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PubMed1987

Postnatal maturation of spindles in deafferented rat soleus muscles.

Whether the motor innervation can direct the morphological and histochemical differentiation of developing muscle spindles in the absence of sensory innervation was investigated by deafferentation of the soleus muscle in immature rats. Dorsal root ganglia containing the cell bodies of afferents from the soleus muscle were removed surgically at a stage of postnatal development when spindles already contain the full complement of intrafusal fibers innervated by both afferents and efferents, but when the fibers are histochemically and structurally immature. Experimental soleus muscles were excised one year after deafferentation and sectioned frozen at a thickness of 8 micron. Sections were stained for enzymes indicative of types of muscle fibers and sites of neuromuscular junctions, and were examined by light microscopy. Spindles of muscles that matured in the absence of sensory innervation were abnormal. They lacked the periaxial fluid space and contained fewer intrafusal fibers than did normal spindles. The morphological and histochemical profiles of the encapsulated fibers present in the deafferented spindles more closely resembled extrafusal rather than intrafusal muscle fibers. These observations suggest that deafferentation of the immature spindles induces disintegration of some intrafusal fibers and alters maturation of others. Moreover, motor axons terminated less frequently along muscle fibers in deafferented spindles than on intrafusal fibers of normal spindles. Thus, maintenance of a full complement of intrafusal fibers in the developing spindle, emergence of histochemical profiles typical of normal intrafusal fibers, and development of adult pattern of fusimotor innervation require intact sensory innervation.

Adenosine Triphosphatases↗

PubMed2004

The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells.

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase-anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 microM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.

Anaphase↗

PubMed2004

A potential tension-sensing mechanism that ensures timely anaphase onset upon metaphase spindle orientation.

The spindle orientation checkpoint (SOC) in fission yeast has been proposed to delay metaphase-to-anaphase transition when the spindle poles are misaligned with respect to the long axis of the cell. This checkpoint is activated in the absence of either an actomyosin division ring or astral microtubules. Although the SOC could be overridden in the absence of the transcription factor Atf1p, its mechanistic nature remained unclear. Here, we show that the SOC-triggered metaphase delay depends on a subset of the spindle assembly checkpoint (SAC) components Mph1p and Bub1p. Based on this finding and a detailed imaging of the spindle orientation process, we hypothesized that the spindle pole might contain proteins capable of sensing the achievement of spindle alignment. We identified the kendrin-like spindle pole body resident Pcp1p as a candidate molecule. A targeted mutation in its central domain specifically triggered the SOC in spite of the presence of oriented spindles, causing a metaphase delay that could be relieved in the absence of Mph1p, Bub1p, and Atf1p. Thus, Pcp1p might provide a link between the mechanical process of spindle alignment and the signal transduction that initiates anaphase.

Cell Cycle↗

PubMed2006

Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function.

The mature mammalian oocyte is highly polarized because asymmetrical spindle migration to the oocyte cortex ensures extrusion of small polar bodies in the two meiotic divisions, essential for generation of the large egg. Actin filaments, myosin motors, and formin-2, but not microtubules, are required for spindle migration. Here, we show that Cdc42, a key regulator of cytoskeleton and cell polarity in other systems , is essential for meiotic maturation and oocyte asymmetry. Disrupting CDC42 function by ectopic expression of its GTPase-defective mutants causes both halves of the first meiotic spindle to extend symmetrically toward opposing cortical regions and prevents an asymmetrical division. The elongated spindle has numerous astral-like microtubules, and aPKCzeta, normally associated with the spindle poles, is distributed along its length. Dynactin is displaced from kinetochores, consistently homologous chromosomes do not segregate, and polar body extrusion is prevented. Perturbing the function of aPKCzeta also causes elongation of the meiotic spindle but still permits spindle migration and polar body extrusion. Thus, at least two pathways appear to be downstream of CDC42: one affecting the actin cytoskeleton and required for migration of the meiotic spindle, and a second affecting the spindle microtubules in which aPKCzeta plays a role.

Animals↗

PubMed2006

The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Gialpha3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells.

We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry.

Amino Acid Motifs↗

PubMed2004

Cell and molecular biology of spindle poles and NuMA.

Mitotic and meiotic cells contain a bipolar spindle apparatus of microtubules and associated proteins. To arrange microtubules into focused spindle poles, different mechanisms are used by various organisms. Principally, two major pathways have been characterized: nucleation and anchorage of microtubules at preexisting centers such as centrosomes or spindle pole bodies, or microtubule growth off the surface of chromosomes, followed by sorting and focusing into spindle poles. These two mechanisms can even be found in cells of the same organism: whereas most somatic animal cells utilize the centrosome as an organizing center for spindle microtubules, female meiotic cells build an acentriolar spindle apparatus. Most interestingly, the molecular components that drive acentriolar spindle pole formation are also present in cells containing centrosomes. They include microtubule-dependent motor proteins and a variety of structural proteins that regulate microtubule orientation, anchoring, and stability. The first of these spindle pole proteins, NuMA, had already been identified more than 20 years ago. In addition, several new proteins have been characterized more recently. This review discusses their role during spindle formation and their regulation in the cell cycle.

Animals↗

PubMed2004

Cell cycle regulation of central spindle assembly.

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.

Adenosine Triphosphatases↗

PubMed2006

Biophysical model of self-organized spindle formation patterns without centrosomes and kinetochores.

Eukaryotic cell division and chromosome segregation depend crucially on the mitotic spindle pattern formation. The usual pathway for spindle production involves microtubule polymerization from two centrosomes. However, experiments using Xenopus extracts with micrometer-sized chromatin-coated beads found, remarkably, that spindle patterns can form in the absence of centrosomes, kinetochores, and duplicated chromosomes. Here we introduce a previously undescribed biophysical model inspired by the heuristic interpretations of the experiments that provides a quantitative explanation and constraints for this type of experiment. The model involves plus-directed (chromokinesin and Eg5) and minus-directed (cytoplasmic dynein oligomers) motors walking on microtubules and the boundary conditions caused by the chromatin-coated spheres. This model combines the effects of the plus-directed cross-linking motor Eg5 and any chromokinesin on the chromatin-covered beads, reflecting current uncertainties in the division of function between the two kinds of motors. The model can nucleate dynamically a variety of self-organized spindle patterns over a wide range of biological parameter values. Our calculations show that spindles will form over a wide range of parameter values. Some parameter values cause a monaster to form instead of a bipolar spindle. Varying the processivity and the dynein microtubule attachment and detachment rates, we find stability parameters for spindle formations. These results not only constrain the possible parameter values, but they point toward the proper division of function between Eg5 and chromokinesin in this spindle formation pathway. The model results suggest experiments that would further enhance our understanding of the basic elements needed for spindle pattern formation in this pathway.

Animals↗

PubMed1994

Metaphase and anaphase in the artificially induced monopolar spindle.

By using monopolar spindles artificially induced in sea urchin embryos, we examined whether or not the presence of two opposing poles was an indispensable condition for keeping chromosomes at a fixed distance from the pole at metaphase and for the anaphase chromosome movement. Chromosomes were stained with Hoechst dye 33342 and their behavior was followed in the monopolar and the control bipolar spindles. In the monopolar spindle, chromosomes were first arranged on a curved metaphase plate and then spread on a part of the imaginary surface of a sphere whose center was the monopole. The estimated chromosome-to-pole distance was similar to that of bipolar spindles at metaphase and remained fixed until chromosomes started to move toward the pole. The average duration of metaphase in the monopolar spindle was 6 times longer than that in the bipolar spindle. The poleward movement of chromosomes in the monopolar spindle was similar to the anaphase A (chromosome-to-pole movement) in the bipolar spindle with respect to the velocity, duration, distance, and synchronization of migration. These results show that even half of the normal spindle has capacities for the arrangement of chromosomes at metaphase and for the anaphase A chromosome movement. Based on these results, we were able to exclude some existing theories of metaphase, such as the one based on the balance of forces between the two poles.

Anaphase↗

PubMed1985

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗

PubMed1986

Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm.

Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle. We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure.

Animals↗

PubMed1990

In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.

Adenylyl Imidodiphosphate↗

PubMed1991

Mitotic spindle assembly by two different pathways in vitro.

We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.

Animals↗

PubMed1992

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.

Animals↗

PubMed1994

Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component.

Genetic evidence suggests that the product of the mei-1 gene of Caenorhabditis elegans is specifically required for meiosis in the female germline. Loss-of-function mei-1 mutations block meiotic spindle formation while a gain-of-function allele instead results in spindle defects during the early mitotic cleavages. In this report, we use immunocytochemistry to examine the localization of the mei-1 product in wild-type and mutant embryos. During metaphase of meiosis I in wild-type embryos, mei-1 protein was found throughout the spindle but was more concentrated toward the poles. At telophase I, mei-1 product colocalized with the chromatin at the spindle poles. The pattern was repeated during meiosis II but no mei-1 product was visible during the subsequent mitotic cleavages. The mei-1 gain-of-function allele resulted in ectopic mei-1 staining in the centers of the microtubule-organizing centers during interphase and in the spindles during the early cleavages. This aberrant localization is probably responsible for the poorly formed and misoriented cleavage spindles characteristic of the mutation. We also examined the localization of mei-1(+) product in the presence of mutations of genes that genetically interact with mei-1 alleles. mei-2 is apparently required to localize mei-1 product to the spindle during meiosis while mel-26 acts as a postmeiotic inhibitor. We conclude that mei-1 encodes a novel spindle component, one that is specialized for the acentriolar meiotic spindles unique to female meiosis. The genes mei-2 and mel-26 are part of a regulatory network that confines mei-1 activity to meiosis.

Adenosine Triphosphatases↗

PubMed1997

Spindle dynamics during meiosis in Drosophila oocytes.

Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro-activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a loss-of-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions.

Animals↗

PubMed2003

The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

Anaphase↗

PubMed2003

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins.

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase-arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.

Animals↗