Gene name - abnormal spindle
Cytological map position - 96A19--20
Function - cytoskeletal element, microtubule binding protein
Keywords - microtubule-associated protein, mitosis, meiosis, spindle, microtubule organizing center
Symbol - asp
FlyBase ID: FBgn0000140
Genetic map position - 3-85.2
Classification - actinin-type actin-binding domain, calmodulin-IQ-binding motifs
Cellular location - nuclear and cytoplasmic
Abnormal spindle (Asp) is a 220-kD microtubule-associated protein (MAP) found at the poles of mitotic spindles in the syncytial embryos of Drosophila melanogaster. It has consensus phosphorylation sites for p34cdc2 and mitogen-activated protein kinases and putative binding domains for actin and calmodulin (Sauders, 1997). abnormal spindle mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, that reflects a requirement for Asp for the integrity of microtubule organizing centers (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 organized 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 localized at its border. It is speculated 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 fertilization, 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. It is concluded that Asp is required to organize microtubules at the central region of the late mitotic spindle to enable cytokinesis (Wakefield, 2001; Riparbelli, 2002).
Asp binds at or near the microtubule minus ends of both the spindle poles and the central spindle. At the spindle poles, Asp may cross-link the microtubules, ensuring both their focusing into polarized arrays and their attachment to centrosomes after release from these nucleating centers. During anaphase and telophase, Asp could also serve to cross-link the minus ends of central spindle microtubules, thus stabilizing this structure and allowing formation of the acto-myosin contractile apparatus required for cytokinesis (Wakefield, 2001).
Meiotic spindles of Drosophila males are much larger than those of mitotic cells and particularly suitable for immunolocalization studies. Thus Asp localization was investigated during Drosophila male meiosis. Wild-type testes were immunostained both with an antibody produced against the NH2-terminal portion of Asp and with an antibody to alpha-tubulin to label the microtubules. During meiotic interphase I, the Asp antibodies stain the nucleus, but upon entry into the first meiotic division the signal relocates to the cytoplasm and weakly illuminates the spindle poles. As meiosis proceeds, the spindle pole staining increases until the spindle has formed completely. After the chromosomes have been segregated, the polar staining of Asp begins to fade. During late anaphase and telophase I, Asp dramatically relocalizes to the minus ends of the central spindle microtubules. This central spindle staining remains until the central spindle is disassembled after cytokinesis. In addition to microtubules, the antibodies also localize to the reforming daughter nuclei during anaphase and telophase I. An identical immunostaining pattern was observed in secondary spermatocytes undergoing the second meiotic division (Wakefield, 2001).
To investigate whether Asp localizes to the minus ends of central spindle microtubules in other cell types, early Drosophila embryos and larval neuroblasts were stained with the anti-Asp antibody. During spindle formation Asp is present at centrosomes and at the area of the spindle adjacent to the centrosome. However, and in addition, Asp exhibits a striking enrichment at the minus ends of central spindle microtubules similar to that seen during male meiosis. Interestingly, in both embryonic and neuroblast telophases the two daughter nuclei are not immunostained by the anti-Asp antibody, indicating that the nuclear staining observed in meiotic telophases is a peculiar feature of this type of cell (Wakefield, 2001).
To determine whether centrosomes are required for Asp localization to the spindle poles and to the outer regions of the central spindle, cells in which functional centrosomes are absent were examined. In Drosophila, two such cell types have been described: oocytes undergoing female meiosis and cells that carry a mutation in the asterless (asl) gene (Wakefield, 2001).
Drosophila female meiosis is mediated by anastral spindles assembled from microtubules nucleated near the chromosomes. During meiosis II, two spindles are tandemly arranged, perpendicular to the oocyte cortex. Between these two spindles there is an aster-like structure that contains centrosomal components such as gamma-tubulin and CP190. In contrast, the two outer poles, although well focused, do not contain any reported centrosomal or microtubule-associated proteins. When oocytes possessing these meiotic II spindles were stained with the Asp antibody, the central aster was recognized only weakly. However, the regions corresponding to the minus ends of the focused spindle poles showed strong Asp staining (Wakefield, 2001).
asl spermatocytes and larval neuroblasts are both defective in centrosome structure and fail to nucleate astral microtubules. In both cell types, the loss of centrosomal integrity leads to the formation of anastral spindles that are built around the chromatin. In asl spermatocytes, meiotic spindles are very poorly focused; chromosomes fail to align on a metaphase plate and often missegregate. Nonetheless, they assemble perfectly normal central spindles that are able to stimulate cytokinesis. The anastral spindles of asl neuroblasts are, in contrast, very well focused and are able to mediate cell division correctly. In metaphase I asl spermatocytes, a clear Asp signal is present at the poorly focused spindle poles. During meiotic telophase I, the anti-Asp antibody immunostains the minus ends of the central spindle microtubules. Similarly, in asl mutant neuroblasts undergoing either metaphase, anaphase, or telophase, Asp localization is comparable to that seen in wild-type controls. Thus, on the basis of observations both of female meiosis and of asl mutant cells it is concluded that the Asp protein does not require centrosomes to accumulate at or near the minus ends of spindle microtubules (Wakefield, 2001).
To understand the basis of Asp localization to the centrosome in greater detail, embryos were treated either with colchicine or taxol (to depolymerize or stabilize microtubules, respectively), and then the localization of Asp was compared to that of gamma-tubulin. When microtubules are depolymerized or stabilized gamma-tubulin staining remains tightly associated with the centrosomes. Similar results were obtained using antibodies to the integral centrosomal protein centrosomin. However, Asp is not visible at the centrosomes in embryos treated with colchicine. Furthermore, in embryos treated with taxol, Asp staining is not restricted to the centrosome but includes a broader region at the minus ends of the stabilized microtubules. Asp is therefore not an integral centrosomal component but is dependent upon microtubules for its localization to the centrosome (Wakefield, 2001).
Thus Asp accumulates at the spindle poles of both mitotic and meiotic Drosophila cells. This result is consistent with those obtained previously on embryonic and larval brain cells. In addition, cells that are devoid of functional centrosomes still accumulate Asp at the spindle poles. Furthermore, in the absence of microtubules Asp does not accumulate at the centrosome. These findings suggest Asp may function at the minus ends of mitotic and meiotic spindles rather than as part of the core centrosome (Wakefield, 2001).
Thus, in Drosophila, asp is involved in meiosis and mitosis, both in the organization and binding together of microtubules at the spindle poles (which causes abnormal spindles when aberrant and in the formation of the central mitotic spindle (whose perturbation leads to disruption of the contractile ring machinery and failure of cytokinesis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, leading to reduced central nervous system development. ASPM (Abnormal spindle-like microcephaly associated) is the human ortholog of the Drosophila abnormal spindle gene. The neuronal progenitor cells of the mammalian cerebral cortical ventricular epithelium have a specific pattern of mitotic activity, as do neuroblasts in the fly: symmetric cell divisions with mitotic spindles in the plane of the neuroepithelium yield two progenitor cells or two neurons, whereas asymmetric divisions yield spindles oriented perpendicular to the neuroepithelium and produce one neuron and regenerate one progenitor cell. Humans with autosomal recessive primary microcephaly (MCPH) show a small but otherwise grossly normal cerebral cortex associated with mild to moderate mental retardation, Mutations in ASPM associated with MCPH suggest that regulation of mitotic spindle orientation may be an important evolutionary mechanism controlling brain size (Bond, 2002 and references therein).
In metazoans, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin filaments that is anchored to the equatorial cortex. Interactions between filaments lead to the contraction of the ring, which constricts the dividing cell in the middle until cleavage is completed. A large body of data suggests that in animal cells the site of cytokinesis is determined by the position of the spindle. However, it is still unclear which part of the spindle provides the signals for cytokinesis. Experiments to manipulate the position of the spindle in invertebrate eggs suggested that the asters are the source of such signals. However, other work indicates that the stimulus is provided by the central spindle. For example, when a barrier is placed between the central spindle and the cortex, cytokinesis is blocked (Riparbelli, 2002 and references therein).
The central spindle is evident as a dense body of microtubules that forms in the region between the two late anaphase-telophase nuclei. The mechanism and dynamics of its formation are still poorly understood. The establishment of the central spindle in Drosophila appears, in part, to depend on at least two kinesin-like proteins: Klp3A and Pavarotti (Pav-KLP). The counterparts of Pav-KLP in other organisms, the zen-4 gene product of C. elegans or the vertebrate Mklp1, have also been shown to be required for cytokinesis. In addition to the contribution made by the spindle, several proteins, known as chromosomal passengers, dissociate from the chromosomes at the metaphase-anaphase transition and are deposited at the cell equator. The inner centromere protein (INCENP) and the associated Aurora B kinase, for example, transfer to the central spindle and the cell cortex and are necessary for completion of cytokinesis. The Polo-like kinases are also required for central spindle formation, and Drosophila Polo-kinase and Pav-KLP are mutually dependent for their localization in the central spindle mid-zone. The analysis of the cytological phenotypes displayed by mutants with disrupted meiotic cytokinesis in Drosophila males has provided insight into an intimate relationship between the formation of the central spindle and the contractile ring. Mutations identified as disrupting the central spindle are found in genes that encode a variety of actin-, microtubule- or septin-associated proteins (Riparbelli, 2002 and references therein).
Mutations in abnormal spindle (asp) have not previously been thought to affect cytokinesis. Male meiotic spindles in asp mutants are bipolar, with particularly long and wavy microtubules (Ripoll, 1985; Casal, 1990). Mitotic cells accumulate at metaphase and show spindles with disorganized broad poles at which gamma-tubulin has an abnormal distribution (do Carmo Avides, 1999). asp encodes a 220 kDa microtubule-associated protein found at the spindle poles and centrosomes from prophase to early telophase. The protein has consensus phosphorylation sites for CDK1 and MAP kinases, an actinin-type actin-binding domain and multiple calmodulin-IQ-binding motifs (Saunders, 1997). Asp and gamma-tubulin are present in partially purified centrosomes and are both required for the organization of microtubules into asters (Avides, 1999). This activity is dependent on the phosphorylation of Asp (Avides, 2001) by the kinase Polo (Riparbelli, 2002 and references therein).
asp function is also required for cytokinesis in male meiosis and the protein becomes localized at late anaphase in a manner consistent with a function in organizing the spindle mid-zone. The distribution of Asp protein was also examined in female meiosis. These divisions are unusual because the first meiotic spindle is acentriolar and appears not to contain any centrosomal proteins, such as gamma-tubulin and CP190. The female meiotic spindle microtubules are initially nucleated from chromatin and require the minus-end motor Ncd to focus the poles of the spindle for meiosis I. At telophase of meiosis I, a central pole body is formed. Microtubules in the central part of the spindle dissociate and reorganize with reverse polarity such that their minus ends are associated with this central pole body. This results in the tandemly linked second meiotic spindles. Asp is a component of this central spindle pole body. It is suggested that Asp has a dual role not only organizing microtubule-nucleating centers at the poles at the onset of M-phase but also participating in organizing the central spindle region at telophase (Riparbelli, 2002).
The Abnormal Spindle protein forms a hemi-spherical cup-like structure overlying the spindle-facing side of centrosomes in larval cells. On the male meiotic spindle it extends much further along the tips of the minus end microtubules at the spindle poles. Moreover, unlike asp mutant neuroblasts where gamma-tubulin is present in poorly organized bodies at broad spindle poles, in asp male meiotic spindles gamma-tubulin has a similar distribution to wild-type and is present at well focused poles. Indeed, the only substantive difference in the localization of centrosomal antigens that was detected in male meiotic spindles was with centrin, which is more tightly restricted to the putative centrioles than in wild-type cells. While these observed differences between mitotic and meiotic centrosomes could reflect the leaky nature of the alleles studied or a more stringent requirement for Asp in one cell type rather than the other, they could also indicate differences in the organization of centrosomes in mitosis and male meiosis. These could be related to the need to produce individual centrioles through the reductive divisions of the latter process that will eventually transform into the basal bodies in spermatids (Riparbelli, 2002).
The spindle poles in female meiosis differ further from those of either mitosis or male meiosis. These spindles are initially nucleated by chromosomes, and the spindle poles are focused by the action of microtubule-associated motor proteins, particularly the ncd-encoded kinesin-like protein. Although the poles of the female meiotic spindle are not stained by antibodies that recognize gamma-tubulin, progression through female meiosis shows some dependence upon this protein. In contrast to the low abundance of gamma-tubulin, Asp is abundant at the acentriolar poles of the female meiotic spindle. Nevertheless, meiosis appears able to proceed, at least for the mutant asp alleles selected for study. These are all hypomorphic and give leaky phenotypes that generally allow the premeiotic mitoses to proceed in both sexes and so permit the progression of both the primary spermatocytes and the oocyte to meiosis (Riparbelli, 2002).
Microtubules are also nucleated in the fertilized egg by a centrosome that develops around the basal body of the sperm following its recruitment of centrosomal proteins such as CP190 and gamma-tubulin. This sperm aster normally comprises short microtubules until metaphase II of female meiosis, at which time the microtubules begin to grow and make contact with the cortex of the egg. Growth of the sperm aster does not take place in asp mutants. A similar phenotype is seen in eggs derived from polo mutant mothers. It is possible that the dramatic effects of both polo and asp mutants on growth of the sperm aster may reflect the ability of Polo kinase to phosphorylate and activate the microtubule organizing properties of Asp (Avides, 2001). In both asp- and polo-derived eggs, as a consequence of the failure of the sperm aster to grow, the female pronucleus cannot migrate to meet its male counterpart, leading to the failure of the first gonomeric division of the zygote. In both types of mutant cytoplasm, the centrioles associated with the sperm aster can dissociate from the male pronucleus and undergo autonomous replication cycles in the syncytium. The male pronuclei can also undergo several rounds of haploid mitoses in both mutants. One noteworthy difference between asp- and polo-derived eggs is that in asp, the female pronuclei remain arrested as the polar body conglomerate whereas in polo eggs they can escape from this arrest and also undergo several rounds of haploid mitosis (Riparbelli, 2002).
In late anaphase-telophase of male meiosis, Asp localizes to the spindle mid-zone, but unlike other centrosomal antigens such as Pavarotti-KLP and Polo kinase that become associated with the central region of the spindle mid-zone, the majority of Asp decorates the very terminal regions of mid-zone microtubules. At this stage the microtubules are positioned between the telophase nuclei and the center of the spindle. The terminal regions are likely to be the minus ends of microtubules that have been released from the centrosomes, which at this stage nucleate independent asters of microtubules. This association of Asp with the spindle mid zone appears to be required for the assembly of the correct structure of the late central spindle and in turn for cytokinesis. The central spindle plays an essential role during cytokinesis and there is a cooperative interaction between this structure and the actinmyosin contractile ring: whenever one of the structures is disrupted the other fails to assemble and function. In keeping with this, many cells within asp mutant cysts have abnormal central spindles lacking the characteristic interdigitating microtubules. Moreover, molecules that participate in forming parts of the contractile ring, Pavarotti-KLP, the septin Peanut, Polo kinase and Actin, do not localize properly in asp mutant spermatocytes (Riparbelli, 2002).
Wakefield (2001) has also analysed the role of the Asp protein at the spindle poles and in cytokinesis. No conflicts are see between his conclusion that Asp plays a role in microtubule bundling at the spindle poles and an earlier report that Asp contributes to the integrity of mitotic MTOCs (Avides, 1999). However, Asp may not be sufficient to bundle microtubules: when centrosomal antigens are dispersed following the disruption of the gamma-tubulin ring complex, spindle poles can be severely splayed even though Asp is present at their minus ends (Barbosa, 2000). The present results confirm and extend the findings of Wakefield (2001) that Asp is required to organize microtubules at the central region of the late mitotic spindle to enable cytokinesis. It is speculated that the minus ends of these microtubules have dissociated from the spindle poles and now rely on the Asp protein for their stability in order to form a mid-zone structure for the central spindle. Moreover, the idea that the central spindle results from de novo nucleation of microtubules by transient organizing centers located in the region between the two daughter nuclei is substantiated by a requirement for gamma-tubulin in the equatorial region at the time of central spindle formation (Riparbelli, 2002).
Female meiosis differs from male meiosis and mitosis in Drosophila not only in that the spindle poles lack centrioles but also in that the first division is not followed by cytokinesis. Instead the central spindle has been postulated to undergo reorganization to reverse the polarity of microtubules around an unusual central spindle pole body and so form two tandemly oriented spindles for meiosis II. A model for how this occurs has been presented by Endow (1998) and is presented here modified in form. It is suggested that Asp is at the acentriolar poles of the female meiotic spindles at metaphase I. The structure of the central MTOC starts to form at early telophase I by a reversal of the polarity of the central spindle microtubules. Asp is recruited to the central region at this time and participates in the nucleation of the minus ends of these central spindle microtubules. The central spindle pole body recruits centrosomal antigens, including CP190 and gamma-tubulin. It is also striking that it additionally recruits molecules that are known to function in cytokinesis. These include Pav-KLP and Asp (Riparbelli, 2002).
This specialized central pole body of female meiosis in Drosophila has several features in common with the central spindle of conventional divisions, and it is speculated that Asp may be one of several centrosomal/central-spindle-associated proteins that play common roles in setting up these structures. In late anaphase, during both the conventional divisions and female meiosis I, a subpopulation of microtubules has to become detached from the initial poles. In effect the two half spindles of female meiosis I separate but remain focused at their original poles. The parallel step in male meiosis or in a conventional mitosis is seen by the ability of the original centrosomes to continue to nucleate asters of microtubules. In contrast, microtubules in the central part of the female meiosis I spindle must be reorganized such that their polarity is reversed to form the linked central poles of the tandemly arranged meiosis II spindles. The Asp-containing central pole body that develops in this region appears first as a ring like structure that nucleates a broad mid-zone region of microtubules that only later becomes independently focused as the meiosis II spindles migrate apart. In mitosis or male meiosis, Asp appears to be at the ends of the reorganized microtubules that form the central spindle. As telophase develops, this mid-zone becomes compacted as it coordinates the formation of the contractile ring. Indeed in male meiosis, it will ultimately be incorporated into the ring canal (Riparbelli, 2002).
Microtubule-nucleating centers that reorganize the late central region of the spindle may be a highly conserved feature of cell division. In fission yeast, a transient MTOC nucleates a post-anaphase array of microtubules in the central part of the cell before the initiation of septation. This is probably the fission yeast counterpart of the central spindle region, since its formation is promoted by overexpression of the fission yeast homolog of Polo kinase encoded by plo1, and it drives septation at inappropriate stages of cell cycle progression (Riparbelli, 2002 and references therein).
Progression through M-phase appears to require the coordination of the activities of several MTOCs. The chromosomes themselves can provide nucleating centers for the plus ends of microtubules, and centrosomes, if present, can contribute a minus-end organizing activity. The results suggest, however, that components of such a minus-end organizing activity may not be restricted to the poles but may later in M-phase be regrouped to participate in organizing the spindle mid-zone in order to successfully execute cytokinesis (Riparbelli, 2002).
abnormal spindle, a gene required for normal spindle structure and function in Drosophila, lies immediately adjacent to the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of ~124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome (Sauders, 1997).
The 6.5-kb asp cDNA encodes a predicted polypeptide of 1,863-amino acid residues. The Asp protein is predominantly hydrophilic and strikingly basic. Its secondary structure is predicted to be mostly alpha-helical. Analysis of the protein using the COILS program shows that short stretches of amino acids near the COOH terminus have the potential to form a coiled coil. There is a small sequence lying between residues 848 and 870 that has significant similarity to the core actin binding domain of a number of actin binding proteins, such as alpha-actinin, fimbrin, spectrin, dystrophin, and the Dictyostelium discoideum ABP120. These proteins either bundle actin filaments together (for example alpha-actinin) or attach actin filaments to other cellular structures. A second sequence lying between residues 938 and 968 corresponds to the conserved calmodulin binding (IQ) motif present in neuromodulin, a neuron-specific membrane-associated protein; neurogranin, a neuron specific protein kinase C substrate; the igloo gene product, a calmodulin-binding protein from the Drosophila central nervous system, and in the 'neck' regions of most forms of conventional and nonconventional myosin. In addition Asp shows six consensus sites for phosphorylation by p34cdc2 and four consensus sites for phosphorylation by MAP kinase. Interestingly, these are all clustered in the NH2-terminal third of the molecule (Sauders, 1997).
date revised: 6 February 2003
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