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PubMed2001

A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.

In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Anaphase↗
PubMed2005

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes.

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.

Amino Acid Sequence↗
PubMed1991

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region. Changes in organization of MTs in the irradiated region were analyzed by EM serial section analysis coupled with 3-D computer reconstruction. Metaphase cells irradiated 2 to 4 microns below the spindle pole (imaged by polarization optics) lost birefringence in the irradiated region. Peripheral spindle fibers, previously curved to focus on the pole, immediately splayed outwards when severed. We demonstrate via serial section analysis that following irradiation the lesion was devoid of MTs. Within 30 s to 1 min, recovery in live cells commenced as the severed spindle pole moved toward the metaphase plate closing the lesion. This movement was concomitant with the recovery of spindle birefringence and some of the severed fibers becoming refocused at the pole. Ultrastructurally we confirmed that this movement coincided with bridging of the lesion by MTs presumably growing from the pole. The non-irradiated half spindle also lost some birefringence and shortened until it resembled the recovered half spindle. Anaphase cells similarly irradiated did not show recovery of birefringence, and the pole remained disconnected from the remaining mitotic apparatus. Reconstructions of spindle structure confirmed that there were no MTs in the lesion which bridged the severed spindle pole with the remaining mitotic apparatus. These results suggest the existence of chromosome-to-pole spindle forces are dependent upon the existence of a MT continuum, and to a lesser extent to the loss of MT initiation capacity of the centrosome at the metaphase/anaphase transition.

Anaphase↗
PubMed1975

Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length.

Changes in birefringence retardation (BR) and length of Chaetopterus meiotic metaphase-arrested spindles produced by increased hydrostatic pressure were observed with polarized-light microscopy using a newly developed optical pressure chamber. Increased pressure produced rapid, reversible decreases in spindle BR and length. Pressures of 3,500 psi or higher at 22 degrees C caused complete disappearance of spindle BR within 3 min. Up to 6,000 psi, the rates of both BR decay and spindle shortening increased progressively with increasing pressure. At 6,000 psi or above, the BR decreased rapidly but there was no evidence of spindle shortening. The general observations are consistent with results of earlier classical experiments on effects of pressure on mitosis, and with experiments that used colchicine or low temperature as microtubule-depolymerizing agents. The kinetics of spindle depolymerization and repolymerization showed two phases: an initial phase of rapid decreases or increase in half-spindle microtubule BR; and a second phase of nearly constant BR during which most of the spindle shortening or growth occurs. BR is assumed to be directly related to the number of microtubules in a spindle cross section. It is hypothesized that microtubules in the spindle have different stabilities depending on the attachment of nonattachment of their ends. This hypothesis is used to explain the two phases of spindle depolymerization and repolymerization as well as several other observations.

Animals↗
PubMed1982

Absence of microtubule sliding and an analysis of spindle formation and elongation in isolated mitotic spindles from the yeast Saccharomyces cerevisiae.

Mitotic spindles were isolated from a cell division cycle mutant of the budding yeast Saccharomyces cerevisiae by the lysis of sphateroplasts on an air:buffer interface and were negatively stained with 1% gold thioglucose. Isolated spindles were incubated under conditions which promoted the sliding disintegration of parallel preparations of Tetrahymena axonemes, namely the addition of ATP to 20 microM. In no experiment was a corresponding change in microtubule organization of the spindle observed even when spindles were first pretreated with either 1-10 microgram/ml trypsin or 0.2-2% Triton X-100. During these experiments a number of spindles were isolated from cells that had passed through the imposed temperature block, and from the images obtained a detailed model of spindle formation and elongation has been constructed. Two sets of microtubules, one from each spindle pole body (SPB), completely interdigitate to form a continuous bundle, and a series of discontinuous microtubules are then nucleated by each SPB. As the spindle elongates, the number of microtubules continuous between the two SPBs decreases until, at a length of 4 micrometer, only one remains. The spindle, composed of only one microtubule, continues to elongate until it reaches the maximal nuclear dimension of 8 micrometer. The data obtained from negatively stained preparations have been verified in thin sections of wild-type cells. We suggest that, as in the later stages of mitosis only one microtubule is involved in the separation of the spindle poles, the microtubular spindle in S. cerevisiae is not a force-generating system but rather acts as a regulatory mechanism controlling the rate of separation.

Cell Cycle↗
PubMed2001

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint.

The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

Amino Acid Sequence↗
PubMed2000

Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

Adenosine Triphosphate↗
PubMed2002

Spindle and cuboidal renal cell carcinoma, a tumour having frequent association with nephrolithiasis: report of 11 cases including a case with hybrid conventional renal cell carcinoma/ spindle and cuboidal renal cell carcinoma components.

AIMS: We present the largest series of an unclassified subtype of renal cell carcinoma, which seems to be a distinct morphological entity and which is sometimes designated as spindle and cuboidal renal cell carcinoma. METHODS AND RESULTS: Eleven cases of spindle and cuboidal renal cell carcinoma were found among 7000 primary renal cell tumours in Pilsen's routine and consultation files. The patients were five men and six women. They ranged in age from 22 to 65 years (mean 56.8). Microscopically, the tumours were composed of two main populations of cells. First, the preponderant type of cells was formed by flattened, spindle cells with sparse cytoplasm. The second cell type was a small cuboidal cell with clear to light eosinophilic cytoplasm. Spindle-shaped cells were arranged in a fascicular pattern often reminiscent of low-grade smooth muscle tumours. Solid areas of spindle cells were also present. Small cuboidal cells formed sparse tubular structures lined by a row of single cells. In addition to all previous published cases of spindle and cuboidal renal cell carcinoma we observed an association of nephrolithiasis in our cases. It was seen in 3/11 of our patients. A previously unreported feature is the occurrence of a conventional renal cell carcinoma component in one of our cases. Seven of our patients are currently well without signs of recurrence or metastasis, one had metastasis in a regional lymph node at the time of nephrectomy, one died of an unrelated condition, and two were lost to follow-up. CONCLUSIONS: We present 11 cases of spindle and cuboidal renal cell carcinoma, which is believed to be a distinctive morphological entity. Our cases were histologically, immunohistochemically and ultrastructurally similar to the previously reported cases of spindle and cuboidal renal cell carcinoma. In contrast to all previously reported cases of spindle and cuboidal renal cell carcinoma, we observed an association with nephrolithiasis in three of our cases; moreover, one of our tumours had a conventional renal cell carcinoma component and another revealed a metastatic focus in a regional lymph node. None of our patients died of the disease. This study confirms that spindle and cuboidal renal cell carcinoma has a low malignant potential.

Adult↗
PubMed1998

DSK1, a kinesin-related protein involved in anaphase spindle elongation, is a component of a mitotic spindle matrix.

DSK1 is a kinesin-related protein that is involved in anaphase spindle elongation in the diatom Cylindrotheca fuisiformis [Wein et al., 1996: J. Cell Biol. 113:595-604]. DSK1 staining appeared to be concentrated in the gap that forms as the two half-spindles separate, suggesting that DSK1 may be part of a non-microtubule spindle matrix. We set out to investigate this possibility using three-dimensional high-resolution fluorescence microscopy, and biochemical methods of tubulin extraction. Three-dimensional fluorescence microscopy reveals that DSK1 remains in the midzone after the bulk of the microtubules from the two half-spindles have left the region. Biochemical studies show that CaCl2 extraction of tubulin from a mitotic spindle preparation does not extract similar proportions of DSK1 protein. Immunofluorescence confirms that this CaCl2 extraction leaves behind spindle-like bars that are recognized by anti-DSK1, but not by anti-tubulin antibodies. We conclude that DSK1 is part of, or attached to, a non-microtubule scaffold in the diatom central spindle. This discovery has implications for both the structural organization of the mitotic spindle and the mechanism of spindle elongation.

Anaphase↗
PubMed1987

Reactivation of spindle elongation in vitro is correlated with the phosphorylation of a 205 kd spindle-associated protein.

Mitotic spindles isolated from the diatom Stephanopyxis turris consist of two half-spindles of closely interdigitating microtubules that slide relative to one another in the presence of ATP, reinitiating spindle elongation (anaphase B) in vitro. Purified spindles that have been exposed to ATP-gamma-S undergo ATP-dependent reactivation more readily than do control spindles. Thiophosphorylated proteins in such spindles are located in the spindle midzone, kinetochores, and a portion of the pole complex. One major thiophosphorylated peptide of 205 kd is detected in extracts prepared from spindles labeled with [35S]ATP-gamma-S, and is also localized in the spindle midzone by using an antibody that recognizes thiophosphorylated proteins. It is likely that this 205 kd peptide is either a positive regulator or mechanochemical transducer of microtubule sliding when it is in a phosphorylated state.

Adenosine Triphosphate↗
PubMed1998

Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis.

BACKGROUND: The Cut1 and Cut2 proteins of the fission yeast Schizosaccharomyces pombe form a complex and are required for the separation of sister chromatids during anaphase. Polyubiquitinated Cut2 degrades at the onset of anaphase and this degradation, like that of mitotic cyclin, is dependent on the anaphase-promoting complex/cyclosome. Expression of Cut2 that cannot be degraded blocks sister chromatid separation and anaphase spindle elongation. Here, we have investigated the role of the Cut1-Cut2 interaction in sister chromatid separation. RESULTS: The carboxyl terminus of Cut2 interacts with the amino terminus of Cut1, and temperature-sensitive Cut2 mutants expressed Cut2 proteins that contain substitutions in the carboxyl terminus and fail to interact with Cut1, resulting in aberrant anaphase. Localization of Cut1 alters dramatically during the cell cycle. Cut1 is retained in the cytoplasm during interphase and moves to the mitotic spindle pole bodies and the spindle upon entry into prophase, when spindles are formed. The association between Cut2 and Cut1 is needed for the localization of Cut1 to the spindles, as Cut1 remains unbound to the spindle if complex formation is impaired. Cut2 degrades during anaphase, but Cut1 remains bound to the anaphase spindle. This association with the anaphase spindle requires the conserved carboxyl terminus of Cut1. CONCLUSIONS: Complex formation between Cut1 and Cut2 is needed for the onset of normal anaphase. Cut2 is required for loading Cut1 onto the spindle at prophase and Cut2 proteolysis is needed for the active participation of Cut1 in sister chromatid separation.

Anaphase↗
PubMed2004

Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles.

During cell division metaphase spindles maintain constant length, whereas spindle microtubules continuously flux polewards, requiring addition of tubulin subunits at microtubule plus-ends, polewards translocation of the microtubule lattice, and removal of tubulin subunits from microtubule minus-ends near spindle poles. How these processes are coordinated is unknown. Here, we show that dynein/dynactin, a multi-subunit microtubule minus-end-directed motor complex, and NuMA, a microtubule cross-linker, regulate spindle length. Fluorescent speckle microscopy reveals that dynactin or NuMA inhibition suppresses microtubule disassembly at spindle poles without affecting polewards microtubule sliding. The observed uncoupling of these two components of flux indicates that microtubule depolymerization is not required for the microtubule transport associated with polewards flux. Inhibition of Kif2a, a KinI kinesin known to depolymerize microtubules in vitro, results in increased spindle microtubule length. We find that dynein/dynactin contribute to the targeting of Kif2a to spindle poles, suggesting a model in which dynein/dynactin regulate spindle length and coordinate flux by maintaining microtubule depolymerizing activities at spindle poles.

Animals↗
PubMed2004

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly.

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

Anaphase↗
PubMed2004

Importin beta is transported to spindle poles during mitosis and regulates Ran-dependent spindle assembly factors in mammalian cells.

Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin beta is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin beta dissociates from poles and is recruited back around reforming nuclei. Importin beta sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin beta accumulation at poles. Importin beta has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin beta mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-beta complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.

Animals↗
PubMed2002

A requirement for the Abnormal Spindle protein to organise microtubules of the central spindle for cytokinesis in Drosophila.

Drosophila abnormal spindle (asp) mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, which reflects a requirement for Asp for the integrity of microtubule organising centres (MTOCs). In male meiosis, the absence of a strong spindle integrity checkpoint enables asp mutant cells to proceed through anaphase and telophase. However, the central spindle region is not correctly organised and cells frequently fail to complete cytokinesis. This contrasts with meiosis in wild-type males where at late anaphase a dense array of microtubules forms in the central spindle region that has Asp localised at its border. We speculate that Asp is associated with the minus ends of microtubules that have been released from the spindle poles to form the central spindle. A parallel situation arises in female meiosis where Asp not only associates with the minus ends of microtubules at the acentriolar poles but also with the central spindle pole body that forms between the two tandem spindles of meiosis II. Upon fertilisation, Asp is also recruited to the MTOC that nucleates the sperm aster. Asp is required for growth of the microtubules of the sperm aster, which in asp mutants remains diminutive and so prevents migration of the pronuclei.

Animals↗
PubMed1985

The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. II. Kinetochore microtubules in non-treated spindles.

We determined the kinetic polarities of chromosomal spindle fibre microtubules in vivo: either the kinetochore or pole ends of chromosomal spindle fibres were irradiated with near-ultraviolet light to prevent depolymerization by colcemid. Irradiations began either just before or just after colcemid addition; cells were continually irradiated and continuously immersed in colcemid. Irradiations of kinetochore ends of chromosomal spindle fibres prevented depolymerization; irradiations of pole ends did not. Therefore, since colcemid acts by binding to the 'on' (assembly) ends of microtubules, the on ends of chromosomal spindle fibre microtubules are at the kinetochores. That is, in untreated chromosomal spindle fibres in vivo tubulin monomers add to kinetochore microtubules at the kinetochore ends. Tubulin diffused from the irradiation sites: irradiations of the cytoplasm sometimes prevented depolymerization of chromosomal spindle fibres. Prevention of chromosomal spindle fibre depolymerization was dependent on the distance of the irradiated region from the nearest chromosome; the longer the distance the less likely was it that the irradiation prevented depolymerization. On the other hand, prevention of chromosomal spindle fibre depolymerization was not dependent on the distance from the irradiated spot to the nearer pole. This analysis, too, we argue, strongly suggests that the kinetochore ends of the chromosomal spindle fibres are the on ends.

Animals↗
PubMed2002

Pigmented spindle cell carcinoid tumour of the thymus with ectopic adrenocorticotropic hormone secretion: report of a rare variant and differential diagnosis of mediastinal spindle cell neoplasms.

AIMS: A variety of histological variants of thymic carcinoid tumour have been described. A rare case of pigmented spindle cell carcinoid tumour of the thymus is documented and compared with the reported cases of thymic pigmented carcinoid tumour in the literature, with a discussion of the differential diagnosis of spindle cell tumours of the mediastinum. METHODS AND RESULTS: A thymic tumour with ectopic adrenocorticotropic hormone (ACTH) secretion was resected from a 24-year-old man suffering from Cushing's syndrome. Histological, immunohistochemical, and ultrastructural studies revealed an ACTH-producing spindle cell carcinoid tumour harbouring pigmented melanocytes. Among four thymic pigmented carcinoid tumours reported before, only one was similar to the present case by being also an ACTH-secreting pigmented spindle cell thymic carcinoid tumour. The clinicopathological features of this tumour distinguish it from a spindle cell thymoma, spindle cell thymic carcinoma, and other mediastinal spindle cell tumours. CONCLUSIONS: This case illustrates an extremely rare variant of thymic carcinoid tumour exhibiting a spindle cell morphology and harbouring pigmented melanocytes. Awareness of this histological variant is important in the differential diagnosis of spindle cell tumours of the mediastinum.

Adrenocorticotropic Hormone↗