abnormal spindle



Mutations in asp affect the morphology of both the mitotic spindle at several developmental stages and the meiotic spindle. At all stages mutant spindle microtubules may be described as having a long and wavy appearance, and it is not uncommon to see the loss of bipolarity in the form of hemispindle structures. To determine whether Asp protein is a constituent of the wild-type spindle, the Rb3133 antibody was used to localize the Asp protein with respect to microtubules in mitosis in syncytial embryos. During interphase, Asp protein appears to be distributed throughout the cytoplasm, but as the syncytium enters mitosis and the bipolar spindle is formed, Asp is seen in association with the polar regions. This polar association becomes tighter throughout metaphase and anaphase, but at telophase, as the chromatin is decondensing and the spindle begins to disassemble, the Asp protein appears to move away from the region occupied by the centrosome onto the central region of the spindle microtubules (Sauders, 1997).


The role of Asp in nucleating microtubules at centrosomes is consistent with its localization at the spindle poles during progression throughout mitosis in the embryonic and larval stages of Drosophila development. However, a functional rationale of the observation that Asp localizes to the spindle midzone at late telophase in embryos has never been offered (Saunders, 1997). This aspect of Asp localization was studied in male meiosis because the larger meiocytes allow greater resolution of subcellular localization. The progenitors of spermatocytes are small cells that undergo four rounds of mitosis within cysts before undertaking a prolonged period of cell growth. Asp is associated with the spindle poles during these mitotic stages and relocates to the mid-zone at late telophase, where it appears to form a ring-like structure. The protein shows a similar localization pattern during the two meiotic divisions and is localized at the spindle poles from prophase through metaphase to early anaphase. A diffuse faint punctuate staining is also shown by the anti-Asp antibody in the central part of the spindle as early as metaphase. By telophase, this central accumulation becomes concentrated at the ends of microtubules that form the mid-zone of the spindle. Thus, at this point there are two prominent regions of Asp localization: at the spindle poles (already duplicated in anticipation of meiosis II), to one side of the telophase nucleus, and at the opposite side of the nucleus where it appears to be on the ends of bundles of microtubules on the borders of the central or mid-zone region of the spindle (Riparbelli, 2002).

Asp co-operates with the gamma-TuRC to correctly to maintain juxtaposed half spindles in spermatocytes

The weak spindle integrity checkpoint in Drosophila spermatocytes has revealed a novel function of the gamma-tubulin ring complex (gammaTuRC) in maintaining spindle bipolarity throughout meiosis. Bipolar and bi-astral spindles form in Drosophila mutants for dd4, the gene encoding the 91 kDa subunit of gammaTuRC. However, these spindles collapse around metaphase and begin to elongate as if attempting anaphase B. The microtubules of the collapsing spindle fold back on themselves, their putative plus ends forming the focused apexes of biconical figures. Cells with such spindles are unable to undergo cytokinesis. A second type of spindle, monopolar hemi-spindles, also forms as a result of either spindle collapse at an earlier stage or failure of centrosome separation. Multiple centrosome-like bodies at the foci of hemi-spindles nucleate robust asters of microtubules in the absence of detectable gamma-tubulin. Time-lapse imaging revealed these to be intermediates that develop into cones, structures that also have putative plus ends of microtubules focused at their tips. Unlike biconical figures, however, cones seem to contain a central spindle-like structure at their apexes and undergo cytokinesis. It is concluded that spermatocytes do not need astral microtubules nucleated by opposite poles to intersect in order to form a central spindle and a cleavage furrow (Barbosa, 2003).

The spindle poles of dd4 primary spermatocytes usually have the expected number of centrioles by the criteria of discrete bodies of Centrosomin (CNN), a component of the pericentriolar material (PCM) that closely surrounds the centrioles in such cells. However, the finding of some spermatocytes with more than four such bodies suggests that there can be failure in centriole separation in the pre-meiotic divisions as has been described in mutant dd4 neuroblast divisions. The CNN-containing bodies in dd4 spermatocytes appear either to have never fully separated or have become reunited after spindle collapse and so the four such bodies are usually at the focus of the astral poles. In common with dd4 mutant neuroblasts, these pole bodies lack the gammaTuRC but are associated with Abnormal spindle (Asp). The ability of these poles to nucleate asters thus goes against the accepted dogma that the proper localization of gamma-tubulin and centrosomal integrity is absolutely required for the function of a polar MTOC to direct the formation of asters. At present it can only be speculated why astral microtubule arrays are not seen in dd4 neuroblasts and yet appear robust in spermatocytes mutant for the hypomorphic allele dd4S. This could reflect a general deterioration of the spindle throughout a prolonged period of metaphase delay due to the more robust spindle integrity checkpoint in neuroblasts. However, it could also reflect underlying differences in spindle structure and function between these cell types. It is possible, for example, that Asp in the focus of asters in dd4S spindles may play more of a role in maintaining astral microtubules in spermatocytes than it does in neuroblasts. This would be consistent with the known function of Asp in the reorganization of radial arrays of microtubules around isolated Drosophila centrosomes. Moreover, meiotic spindles in asp spermatocytes are abnormal in shape, and the morphology of their asters is considerably affected However, it would seem that Asp may not be as efficient at stabilizing asters in the dd4 larval CNS as in dd4 spermatocytes (Barbosa, 2003).

Many of the astral structures revealed by the immunostaining of dd4 testes appeared sufficiently asymmetric to have the appearance of hemi-spindles. These were truly monopolar by the criteria of having Asp at the focused putative minus ends of microtubules and with Pav-KLP located at their periphery, the putative plus ends. Such hemi-spindles are quite different structures from the asymmetric spindles sometimes observed in dd4 mutant neuroblasts in which one Asp containing pole can be focused and the other comprised of scattered bundles of microtubules whose putative minus ends are associated with Asp. However, real-time imaging suggests the hemi-spindles seen in dd4 meiocytes are an intermediary in the development of cones. In this process it seems that bipolarity is developed by the chromatin apparently acting to stabilize the diverging microtubules. Such spindles have one pole with multiple centrioles and the other with none (Barbosa, 2003).

The normal origin of the central spindle microtubules in wild-type cells is obscure. Treatment of cells with microtubule destabilizing agents after the onset of anaphase suggests that the central spindle may be assembled from newly nucleated microtubules and not from remains of the mitotic spindle material left in the cell equator. However, although the localization of gamma-tubulin in the central spindle of mammalian dividing cells has been reported by several groups, the presence of gamma-tubulin in Drosophila central spindle is still a matter of debate. The spindle collapse that occurs in dd4 meiocytes could be related to the onset of reorganization of the spindle that occurs at the metaphase-anaphase transition when some microtubules appear to detach from the centrosomes as the central spindle structure begins to form. In wild-type meiocytes this is seen by the generation of a new set of central spindle microtubules with Asp at their putative minus ends. Central spindle microtubules never become fully organized in the dd4 spermatocytes although this seems to progress further in cones. Consistently, Asp never undertakes its normal redistribution but rather adopts a fibrous pattern of organization extending from the spindle poles. If as it has been suggested, Asp works as an anchor to the putative minus ends of microtubules, it is possible that microtubules are released from the spindle poles and rather dispersed throughout the conical microtubule structure in dd4 meiocytes. But the lack of Asp capped microtubules of central spindle-like structures in these cells suggests some degree of co-operation with the gamma-TuRC is necessary to correctly co-ordinate this transition in spindle structure (Barbosa, 2003).

In summary these observations indicate that the gammaTuRC may provide several functions to the spindle. It is not absolutely essential for microtubule nucleation to form asters in all cell types. Rather, it may be required for the specific function of subsets of spindle microtubules that maintain pole separation. It appears to co-operate with other proteins associated with the minus ends of microtubules, notably Asp in Drosophila cells, and this appears to be important in the reorganization of the spindle that occurs following the metaphase-anaphase transition. Further work will be required to determine the extent to which defects in the reorganization of the central spindle at this stage reflect a direct requirement for the gammaTuRC or are a consequence of earlier defects in spindle organization (Barbosa, 2003).

Effects of Mutation or Deletion

In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, the role of Asp has been determined in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. The data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis (Wakefield, 2001).

To define the functional role of Asp in neuroblast spindle formation, cell division was examined in larval brains of asp1/asp1 homozygotes and asp1/aspE3 heterozygotes. The asp1 and aspE3 alleles are both semilethal mutations; they are two of the strongest extant asp mutant alleles and exhibit comparable mitotic phenotypes. When brains of asp1/asp1 and asp1/aspE3 larvae were examined, very similar phenotypes were observed. Thus, focus was placed on asp1/asp1 neuroblasts for detailed cytological analysis (Wakefield, 2001).

In asp1 brains, most dividing neuroblasts arrest in metaphase. To analyze the structure of the spindle poles, both wild-type and asp1 mutant brains were double stained for either alpha- and gamma-tubulin or alpha-tubulin and centrosomin. The gamma-tubulin and centrosomin signals observed in asp metaphases are of similar intensity to those of controls. However, although there is always a single signal at the spindle poles of control metaphases, in ~40% of asp metaphases two centrosomal signals are seen at each pole. These results indicate that asp mutants normally accumulate gamma-tubulin and centrosomin at their centrosomes. In addition, they show that in a substantial fraction of the asp metaphases there is a precocious centrosome splitting (Wakefield, 2001).

The precocious centrosome separation observed in asp metaphases is not surprising, since asp-dividing neuroblasts remain arrested in metaphase for a long time. This is likely to ungear the centrosome cycle from the spindle dynamics, leading to centrosome splitting in metaphase. These results are in apparent contrast with those of Avides (1999) who reported that asp mutant neuroblasts have disorganized gamma-tubulin clumps at the spindle poles. However, to document their data they showed an asp neuroblast metaphase with two gamma-tubulin spots at each pole. It is thus likely that they interpreted the precocious centrosome separation that occurs in asp metaphases as an aberrant gamma-tubulin distribution at the spindle poles caused by its dissociation from the centrosome (Wakefield, 2001).

Although asp and wild-type metaphases contain similar amounts of centrosomal material, mutant metaphases have fewer and generally shorter astral microtubules than their wild-type counterparts. Moreover, in asp metaphases many spindle microtubules are poorly focused and do not appear to terminate at the centrosome. These observations are interpreted to mean that Asp is required for correct microtubule nucleation from the centrosome. Alternatively, Asp could function directly at the microtubule minus ends, mediating microtubule focusing and centrosome attachment to the spindle poles (Wakefield, 2001).

By examining the cytological phenotype of asl2 asp1 double mutants, attempts were made to discriminate between these possibilities. In asl2 mutants, despite the absence of functional centrosomes and astral microtubules, metaphase spindles of larval neuroblasts are very well focused at their poles. In contrast, in neuroblast metaphases of asl asp double mutants, the microtubule minus ends are splayed apart and fail to converge into focused spindle poles. This suggests that the poorly focused spindles observed in asp1 mutants are not a consequence of centrosome abnormalities. Instead, the above results indicate that Asp is a microtubule minus end-associated protein that mediates spindle pole formation independently of centrosomes (Wakefield, 2001).

Drosophila male meiosis offers a significant advantage over mitotic larval brain cells for the phenotypic analysis of mutants defective in spindle formation. In larval brain cells, the presence of disorganized spindles activates the spindle integrity checkpoint, precluding the observation of cell division subsequent to arrested metaphases. However, in spermatocytes the spindle checkpoint is not stringent and causes only a small delay in progression through meiosis. Thus, by analyzing meiotic divisions in asp males, in addition to the role of asp in spindle pole formation, its role in central spindle assembly could be addressed (Wakefield, 2001).

As an initial step to characterize male meiosis in asp mutants, 'onion stage' spermatids were examined in living preparations of asp1/asp1 and asp1/aspE3 mutant testes by phase-contrast microscopy. Because these males displayed comparable meiotic abnormalities, focus was placed on asp1/asp1 testes for detailed cytological analysis. In wild type, each spermatid consists of one phase-light nucleus and one phase-dense mitochondrial derivative (called the Nebenkern) of similar size. The regular size of both nuclei and Nebenkern depends on the correct execution of both meiotic divisions. Errors in chromosome segregation result in the formation of nuclei of abnormal size. Failures in cytokinesis produce aberrant spermatids composed of an abnormally large Nebenkern associated with two or four nuclei of regular size (Wakefield, 2001).

In asp mutant males, 41% of the spermatids consist of a normal-sized nucleus associated with a normal Nebenkern and are likely to be the product of regular meiotic divisions. 15% of the spermatids contain a normal Nebenkern associated with an abnormally sized nucleus and are probably the consequence of a failure in chromosome segregation but not in cytokinesis. Conversely, 23% of spermatids result from failures in cytokinesis but not in chromosome segregation, since they exhibit two or four normal-sized nuclei associated with a single Nebenkern of twice or four times the size of a normal Nebenkern, respectively. Finally, spermatids consisting of a single large Nebenkern associated with two (12%), four (3%), or more than four (6%) nuclei of different sizes were observed. These peculiar spermatids are likely to be the consequence of failures in both chromosome segregation and cytokinesis. Taken together, these results strongly suggest that asp mutants are defective in both processes (Wakefield, 2001).

To determine the primary defects that cause the formation of aberrant spermatids, meiotic cell division was examined in asp1 males by staining fixed preparations for chromatin, alpha-tubulin, and either gamma-tubulin or centrosomin. Several phenotypes were observed. (1) During prometaphase the asters are smaller than their wild-type counterparts and often are not attached to the nuclear envelope as in wild type. In addition, the two asters are usually close to each other, suggesting a delayed migration to the opposite sides of the nucleus. (2) asp spermatocytes form abnormally shaped and poorly focused bipolar spindles. (3) In many cells the centrosomes do not appear to be attached to the ends of these spindles but float free in the cytosol. (4) Gamma-tubulin is present at the centrosomes in asp spermatocytes throughout meiosis, and no difference could be detected in immunostaining intensity between asp and wild-type cells. Staining with antibodies to centrosomin gave similar results. Thus, asp spermatocytes behave similarly to asp neuroblasts. Although they have small asters and poorly focused spindle poles, their centrosomes recruit normal amounts of gamma-tubulin and centrosomin (Wakefield, 2001).

To determine whether the Asp protein plays a role in central spindle assembly, asp1 telophases were examined for the presence and normality of the central spindle. Although central spindle morphology looked rather normal in about one half of the telophases, in the other half it was severely affected. In a fraction of the abnormal telophases, the central spindle fails to form completely, whereas in the remaining cells some interzonal microtubules can be discerned that are organized in small and irregular bundles that do not completely traverse the cell. To better define central spindle structure in asp mutant telophases, asp1 testes were immunostained for KLP3A, a plus end-directed microtubule motor that localizes to the equatorial region of the central spindle and is required for meiotic cytokinesis. A KLP3A distribution was observed that reflects the organization of interzonal microtubules; this protein was not detected in areas with sparse and unbundled microtubules but accumulates in the center of the irregular microtubule bundles. The actin-enriched contractile ring of asp telophases was also examined by staining cells with rhodamine-phalloidin. In wild type, this procedure reveals a clear acto-myosin ring that surrounds the central spindle midzone. In asp telophases with a normal central spindle, actin localized correctly at the cell equator. However, in cells where the central spindle failed to form properly, actin localization was severely disrupted, and where the central spindle was completely absent, no actin staining was observed (Wakefield, 2001).

The defects in central spindle formation observed in asp mutants could be either a secondary consequence of a global disorganization of the meiotic spindle or a direct consequence of an impairment of the Asp function. To discriminate between these possibilities, larval testes of asl2 asp1 double mutants were examined. In asl2/asl2 testes, telophases display a morphologically normal central spindle and cytokinesis occurs normally. In the asl2 asp1 double mutant, the frequency of cells undergoing meiosis is similar to that observed in asl2/asl2 homozygotes. However, in contrast to asl single mutants most if not all asl2 asp1 double mutant telophase-like figures have highly disorganized central spindles, which exhibit only small and irregular KLP3A and F actin patches. These results support the hypothesis that Asp contributes towards central spindle organization during anaphase and telophase. The defects in central spindle formation could then disrupt the assembly of the contractile apparatus, causing defective cytokinesis (Wakefield, 2001).

Both centrosome-containing asp mutant neuroblasts and spermatocytes display common phenotypical features. They form smaller astral microtubule arrays than their wild- type counterparts, have poorly focused spindle poles, and accumulate normal amounts of gamma-tubulin and centrosomin at their centrosomes. In addition, in a substantial fraction of mutant cells centrosomes are disconnected from the rest of the spindle. Free centrosomes can also be observed in embryos produced by homozygous asp mothers (Gonzalez, 1990 ). An interpretation consistent with all the phenotypes observed in asp mutant cells is that the Asp protein mediates interactions between microtubule minus ends and the centrosome. In asp mutants, the microtubules would be normally nucleated by the centrosome but would not be held in its vicinity after their release from this organelle. This would account for the small size of asters and for the detachment of the centrosomes from the spindle poles (Wakefield, 2001).

This interpretation is supported by the analysis of neuroblast division in asl asp double mutants that provides a trenchant demonstration of Asp function in spindle pole formation in the absence of centrosomes. In asl mutant neuroblasts, despite the absence of functional centrosomes and astral microtubule arrays, microtubule minus ends converge into well-focused spindle poles. However, when the asl and asp functions are simultaneously impaired, the microtubule minus ends are splayed outward and fail to converge into poles. This indicates that Asp plays a role in microtubule focusing at the spindle poles that is independent of any putative function in microtubule nucleation (Wakefield, 2001).

If these results indicate that Asp plays an essential role in microtubule bundling at the spindle poles, how can they be reconciled with the in vitro experiments using purified centrosomes that point towards a microtubule nucleating function for Asp (Avides, 1999)? In those in vitro experiments, embryo extracts were added to purified salt-stripped centrosomes, and microtubule nucleation from the centrosome was measured. When wild-type extract was added, asters were formed. However, when either asp extracts or extracts immunodepleted of Asp were used, asters did not form. Instead, many linear arrays of microtubules were seen that did not appear to have a focus. This led the authors to suggest that Asp functions by holding together gamma-TuRCs at the centrosome, thus facilitating microtubule nucleation. Clearly, this hypothesis cannot fully account for the results described in this study. An alternative model is that purified centrosomes do nucleate microtubules in an asp mutant background but that the microtubules are released very soon afterwards where they could continue to grow, producing the linear arrays of microtubules (Wakefield, 2001).

The functions proposed for Asp during spindle formation are very similar to those thought to be played by the dynein-dynactin-NuMA complex in a variety of vertebrate cell systems. Disruption of the activity of either dynein or NuMA during the in vitro assembly of Xenopus acentrosomal spindles results in splayed spindle poles comparable to those seen in asl asp double mutants. When the function of either dynein, dynactin, or NuMA is disrupted in centrosome-containing systems, the centrosomes retain their nucleating ability and form small asters, but spindle pole organization is disrupted; spindles display unfocused poles, which are often disconnected from the centrosomes. These findings suggest that vertebrates and Drosophila exploit similar mechanisms for microtubule tethering at the spindle poles and that NuMA and Asp play similar roles in these processes (Wakefield, 2001).

In addition to its function at the spindle poles, Asp appears to perform an additional function during central spindle assembly. Asp is not uniformly distributed along the central spindle but exhibits a striking enrichment at the microtubule minus ends of this structure. Many proteins have been found to bind to the equatorial region of the central spindle, and some of them have been shown to be necessary for central spindle stability. In contrast, only two proteins, Asp and gamma-tubulin, are known to accumulate at the minus ends of central spindle microtubules. The localization of gamma-tubulin to the central spindle extremities has been seen only in mammalian cells. Accordingly, gamma-tubulin depletion by either antibody injection or antisense RNA disrupts the central spindle and causes failure in cytokinesis. However, the presence of gamma-tubulin at the central spindle extremities in various Drosophila cell types has never been demonstrated. This suggests that in Drosophila the role played by gamma-tubulin in mammalian central spindle assembly is fulfilled by other proteins, one of which could be Asp (Wakefield, 2001).

When Asp function is disrupted, a large fraction of spermatocyte telophases display severe defects in central spindle morphology. These defects are unlikely to be an indirect consequence of abnormal microtubule organization in earlier stages of meiotic cell division in asp mutants. asl spermatocytes, which exhibit highly disorganized metaphase and anaphase spindles, nonetheless form central spindles that are undistinguishable from their wild-type counterparts. However, in asl asp double mutants, although metaphase and anaphase spindles are comparable to those of asl single mutants, central spindles either fail to form or are severely defective. Thus, it is concluded that the Asp protein plays a specific role in central spindle organization and stabilization (Wakefield, 2001).

The abnormalities seen in the central spindle of asp spermatocytes suggest that the Asp protein may help to cross-link the minus ends of central spindle microtubules, preventing them from sliding and splaying apart. Central spindle assembly during male meiosis also requires KLP3A, a kinesin-like protein that accumulates in the central spindle midzone. It is therefore likely that central spindle formation depends on both plus end- and minus end-associated proteins, which work in concert to ensure proper orientation, alignment, and stabilization of central spindle microtubules (Wakefield, 2001).

There is growing evidence that the assembly of the contractile ring depends on the integrity of the central spindle. The observations on asp mutant telophases provide additional support for this view. In asp telophases with a normal central spindle, an apparently regular F actin-enriched ring is also observed. In cells where the central spindle is completely disrupted, there is no F actin accumulation at the cell equator. Finally, in asp telophases that possess irregular bundles of microtubules between the two daughter nuclei, patches of F actin are usually observed that are always associated with bundled microtubules but which never form a continuous contractile ring. It is thus concluded that the failures in cytokinesis observed in asp spermatids are brought about by a primary defect in central spindle organization that leads to a partial or complete failure in the acto-myosin ring assembly (Wakefield, 2001).

Since spermatogenesis in Drosophila is a series of interconnected and interdependent steps and most of the spermatogenic events take place in the absence of transcription, failures at a given stage can give rise to a cascade of defects later on. The asp locus of Drosophila melanogaster codes for a non-tubulin component implicated in proper spindle structure and/or function. Homozygous asp males exhibit abnormal meiotic spindles giving rise to altered segregation of chromosomes and mitochondria and failures in cytokinesis. Postmeiotic spermatogenic stages of asp males show a series of alterations that has been interpreted as due to the previously occurring defective meiosis because meiotic spindles are the only microtubular structure altered in mutant testes. The most conspicuous alterations are: (1) variable size of nuclei and nebenkerns of early spermatids, which are also multinucleate instead of having single and uniformly sized nuclei; (2) elongating spermatids in which abnormal-sized mitochondrial derivatives elongate alongside more than one axoneme; (3) failures in the individualization process, where abnormal spermatids remain syncytial, and seem to be eliminated during the coiling stage (Casal, 1990).

Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilized eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilized homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularization, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure (Gonzalez, 1990).

A requirement for the Abnormal spindle protein to organize microtubules of the central spindle for cytokinesis in Drosophila

Whereas meiotic spindle poles appear well focused in asp mutant meiosis, central spindle defects lead to a failure of cytokinesis. Since mutations in asp are associated with disruptions in the compact nature of the MTOCs in third instar larval neuroblasts, resulting in broad spindle poles, pole integrity during male meiosis was studied in asp mutant alleles. Mutant males were studied by immunostaining with antibodies that revealed tubulin and the centrosomal proteins Centrin, gamma-tubulin and Centrosomin. The aspdd4 allele is a weak hypomorph that permits homozygotes to survive to adulthood. Such flies show rough eyes and wing defects characteristic of cell division cycle mutants, and both sexes show reduced fertility. The asp allele has previously been placed in an allelic series on the basis of the phenotypes seen in the sterile hemizygotes (White-Cooper, 1996). Comparable phenotypes were observed in male meiosis in each of the allelic combinations studied. The observations confirmed the meiotic spindle defects that have previously been reported for asp, principally, the long, wavy microtubules that are particularly evident at metaphase. Examination of the spindle poles by immunostaining against Centrin, gamma-tubulin and Centrosomin reveals that they are well organized in discrete structures but are often irregularly positioned. Moreover, in contrast to the wild-type, the antibody against Centrin fails to recognize pericentiolar material and only stains the centrioles (Riparbelli, 2002).

Whereas all cells in wild-type cysts are generally at similar stages of meiotic progression, meiocytes in asp cysts were found at a range of stages. Indeed a greater proportion of cells was consistently observed in all stages of the first meiotic division in asp mutants and a reduction of the proportion in the second division in comparison to wild type. This observation is consistent with the abnormal spindles leading to a checkpoint response, which in male meiosis is seen as a delay rather than an arrest in the progression through prophase and metaphase (Savoian, 2000; Rebollo, 2000). It may also reflect difficulties in exit from the first meiotic division cycle. In any event, excluding the unlikely possibility that the phenotype is a direct consequence of earlier defects, the ability of these cells to progress beyond metaphase reveals mutant defects later in the meiotic cycle that suggest additional functions of the Asp protein other than at the spindle poles. In contrast to the well organized poles, the central regions of the spindles of the late meiotic spindles are not correctly organized at anaphase and telophase in asp testes. Depending upon the allelic combination, mid-zone microtubules were found to be poorly organized in 20%-40% of cells within the cyst and absent in 5%-15% of cells. The absence of the mid-zone correlates with a failure of cytokinesis, which in turn leads to tetranucleate cells at the end of meiosis II. It was possible to quantify the extent of cytokinetic failure by counting the numbers of multinucleate spermatids. In each allelic combination studied, up to approximately half of the cells complete cytokinesis in both meiotic divisions, but about 35% fail one and up to 10% fail both divisions. The leakiness of this mutant phenotype probably reflects the hypomorphic nature of the alleles under study (Riparbelli, 2002).

To further correlate the central spindle defects described above with abnormal cytokinesis, the distribution of three proteins that associate with the contractile furrow was studied: Actin, Pav-Klp and Peanut. In wild-type cells, all three proteins are found in a ring-like structure at the central region of the spindle. A spectrum of localization patterns of these proteins was seen in the asp mutant cells; this reflects the extent to which the central spindle had formed again, probably reflecting the leaky nature of the hypomorphic mutant combinations studied. Localization of Actin was found to be most dramatically disrupted and scattered throughout the central part of the cell in fibrous aggregates. Nevertheless, some cells managed to produce a ring-like structure able to constrict the telophase cell. Similarly when stained to reveal Pavarotti, some meiotic cells had normal contractile rings, whereas in others in the same field Pav-KLP was more diffuse. The presence of Pav-KLP in ring canals indicated that cytokinesis had occurred correctly within the pre-meiotic rounds of mitosis. Staining to reveal the septin Peanut also showed that some cells were able to construct an apparently normal contractile ring in association with an organized central spindle region. In other cells at a similar stage, however, both the distribution of Peanut and the central spindle microtubules were disorganized (Riparbelli, 2002).

In Drosophila female meiosis, an unusual microtubule organizing structure known as the central spindle pole body is formed between the daughter nuclei at the end of the first division. Female meiosis occurs without cytokinesis, and the shared pole of the second meiotic spindles is formed at the end of meiosis I in a position at which cytokinesis would be expected to occur in any other cell type. It was therefore of interest to determine the localization of Asp with respect to the spindle poles at this unusual central microtubule organizing center (MTOC). In female meiosis, the spindle is organized primarily by the condensed chromosomes and becomes focused at the poles by the concerted action of minus-end-directed motor proteins such as Ncd. Although these poles appear to lack gamma-tubulin, by the criterion of immunostaining Asp protein was found at the poles at metaphase and anaphase. At late anaphase or early telophase of meiosis I, Asp was seen as a well defined ring in the array of centrally nucleated microtubules. Prior to meiosis II, the region previously occupied by the plus ends of microtubules in contact with chromosomes has to become the site of microtubule minus ends at the shared central pole of the second meiotic spindle. At anaphase II, Asp also becomes concentrated at the innermost spindle poles, and the ring-like staining at the central MTOC becomes more dispersed as meiosis II proceeds (Riparbelli, 2003).

Meiotic progression and behavior of the pronuclei were studied in eggs derived from asp mothers. It was only possible to do this in oocytes derived from mothers homozygous for aspdd4 and transheterozygous for aspdd4/asp1. Meosis I progresses normally in such eggs, as can be seen by the spindle at early telophase, and the aster of microtubules that forms around the basal body (contributed by the incoming sperm) has begun to form. The second meiotic division also appears to proceed normally. However, the sperm aster does not increase in dimension. This should be contrasted with the sperm aster in wild-type eggs, where centrosomal proteins such as gamma-tubulin and CP190 are recruited from the egg cytoplasm around the spindle basal body and the microtubules grow to reach the cortex of the egg cytoplasm. Immunostaining of wild-type eggs reveals that Asp is also recruited to this MTOC, which eventually contributes to both poles of the gonomeric spindles. In the asp mutant cytoplasm, the microtubules of the sperm aster never extend to the egg cortex. The sperm aster duplicates in the mutant eggs as it does in wild type, but it was frequently found to detach from the spindle that forms around the haploid male pronucleus. These centrosome-associated asters continue to duplicate as do the haploid male-derived nuclei that can undergo multiple rounds of haploid mitosis. However, the haploid products of female meiosis frequently appear to condense to form typical polar body-like structures (Riparbelli, 2003).

Genetic interactions are described between mutations in (merry-go-round) (mgr), asp, and polo, genes required for the correct behavior of the spindle poles in Drosophila. The phenotype of a polo1 mgr double mutant is more similar to mgr than polo1, but the frequency of circular monopolar figures (CMFs) seen with either mutant alone is additive, suggesting that the two gene products are required for independent functions in the formation of bipolar spindles. The aspE3;mgr double mutant arrests much earlier in development than either mutant alone, indicative of a strong block to cell proliferation. Whether the lack of microtubular structures in these cells reflects an extended mitotic arrest, or if it is a more direct consequence of the double mutant combination, is discussed. A polo1;aspE3 double mutant shows a dramatic synergistic increase in mitotic frequency. The loss of CMFs normally associated with the polo1 phenotype suggests that the Asp microtubule-associated protein is required to maintain the structure of spindle poles. It is speculated that Asp protein might be a substrate for the serine-threonine protein kinase encoded by polo (Gonzalez, 1998).

The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II

Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. This study characterized the function of the Drosophila ASPM orthologue, Abnormal spindle (Asp), and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. Size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. It is proposed that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness (Rujano, 2013).

Epithelial morphogenesis involves the action of several key molecules that coordinate cell division, tissue growth, establishment and maintenance of cell polarity, the generation of mechanical forces and the organization of cytoskeleton architecture. This study shows that Asp is a key regulator of neuroepithelium morphogenesis, as it coordinates and integrates at least two essential morphogenetic events. First it regulates tissue size and layering, by contributing to accurate cell division, spindle positioning and Interkinetic nuclear migration INM, and second, controls cell and tissue shape, by promoting apico-basal myo-II tension distribution and tissue cohesiveness during development of the optic lobe (Rujano, 2013).

In Drosophila asp mutant brain, central brain neuroblasts present defects in spindle morphology and prometaphase arrest. This study shows that in the fly neuroepithelium, cells do not arrest in prometaphase. Instead they divide with defects in chromosome segregation. These defects result in the generation of aneuploid cells that most likely die by apoptosis, as occurs in aneuploid mouse neuroepithelial cells. Furthermore, it was shown that in the fly, Asp is required for correct spindle positioning in the neuroepithelium as ASPM. Together, correct spindle assembly and positioning contribute to size determination and tissue organization in fly neuropithelium (Rujano, 2013).

As in other pseudostratified epithelia, in the fly neuroepithelium mitotic nuclei undergo INM. Myo-II cortical contractility is known to contribute to the translocation movement that allows G2 nuclei to move towards the apical cortex and the current observations support the idea that actomyosin drives apical nuclear movement during INM in the fly neuroepithelium. An even distribution of the Asp-dependent basally localized myo-II is interpreted as allowing the right amount of forces to be distributed across the basal membrane. The generation of tension and contractility then contributes to move the mitotic nucleus to the apical side of the cell. In asp, the myo-II distribution is irregular, which generates regions of high tension and contractility where the nucleus can move upwards to divide (even if too high), as opposed to regions of low tension and contractility in which low force generation impedes the upward movement of nuclei. As a consequence, the distribution of nuclei is altered and with it tissue organization (Rujano, 2013).

One of the most surprising findings in this study is the localization of Asp to interphase microtubules and to the neuroepithelium basal cortex. The fact that Asp interacts with myo-II and is required for its proper apico-basal polarization, together with the observation that myo-II is (like Asp) also enriched at the basal cortex, suggests that these proteins form part of a complex at the basal domain. Basal accumulation of myo-II most likely provides rigidity and tension to basal membranes, contributing to cell shape and tissue architecture. Thus, Asp interaction with myo-II facilitates the maintenance of a pool of myo-II at the basal side of the neuroepithelium, thereby creating a polarized distribution of myo-II within these cells, which could serve several purposes: to provide the necessary forces to push mitotic nuclei apically during mitosis; to organize a basal structural constraint that ensures tissue integrity and shape; and to prevent myo-II-mediated apical constriction. Several studies have shown the importance of apical membrane and adherens junctions in the organization of an epithelium. This work shows that basal membrane organization and the balance of forces between these two domains are of the utmost importance for the maintenance of epithelium integrity (Rujano, 2013).

Mutations in ASPM are the most common cause of MCPH. Intriguingly, ASPM knockout mouse present only mild brain size reduction. Detailed sequence analysis has shown that this knockout still contains an intact CH1 domain, which in the fly was found to be essential for Asp function. This work shows that in flies Asp is a major regulator of brain size, but in addition also of neuroepithelium morphogenesis. Microcephaly has been mainly attributed to mutations that exclusively influence the balance between neural stem cell division and differentiation by regulating mitotic spindle positioning. This study identifies however, a non-mitotic function of a microcephaly gene in brain development and neuroepithelium morphogenesis and reveals that Asp plays a role beyond spindle organization. Future work should address whether these finding can be extended to other microcephaly proteins (Rujano, 2013).


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abnormal spindle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 2 January 2016

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