscle actin immunohistochemically. Pathological examination indicated that PEComa originated from the pancreas, but symptoms related to tuberous sclerosis were absent. Since PEComa is extremely rare in the pancreas, it is likely to be ignored in differential diagnosis. In conclusion, our article highlighted the clinicopathological features of PEComa, and we conducted a literature review focusing on PEComa so as to deepen the understanding of this tumor type. PMID:27053862

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.

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

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.

Angiofibroma of soft tissue: clinicopathologic study of 2 cases of a recently characterized benign soft tissue tumor

PubMed Central

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. PMID:24133600

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.

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.

Dedifferentiated Liposarcoma: Updates on Morphology, Genetics, and Therapeutic Strategies.

PubMed

Thway, Khin; Jones, Robin L; Noujaim, Jonathan; Zaidi, Shane; Miah, Aisha B; Fisher, Cyril

2016-01-01

Well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL) form the largest subgroup of liposarcomas, and represent a morphologic and behavioral spectrum of 1 disease entity, which arises typically in middle to late adult life, most frequently within the retroperitoneum or extremities. DDL is defined as nonlipogenic sarcoma that is juxtaposed to WDL, occurs as a recurrence of WDL or which can arise de novo, and typically has the appearance of undifferentiated pleomorphic or spindle cell sarcoma. DDL have a propensity for local recurrence, whereas distant metastasis is rarer, and behavior is related to anatomic site, with retroperitoneal neoplasms showing a significantly worse prognosis. Surgical resection remains the mainstay of treatment, and medical options for patients with aggressive recurrent or metastatic disease are limited. DDL share similar genetic abnormalities to WDL, with high-level amplifications of chromosome 12q14-15, including the MDM2 and CDK4 cell cycle oncogenes, and DDL harbor additional genetic changes, particularly coamplifications of 6q23 and 1p32. Novel therapies targeted at the gene products of chromosome 12 are being tested in clinical trials. We review the pathology and genetics of DDL, discussing morphologic patterns, immunohistochemical and genetic findings, the differential diagnosis, and future therapeutic strategies.

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

Using MEG to Understand the Progression of Light Sleep and the Emergence and Functional Roles of Spindles and K-Complexes

PubMed Central

Ioannides, Andreas A.; Liu, Lichan; Poghosyan, Vahe; Kostopoulos, George K.

2017-01-01

We used tomographic analysis of MEG signals to characterize regional spectral changes in the brain at sleep onset and during light sleep. We identified two key processes that may causally link to loss of consciousness during the quiet or “core” periods of NREM1. First, active inhibition in the frontal lobe leads to delta and theta spectral power increases. Second, activation suppression leads to sharp drop of spectral power in alpha and higher frequencies in posterior parietal cortex. During NREM2 core periods, the changes identified in NREM1 become more widespread, but focal increases also emerge in alpha and low sigma band power in frontal midline cortical structures, suggesting reemergence of some monitoring of internal and external environment. Just before spindles and K-complexes (KCs), the hallmarks of NREM2, we identified focal spectral power changes in pre-frontal cortex, mid cingulate, and areas involved in environmental and internal monitoring, i.e., the rostral and sub-genual anterior cingulate. During both spindles and KCs, alpha and low sigma bands increases. Spindles emerge after further active inhibition (increase in delta power) of the frontal areas responsible for environmental monitoring, while in posterior parietal cortex, power increases in low and high sigma bands. KCs are correlated with increase in alpha power in the monitoring areas. These specific regional changes suggest strong and varied vigilance changes for KCs, but vigilance suppression and sharpening of cognitive processing for spindles. This is consistent with processes designed to ensure accurate and uncorrupted memory consolidation. The changes during KCs suggest a sentinel role: evaluation of the salience of provoking events to decide whether to increase processing and possibly wake up, or to actively inhibit further processing of intruding influences. The regional spectral patterns of NREM1, NREM2, and their dynamic changes just before spindles and KCs reveal an edge effect facilitating the emergence of spindles and KCs and defining the precise loci where they might emerge. In the time domain, the spindles are seen in widespread areas of the cortex just as reported from analysis of intracranial data, consistent with the emerging consensus of a differential topography that depends on the kind of memory stored. PMID:28670270

The many faces and mimics of papillary thyroid carcinoma.

PubMed

Albores-Saavedra, Jorge; Wu, Jianhua

2006-01-01

This article provides an overview of the 15 histologic variants of papillary thyroid carcinoma listed by the 2004 World Health Organization (WHO) monograph on endocrine tumors. The histologic features, differential diagnosis, and clinical course of each variant are discussed in some detail. The follicular variants (conventional and macrofollicular) constitute a morphologic challenge because the majority of these tumors are encapsulated and, also, because, in many tumors, not all neoplastic cells show the nuclear features considered to be diagnostic of papillary carcinoma. As a result, most of these tumors are missed even by experienced pathologists. Moreover, hyperplastic thyroid lesions, follicular adenomas, and Hashimoto's thyroiditis may contain cells with clear nuclei resembling those of papillary carcinoma. Papillary carcinomas composed entirely of hyperchromatic cells have been overlooked. The WHO monograph defines papillary carcinoma with focal spindle and giant cell carcinoma components but its clinical behavior is unknown. Papillary carcinoma with an insular pattern that does not show the artifactual separation of the cell nests has been misinterpreted as the solid variant of papillary carcinoma. Papillary microcarcinomas include not only the conventional type and the follicular variants but also the tall cell and columnar cell variants.

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

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

Sinonasal haemangiopericytoma: histomorphology and differential diagnoses.

PubMed

Leow, Wei Qiang; Sng, Ivy

2015-04-01

A 39-year-old female presented with a fleshy nasal polyp occluding the left nasal cavity, associated with haemopurulent discharge. Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) scans of the paranasal cavities revealed a large polypoid tumour arising from the left middle turbinate and obstructing the left maxillary sinus ostium. However, no bony or intracranial involvement was identified. A biopsy revealed a tumour with small blue round cell morphology. The tumour cells showed diffuse strong membranous CD99 positivity and patchy CD34 positivity. Ancillary cytogenetic tests for the EWSR1 and SS18/SYT gene translocations were negative. In view of the non-invasive nature of the tumour and the low cell proliferative index (Ki-67) of 5%, a medial maxillectomy resection was performed. The resection revealed additional areas with spindle-cell morphology and focal haemangiopericytic vasculature. The tumour continued to show immunoreactivity to CD99 and CD34, as well as Smooth Muscle Actin (SMA) and Muscle Specific Actin (MSA). The overall findings are in keeping with a sinonasal haemangiopericytoma. With clear surgical resection margins, the patient is on routine follow-up and is currently disease-free.

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.

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

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

Nuclear β-Catenin Expression is Frequent in Sinonasal Hemangiopericytoma and Its Mimics.

PubMed

Jo, Vickie Y; Fletcher, Christopher D M

2017-06-01

Sinonasal hemangiopericytoma (HPC) is a tumor showing pericytic myoid differentiation and which arises in the nasal cavity and paranasal sinuses. CTNNB1 mutations appear to be a consistent aberration in sinonasal HPC, and nuclear expression of β-catenin has been reported. Our aim was to evaluate the frequency of β-catenin expression in sinonasal HPC and its histologic mimics in the upper aerodigestive tract. Cases were retrieved from the surgical pathology and consultation files. Immunohistochemical staining for β-catenin was performed on 50 soft tissue tumors arising in the sinonasal tract or oral cavity, and nuclear staining was recorded semiquantitatively by extent and intensity. Nuclear reactivity for β-catenin was present in 19/20 cases of sinonasal HPC; 17 showed moderate-to-strong multifocal or diffuse staining, and 2 had moderate focal nuclear reactivity. All solitary fibrous tumors (SFT) (10/10) showed focal-to-multifocal nuclear staining, varying from weak to strong in intensity. Most cases of synovial sarcoma (9/10) showed nuclear β-catenin expression in the spindle cell component, ranging from focal-weak to strong-multifocal. No cases of myopericytoma (0/10) showed any nuclear β-catenin expression. β-catenin expression is prevalent in sinonasal HPC, but is also frequent in SFT and synovial sarcoma. Our findings indicate that β-catenin is not a useful diagnostic tool in the evaluation of spindle cell tumors with a prominent hemangiopericytoma-like vasculature in the sinonasal tract and oral cavity, and that definitive diagnosis relies on the use of a broader immunohistochemical panel.

Substrate Curvature Restricts Spreading and Induces Differentiation of Human Mesenchymal Stem Cells.

PubMed

Lee, Sang Joo; Yang, Shengyuan

2017-09-01

While cells attach, spread, migrate, proliferate, and differentiate in three-dimensional (3D) micromechanical environments, the mechanical factors of these environments influence the shapes, sizes, and adhesion forces of the cells. Here, the authors culture human mesenchymal stem cells (hMSCs) on a unique class of curvature-defined substrates, micro glass ball embedded polyacrylamide gels, prepared with an improved protocol, and investigate the spreading responses of the hMSCs on the glass balls to study the effects of substrate curvature on the spreading of hMSCs. The authors find that, among the used diameters of glass balls, the minimum diameter of a glass ball on which an hMSC can attach and spread is 500 μm. In contrast to the well-spread morphologies with randomly-multiple lamellipodia for the hMSCs growing on the flat glass plates, the morphologies of the hMSCs growing on the glass balls are almost uniformly spindle-shaped with two lamellipodia. The sensitivities of the attachment and spreading morphology of an hMSC to substrate curvature are very different from those of a fibroblast. The RT-PCR analysis reveals that the substrate curvature alone can induce adipogenesis of the hMSCs. These findings imply that substrate curvature has profound effects on stem cell behaviors, and detailed and in-depth studies on these effects and their underlying biophysical mechanisms are necessary. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CRYOPRESERVATION STRATEGY FOR TISSUE ENGINEERING CONSTRUCTS CONSISTING OF HUMAN MESENHYMAL STEM CELLS AND HYDROGEL BIOMATERIALS.

PubMed

Wu, Y; Wen, F; Gouk, S S; Lee, E H; Kuleshova, L

2015-01-01

The development of vitrification strategy for cell-biomaterial constructs, particularly biologically inspired nanoscale materials and hydrogels mimicking the in vivo environment is an active area. A cryopreservation strategy mimicking the in vivo environment for cell-hydrogel constructs may enhance cell proliferation and biological function. To demonstrate the efficacy of vitrification as a platform technology involving tissue engineering and human mesenchymal stem cells (hMSCs). Microcarriers made from alginate coated with chitosan and collagen are used. Conventional freezing and vitrification were compared. The vitrification strategy includes 10 min step-wise exposure to a vitrification solution (40% v/v EG, 0.6M sucrose) and immersion into liquid nitrogen. Confocal imaging of live/dead staining of hMSCs cultured on the surface of microcarriers demonstrated that vitrified cells had excellent appearance and prolonged spindle shape morphology. The proliferation ability of post-vitrified cells arbitrated to protein Ki-67 gene expression was not significantly different in comparison to untreated control, while that of post-freezing cells was almost lost. The ability of hMSCs cultured on the surface of microcarriers to proliferate has been not affected by vitrification and it was significantly better after vitrification than after conventional freezing during continuous culture. Collagen II related mRNA expression by 4 weeks post-vitrification and post-freezing showed that ability to differentiate into cartilage was sustained during vitrification and reduced during conventional freezing. No significant difference was found between control and vitrification groups only. Vitrification strategy coupled with advances in hMSC-expansion platform that completely preserves the ability of stem cells to proliferate and subsequently differentiate allows not only to reach a critical cell number, but also demonstrate prospects for effective utilization and transportation of cells with their support system, creating demand for novel biodegradable materials.

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

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.

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.

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.

Mesenchymal stem cells with osteogenic potential in human maxillary sinus membrane: an in vitro study.

PubMed

Berbéri, Antoine; Al-Nemer, Fatima; Hamade, Eva; Noujeim, Ziad; Badran, Bassam; Zibara, Kazem

2017-06-01

The aim of our study is to prove and validate the existence of an osteogenic progenitor cell population within the human maxillary Schneiderian sinus membrane (hMSSM) and to demonstrate their potential for bone formation. Ten hMSSM samples of approximately 2 × 2 cm were obtained during a surgical nasal approach for treatment of chronic rhinosinusitis and were retained for this study. The derived cells were isolated, cultured, and assayed at passage 3 for their osteogenic potential using the expression of Alkaline phosphatase, alizarin red and Von Kossa staining, flow cytometry, and quantitative real-time polymerase chain reaction. hMSSM-derived cells were isolated, showed homogenous spindle-shaped fibroblast-like morphology, characteristic of mesenchymal progenitor cells (MPCs), and demonstrated very high expression of MPC markers such as STRO-1, CD44, CD90, CD105, and CD73 in all tested passages. In addition, von Kossa and Alizarin red staining showed significant mineralization, a typical feature of osteoblasts. Moreover, alkaline phosphatase (ALP) activity was significantly increased at days 7, 14, 21, and 28 of culture in hMSSM-derived cells grown in osteogenic medium, in comparison to controls. Furthermore, osteogenic differentiation significantly upregulated the transcriptional expression of osteogenic markers such as ALP, Runt-related transcription factor 2 (Runx-2), bone morphogenetic protein (BMP)-2, osteocalcin (OCN), osteonectin (ON), and osteopontin (OPN), confirming that hMSSM-derived cells are of osteoprogenitor origin. Finally, hMSSM-derived cells were also capable of producing OPN proteins upon culturing in an osteogenic medium. Our data showed that hMSSM holds mesenchymal osteoprogenitor cells capable of differentiating to the osteogenic lineage. hMSSM contains potentially multipotent postnatal stem cells providing a promising clinical application in preimplant and implant therapy.

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

Primary colonic well-differentiated / dedifferentiated liposarcoma of the ascending colon: a case report.

PubMed

Sawayama, Hiroshi; Yoshida, Naoya; Miyamoto, Yuji; Uchihara, Tomoyuki; Toihata, Tasuku; Yagi, Taisuke; Hiyoshi, Yukiharu; Iwatsuki, Masaaki; Baba, Yoshifumi; Baba, Hideo

2017-08-30

Primary colonic and dedifferentiated liposarcomas are both remarkably rare. This work describes a case of primary colonic well-differentiated/dedifferentiated liposarcoma and reviews the clinical characteristics and current therapies for liposarcoma tumors. A 52-year-old woman was referred to our hospital with a submucosal tumor of the ascending colon. Clinical analysis by ultrasound colonoscopy and computed tomography revealed a partially ossified tumor with irregular edges continuous with the muscular layer. High F-18 deoxyglucose uptake was detected by positron emission tomography. Radical resection with lymph node dissection was performed, yielding a tumor specimen approximately 6.5 × 4.0 × 3.2 cm. Neoplastic spindle cell proliferation was found from submucosa to subserosa. Well-differentiated adipose tissue surrounded the tumor, but contained atypical nuclei with condensed chromosomes. Immunohistochemical staining was positive for p16, CDK4, and MDM2 expression. Based on these findings, a diagnosis of well-differentiated/dedifferentiated liposarcoma was given. Dedifferentiated liposarcomas are more aggressive than their well-differentiated, low-grade counterparts. While local recurrence can occur with both tumor types, dedifferentiated liposarcomas are more prone to developing distant metastases. The patient received no postoperative therapy, and no recurrences have been observed 12 months after surgery. Here we report an extremely rare case of well-differentiated/dedifferentiated liposarcoma of the ascending colon. The dedifferentiated component was high-grade liposarcoma and well-differentiated liposarcoma was detected around the main tumor.

Interpretation of the Precision Matrix and Its Application in Estimating Sparse Brain Connectivity during Sleep Spindles from Human Electrocorticography Recordings

PubMed Central

Das, Anup; Sampson, Aaron L.; Lainscsek, Claudia; Muller, Lyle; Lin, Wutu; Doyle, John C.; Cash, Sydney S.; Halgren, Eric; Sejnowski, Terrence J.

2017-01-01

The correlation method from brain imaging has been used to estimate functional connectivity in the human brain. However, brain regions might show very high correlation even when the two regions are not directly connected due to the strong interaction of the two regions with common input from a third region. One previously proposed solution to this problem is to use a sparse regularized inverse covariance matrix or precision matrix (SRPM) assuming that the connectivity structure is sparse. This method yields partial correlations to measure strong direct interactions between pairs of regions while simultaneously removing the influence of the rest of the regions, thus identifying regions that are conditionally independent. To test our methods, we first demonstrated conditions under which the SRPM method could indeed find the true physical connection between a pair of nodes for a spring-mass example and an RC circuit example. The recovery of the connectivity structure using the SRPM method can be explained by energy models using the Boltzmann distribution. We then demonstrated the application of the SRPM method for estimating brain connectivity during stage 2 sleep spindles from human electrocorticography (ECoG) recordings using an 8 × 8 electrode array. The ECoG recordings that we analyzed were from a 32-year-old male patient with long-standing pharmaco-resistant left temporal lobe complex partial epilepsy. Sleep spindles were automatically detected using delay differential analysis and then analyzed with SRPM and the Louvain method for community detection. We found spatially localized brain networks within and between neighboring cortical areas during spindles, in contrast to the case when sleep spindles were not present. PMID:28095202

Results of Accelerated Life Testing of LCLS-II Cavity Tuner Motor

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

Huque, Naeem; Daly, Edward; Pischalnikov, Yuriy

An Accelerated Life Test (ALT) of the Phytron stepper motor used in the LCLS-II cavity tuner has been conducted at JLab. Since the motor will reside inside the cryomodule, any failure would lead to a very costly and arduous repair. As such, the motor was tested for the equivalent of 30 lifetimes before being approved for use in the production cryomodules. The 9-cell LCLS-II cavity is simulated by disc springs with an equivalent spring constant. Plots of the motor position vs. tuner position ' measured via an installed linear variable differential transformer (LVDT) ' are used to measure motor motion.more » The titanium spindle was inspected for loss of lubrication. The motor passed the ALT, and is set to be installed in the LCLS-II cryomodules.« less

Ultrasound-guided fine-needle aspiration of a posterior neck dedifferentiated liposarcoma with MDM2 fluorescence in situ hybridization performed on a Pap-stained smear.

PubMed

Zreik, Riyam; Soyalp, Krystal; Ruiz, Steve; Ward, Russell; Dobin, Sheila; Chen, Xiangbai; Liu, Lina; Rao, Arundhati

2015-04-01

Head and neck liposarcomas, while rare, tend to be subcutaneous and well-differentiated. Dedifferentiated liposarcomas of the head and neck are exceedingly rare in the literature. We present a case of a dedifferentiated liposarcoma arising in the soft tissue of the posterior neck of an 86-year-old man and diagnosed by fine-needle aspiration. Aspirate smears showed a dual population of atypical lipomatous and spindled cells. MDM2 (murine double minute 2) amplification was demonstrated on a Pap-stained smear using fluorescence in situ hybridization (FISH). To the best of our knowledge, this is the first report of MDM2 FISH amplification in a liposarcoma performed on an aspirate smear. © 2014 Wiley Periodicals, Inc.

Gastrointestinal tract spindle cell lesions--just like real estate, it's all about location.

PubMed

Voltaggio, Lysandra; Montgomery, Elizabeth A

2015-01-01

Interpretation of gastrointestinal tract mesenchymal lesions is simplified merely by knowing in which anatomic layer they are usually found. For example, Kaposi sarcoma is detected on mucosal biopsies, whereas inflammatory fibroid polyp is nearly always in the submucosa. Gastrointestinal stromal tumors (GISTs) are generally centered in the muscularis propria. Schwannomas are essentially always in the muscularis propria. Mesenteric lesions are usually found in the small bowel mesentery. Knowledge of the favored layer is even most important in interpreting colon biopsies, as many mesenschymal polyps are encountered in the colon. Although GISTs are among the most common mesenchymal lesions, we will concentrate our discussion on other mesenchymal lesions, some of which are in the differential diagnosis of GIST, and point out some diagnostic pitfalls, particularly in immunolabeling.

RESULTS OF ACCELERATED LIFE TESTING OF LCLS-II CAVITY TUNER MOTOR

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

Huque, Naeem; Daly, Edward F.; Pischalnikov, Yuriy

An Accelerated Life Test (ALT) of the Phytron stepper motor used in the LCLS-II cavity tuner has been conducted at JLab. Since the motor will reside inside the cryomodule, any failure would lead to a very costly and arduous repair. As such, the motor was tested for the equivalent of 30 lifetimes before being approved for use in the production cryomodules. The 9-cell LCLS-II cavity is simulated by disc springs with an equivalent spring constant. Plots of the motor position vs. tuner position ' measured via an installed linear variable differential transformer (LVDT) ' are used to measure motor motion.more » The titanium spindle was inspected for loss of lubrication. The motor passed the ALT, and is set to be installed in the LCLS-II cryomodules.« less

Spontaneous multicentric soft tissue sarcoma in a captive African pygmy hedgehog (Atelerix albiventris): case report and literature review.

PubMed

Díaz-Delgado, Josué; Pool, Roy; Hoppes, Sharman; Cerezo, Argine; Quesada-Canales, Óscar; Stoica, George

2017-05-18

This report describes the clinical, macroscopic, histopathological and immunohistochemical features of a spontaneous multicentric extraskeletal sarcoma in an adult male African hedgehog (Atelerix albiventris). It also provides a succinct up-to-date review on neoplasia in this species. On autopsy examination, main gross findings included a moderately demarcated cranial mass and a multilobulated, caudal intra-abdominal mass. The cranial mass had perforated the underlying temporal and occipital bones and had extended into the cranial vault and was compressing the surface of the cerebellum and cerebrum. Histologic, histochemical and immunohistochemical analyses supported a diagnosis of multicentric poorly differentiated spindle cell sarcoma with fibrosarcomatous, storiform and myxoid foci. The high incidence of neoplasia and cross similarities renders the African hedgehog a suitable species for comparative pathology studies.

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.