InteractiveFly: GeneBrief

asterless: Biological Overview | References

Centrosomes, the major organizers of the microtubule network in most animal cells (see The centrosome cycle in mammalian cells from Azimzadeh, 2007), are composed of centrioles embedded in a web of pericentriolar material (PCM). Recruitment and stabilization of PCM on the centrosome is a centriole-dependent function. Compared to the considerable number of PCM proteins known, the molecular characterization of centrioles is still very limited. Only a few centriolar proteins have been identified so far in Drosophila, most related to centriole duplication. asterless (asl) has been cloned and found to encode a 120 kD highly coiled-coil protein that is a constitutive pancentriolar and basal body component. Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. In embryos deficient for Asl, development is arrested right after fertilization. Asl shares significant homology with Cep 152, a protein described as a component of the human centrosome for which no functional data is yet available. The cloning of asl offers new insight into the molecular composition of Drosophila centrioles and a possible model for the role of its human homolog. In addition, the phenotype of asl-deficient flies reveals that a functional centrosome is required for Drosophila embryo development (Varmark, 2007).

As the major organizer of the microtubule cytoskeleton in most animal cells, the centrosome provides essential functions required for cell proliferation, differentiation, and development. Centrosomes consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that contains the proteins involved in microtubule nucleation and other cellular processes regulated by the centrosome. A significant number of PCM proteins have been identified in different experimental species by genetic analysis, via antibodies raised against purified centrosomes, or by homology with centrosomal proteins identified in other species. In Drosophila, examples include the Polo and Aurora-A kinases, founding members of the Polo/Plk and Aurora families; CP190 and CP60; Cnn; DTACC and Msps; and components of the γTURC (Varmark, 2007).

Data on the molecular composition of centrioles and the pathways that control their assembly are very limited. Most of our current knowledge on centrioles comes from studies in C. elegans where genome-wide RNAi and genetic screens have identified a number of proteins essential for centriole duplication. These include SAS-4, SAS-5, SAS-6, ZYG-1, and SPD-2 (see Drosophila Spd-2). Some of these proteins seem to be conserved in evolution. Drosophila SAK and its human orthologs Plk4 are related to C. elegans ZYG-1, and Sas-4 and CenpJ/CPAP are the suspected orthologs of C. elegans SAS-4 in Drosophila and humans. Orthologs of SAS-6 have also been identified in humans and Drosophila. Other proteins necessary for centriole duplication in Drosophila are Ana1 and Ana2 that were identified in an RNAi screen in S2 cells. In addition to these, the only other known centriolar proteins in Drosophila are Unc and D-Plp, reported to be required mostly for ciliogeneis (Varmark, 2007).

The gene asterless (asl) was identified by B. Wakimoto in a screen for mutants that affect male fertility (Bonaccorsi, 1998). Cytological studies showed that in larval neuroblasts and spermatocytes mutant for asl, γTUB accumulation and aster nucleation are highly defective (Bonaccorsi, 1998; Bonaccorsi, 2000; Giansanti, 2001). asl encodes a constitutive pancentriolar protein. The asl mutant phenotype reveals that Asl function is required for PCM recruitment and that a functional centrosome is mandatory for embryo development. The asl gene encodes a large, highly coiled-coil protein that shares significant homology with Cep152, one of the proteins identified in a proteomic analysis of human centrosomes. No functional data have yet been published on CEP152. The cloning of asl now offers a new tool to further characterize centriole function in Drosophila and a possible model for the role of its human homolog (Varmark, 2007).

The gene asl was originally mapped by meiotic recombination 0.03 cM proximal to Ki (47.3), within region 83A1-83D3 of the polytene chromosomes. Three alleles of asl have been described: asl¹ (or asl), asl², and asl³, all three EMS induced (Bonaccorsi, 1998). As a first step toward cloning the asl gene, it was mapped more precisely by P element-induced recombination of the asl¹ allele in the male germline. By taking advantage of a series of available P elements inserted in this region, it was possible to narrow down the position of asl to a short stretch of 11,735 base pairs, between the insertion points of EP(3)3591 and EP(3)3713. Only two genes have been predicted to be encoded by this region: CG1427 and CG2919. Further positional cloning results confirmed that Asl is encoded by CG2919 (Varmark, 2007).

In extracts made from S2 cells or from embryos derived from wild-type females, western blotting with Rb5110, an antibody raised against the C-terminal 16 amino acid peptide of CG2919, recognizes one band of about 120 kD that corresponds to the MW predicted from the asl coding sequence and a second band of about 95 kD. A third band with a rMW of 54 kD is recognized in extracts from embryos, but not in S2 cells. The 120 kD band is absent in embryos laid by asl¹/Df(3R)ED1557 females. These and other results strongly suggest that the 120 kD protein recognized by the Rb5110 antibody is Asl. Upon sequencing of the genomic region of the asl¹ and asl² alleles covered by the P[asl^GR] transgene, no significant polymorphism were identified. This is a surprising result given the fact that asl^GR contains all sequences required to provide asl function. However, a 71 base pair deletion (from bp 2381 to 2452) was observed in the coding sequence of asl³. This deletion shifts the reading frame and introduces a premature stop codon at position 795 (Varmark, 2007).

CG2919 encodes a 994 amino acid protein with six predicted coiled-coil motifs that span 86.7% of the protein. Stretches of low sequence complexity link these domains. Comparison with public protein databases by BLAST identified Asl homologs in different Drosophila species like D. pseudoobscura (64% sequence identity) as well as in mosquito. PSI-BLAST search based on the cluster of highly conserved Asl homologs in insects identified the human centrosomal protein Cep152 as an Asl homolog in humans. In addition, Drosophila Asl and human Cep152 were identified as putative orthologs by reciprocal best hit analysis with BLAST, as well as by the orthology prediction software of the Ensembl Genome Browser and the eukaryotic ortholog database Inparanoid. Identity throughout the entire protein sequence between Asl and Cep152 is 13% (Varmark, 2007).

To determine the subcellular localization of Asl, wild-type testes were immunostained with the Asl antibody Rb5110. PCM and chromatin were counterstained with γTUB antibodies and DAPI. A figure in the paper summarizes the main stages of PCM reorganization that take place through spermatogenesis. During the four rounds of mitosis in spermatogonia, two dots of γTUB signal, one at each side of the metaphase plate, reveal the PCM of each centrosome in these cells. At this stage, Asl signal is located at the core of each centrosome, surrounded by the PCM. During prophase of meiosis I, the PCM is dramatically reshaped and enlarged, appearing as two rods, about 2 μm in length each, joined to form a V-shaped structure. At this stage, Asl largely colocalizes with the PCM. A second reorganization of the PCM is observed during prometaphase, as the PCM coalesces back into a compact mass. At this stage, Asl remains as a V-shaped figure, the vertex of which overlaps with the PCM. After completion of meiosis, early spermatids contain a spot of γTUB that is close to the nucleus and is made of two domains of different γTUB concentrations, the proximal being more heavily labeled by the γTUB antibodies than the distal. In these cells, the Asl antibody stains one rod that spans the entire length of the low-γTUB density domain and partially colocalizes with the high-γTUB density domain. Asl was also detected in individualized, fully mature sperm where γTUB cannot be observed. EM studies have shown that in spermatocytes, before meiosis, the centriole pair acquires a distinctive V shape that is large enough to be observed by light microscopy and remains such until the end of meiosis I, when each of the two rods are segregated apart in preparation for meisosis II. Thus, throughout spermatogenesis, Asl colocalizes with centrioles and remains basal body bound in fully matured sperm. No signal could be detected by immunofluorescence with the Asl antibody Rb 5110 in spermatocytes from asl¹ males (Varmark, 2007).

YFP-Asl^FL localization was followed in embryos during early cleavage mitosis. At metaphase, YFP-Asl^FL reveals a core structure surrounded by PCM. The centrosome cycle during these nuclear divisions, which proceed very rapidly without intervening gap phases, is somewhat different from the canonical cycle. In these syncytial cycles, centrosomes split apart and start to segregate by late anaphase. At this stage, YFP-Asl^FL labels two dots at the core of the replicating centrosomes. Asl also localizes to the centrioles throughout the cell cycle in larval discs and brains (Varmark, 2007).

To determine the precise localization of Asl at the ultrastructural level, immuno-electron-microscopy was used. Three different experimental conditions were tested. First, Schneider's S2 cells were immunostained with the Asl antibody Rb5110. Second, sections of testes expressing YFP-Asl^FL were immunostained with an GFP antibody. Finally, whole-mounted embryo centrosomes purified by centrifugation through sucrose gradients were immunostained with the Asl antibody (not shown). All three tests revealed that Asl and the YFP-Asl^FL fusion are closely bound to centrioles, largely located on the periphery of the centriole barrel along its entire length. All centrosomes studied, whether by immunofluorescence or immuno-EM, contained Asl. Altogether, these data strongly suggest that Asl is a constitutive component of Drosophila centrioles and basal bodies and that the centriolar localization of Asl is not cell cycle dependent (Varmark, 2007).

FRAP analysis in living embryos expressing YFP-Asl^FL showed that photobleached centrioles recover endogenous levels of centriole-bound YFP-Asl^FL signal with a half-turnover rate of ~5 min. A similar rate of YFP-Asl^FL turnover was observed in larval neuroblasts. This dynamic behavior of Asl is markedly different from that of the other two centriolar proteins subjected to FRAP analysis so far in Drosophila. In the case of PACT-GFP, a fusion between GFP and the Pericentrin/AKAP450 centrosomal targeting (PACT) domain of Drosophila Pericentrin, FRAP of the PCM-bound signal occurs rapidly, with a half-turnover rate of 1-2 min, while the centriole-bound signal recovers only in the following round of centriole replication (Martinez-Campos, 2004). The same applies to Unc-GFP (Baker, 2004) that shows no sign of recovery 1 hr after photobleaching (Varmark, 2007).

To further characterize the function of Asl, asl¹ mutant spermatocytes expressing the centriolar marker PACT-GFP were analyzed; these mutants were immunostained with antibodies against γTUB to reveal the PCM. In wild-type spermatocytes from prophase to prometaphase, PACT-GFP reveals the centrioles as two pairs of rods that partially overlap with the PCM. In asl¹ spermatocytes, where no Asl protein could be detected, the centrosomes were severely perturbed in two regards. First, two PACT-GFP-decorated, V-shaped centriole pairs were rarely observed. Rather, the PACT-GFP signal in most asl¹ spermatocytes appeared as one cluster of irregularly shaped structures. In some unfixed, living asl¹ spermatocytes where the PACT-GFP signal is sharper, these clusters seems to be made of rods like those seen in wild-type cells, but randomly arranged. Consistent with these observations, nonserial EM sectioning of these cells showed that most asl¹ spermatocytes contained a single cluster of up to four centrioles. Centriole ultrastucture was normal in most cells, with only a very small fraction of the sections showing minor alterations at this level. Therefore, the irregularly shaped centriolar material revealed by PACT-GFP in asl¹ spermatocytes, rather than reflecting gross structural centriole abnormalities, seem to correspond to groups of up to four clustered centrioles that fail to segregate and may have lost the geometric arrangement stereotypical of wild-type centriole pairs. Loss of D-plp (Martinez-Campos, 2004) has also been reported to compromise the orthogonal orientation of mother and daughter centrioles (Varmark, 2007).

The centrosomes in asl spermatocytes were also abnormal in that the amount of PCM material associated with the centrioles was highly reduced: in 70% of the cells, the γTUB signal was below 25% of the average signal observed in control cells, the remaining 30% of the cells showing no significant accumulation of γTUB around the centrioles. Such loss of PCM recruitment and the resulting failure to nucleate microtubules have direct consequences in spindle assembly. In cells where the centrosomes do not recruit detectable amounts of PCM, microtubule organization is anastral and chaotic. In cells where some PCM is recruited, loss of centrosome segregation results in either one single aster that remain very close to each other. In a few instances, the asters separate and bipolarity is established. Thus, the expressivity of the asl¹ phenotype varies from virtually acentrosomal cells to cells that contain two segregated asters before NEB. However, even in those cases where PCM was recruited and microtubule asters were organized, the asters were always much smaller and less dense than in control cells. Video recording in living asl¹ spermatocytes expressing GFP-α-tubulin revealed that in the few cells that initiated spindle assembly with well-separated centrosomes, biastral bipolar spindles were organized. In the case of cells with nonseparated centrosomes, the microtubule arrays organized into a monastral spindle. Monastral spindle figures were the main spindle type observed in asl¹ spermatocytes. Thus, the abnormal centrosome function caused by the asl mutation results in reduced microtubule nucleation and severe defects in meiotic spindle assembly, which in turn results in a high incidence of aneuploidy (Varmark, 2007).

Fertilization contributes the first centriole of the developing Drosophila embryo via the sperm basal body. Asl is be a constitutive element of the sperm basal body and hence to be paternally contributed. However, Asl is also maternally provided in quantities that significantly exceed the amount of paternal centriole-bound Asl. This fact, together with the high turnover rate of centriolar Asl, results in the fast replacement of the basal body-bound Asl by the maternal pool. Thus, in unlabelled eggs fertilized by males expressing the YFP-Asl^FL fusion, the basal body loses its YFP signal soon after fertilization and becomes untraceable by fluorescence microscopy. Likewise, when YFP-Asl^FL-expressing females are fertilized by wild-type males, the basal body incorporates YFP-Asl^FL immediately after sperm entry. In the following telophase, after a further round of replication, the rod-like, basal body-derived centriole can still be seen together with the three dot-like new centrioles. Indeed, the long centriole that originates during spermatogenesis perdures and serves as a centriole beyond the first zygotic mitosis. These observations show that Asl is associated with centrioles from the first stages of development and that paternal basal body-bound Asl is quickly exchanged with the maternally provided Asl pool (Varmark, 2007).

It was then determined whether Asl plays a function during these first stages of zygotic development. After sperm entry and activation, female meiosis is resumed and four haploid nuclei are produced in wild-type eggs. The most internal of these nuclei, which is the closest to the sperm nucleus, becomes the female pronucleus, while the others, usually clustered into a polar body, remain inactive and eventually disappear. Recruitment of PCM around the paternally contributed centriole results in the assembly of the first zygotic centrosome and in the organization of a prominent aster, which is thought to mediate male and female pronuclear fusion. Soon afterwards, the duplicated centrosomes migrate apart over the male pronucleus, and fusion with the female pronucleus takes place. The first mitotic spindle is then assembled, and repeated rounds of nuclear division cycles result in the exponential proliferation of syncytial nuclei. In embryos derived from asl¹/Df(3R)ED1557 mothers, female meiosis proceeds normally: distinct polar bodies and female pronuclei can be identified, sperm entry takes place, and upon fertilization, all five nuclei are present and arranged in a seemingly wild-type configuration. Cnn accumulates at a point near the male pronucleus, presumably around the paternally provided basal body that is wild-type for Asl, but a functional MTOC is not organized, development is brought to a halt, and the first zygotic mitosis never occurs. Instead, nonfunctional, anastral spindle-shaped structures are organized around the chromatin. These spindles, which do not segregate chromosomes, persist in embryos aged for 1-2 hr, a stage at which wild-type embryos contain hundreds of nuclei. Thus, maternal Asl is needed to facilitate the centrosome function required for initiation of cleavage cycles in the fly. This terminal phenotype is indistinguishable from the phenotype of embryos derived from γTUBTW1 homozygous females, which lack the maternal γTUB37C gene (Varmark, 2007).

In terms of the possible roles that centrioles may play during development, these results show that the first zygotic division never occurs in a cytoplasm deficient for Asl, strongly suggesting that functional centrosomes are needed for embryogenesis in Drosophila. The same conclusion was suggested by the observation that in eggs derived from females lacking PCM components like γTUB37C or D-TACC, the first mitotic division does not take place. However, the possibility remained that this early developmental arrest in γTUB37C or D-TACC-deficient embryos could be a downstream consequence of the meiotic defects caused by mutations in these genes. This caveat is now largely circumvented by the phenotype of embryos derived from asl mutant females in which both meiotic divisions proceed normally. Thus, although a possible noncentrosomal function of Asl cannot be rule out, the phenotype of embryos derived from asl mutant females is consistent with the hypothesis that centrosomes are required for Drosophila embryo development (Varmark, 2007).

Previous reports have shown that zygotic loss of key centrosomal proteins such as D-plp, Sas4, Sak/Plk4, or Cnn does not block progression of development into adult flies. However, the centrosome-less females that hatch are sterile, strongly suggesting that eggs defective for these centrosomal components cannot support embryogenesis. How development can proceed in zygotic loss-of-function conditions for these genes is not entirely clear. However, the initial stages of development of individuals homozygous for mutations in these centrosomal proteins are likely to be sustained by the wild-type RNA/protein contributed to the egg by the heterozygous females from which they derive. Thus, until a certain stage that is hard to specify, development in these mutant individuals actually takes place when cells still have centrosomes. The hatching of adults that have undergone the last stages of development without centrosomes certainly proves a certain level of centrosome dispensability in Drosophila development, even though such adult flies are uncoordinated and sterile and die only hours after eclosion (Varmark, 2007).

Loss of centrosome function has been reported to impair a number of developmental stages in vertebrates. In humans, for instance, abnormal centrosomes have been linked to impaired neuronal migration, hereditary spastic paraplegia, Bardet-Biedl syndrome, development of cystic kidneys, perturbed left-right asymmetry, microcephaly, and cancer. In mice, lack of function for Sak/Plk4 is a lethal condition, and haploinsuficiency for this gene results in a high incidence of tumors. The molecular dissection of centrioles in Drosophila may help to model the cellular basis of some of these processes (Varmark, 2007).

Spindle self-organization and cytokinesis during male meiosis in asterless mutants of Drosophila

While Drosophila female meiosis is anastral, both meiotic divisions in Drosophila males exhibit prominent asters. A gene has been identified, asterless (asl), that is required for aster formation during spermatogenesis. Ultrastructural analysis showed that asl mutants have morphologically normal centrioles. However, immunostaining with antibodies directed either to γ tubulin or centrosomin revealed that these proteins do not accumulate in the centrosomes, as occurs in wild-type. Thus, asl appears to specify a function required for the assembly of centrosomal material around the centrioles (Bonaccorsi, 1998).

Despite the absence of asters, meiotic cells of asl mutants manage to develop an anastral spindle. Microtubules grow from multiple sites around the chromosomes, and then focus into a peculiar bipolar spindle that mediates chromosome segregation, although in a highly irregular way (Bonaccorsi, 1998).

Surprisingly, asl spermatocytes eventually form a morphologically normal ana-telophase central spindle that has full ability to stimulate cytokinesis. These findings challenge the classical view on central spindle assembly, arguing for a self-organization of this structure from either preexisting or newly formed microtubules. In addition, these findings strongly suggest that the asters are not required for signaling cytokinesis (Bonaccorsi, 1998).

asterless specifies a function necessary for aster formation during Drosophila male meiosis. In interphase primary spermatocytes and meiotic cells of wild-type males, centrosomes are enriched in γ tubulin. In contrast, in the same cell types of asl mutants this protein does not accumulate in the centrosomes but remains dispersed in multiple cytoplasmic aggregates that do not have microtubule-nucleating ability. Most likely, this primary defect in centrosome assembly prevents aster formation throughout meiotic cell division in asl mutants (Bonaccorsi, 1998).

A similar but not identical situation has been observed in the acentriolar Drosophila cell line 1182-4, established from aploid embryos produced by the female sterile mutant mh 1182. In control embryonic cell lines, γ tubulin accumulates in both interphase and mitotic centrosomes. In 1182-4 acentriolar cells γ tubulin fails to associate with the interphase centrosomes, but it concentrates in the spindle poles where it exhibits different patterns of accumulation. However, the γ tubulin polar spots seen in the acentriolar cells are not true centrosomes in that they readily disappear upon microtubule disassembly with either cold or colchicine treatment. Based on these results, it has been suggested that centrioles play an important role in the assembly of centrosomal material (Bonaccorsi, 1998 and references therein).

This study has shown that in wild-type testes, antibodies directed to centrosomin immunostain the centrosomes in mature primary spermatoytes and throughout meiosis. In asl mutants these antibodies detect either doublets or quartets of discrete structures that are present in all late prophase/prometaphase primary spermatocytes, but are transmitted to only one half of the secondary spermatocytes and to one fourth of the spermatids. The behavior of these centrosomin-enriched bodies seen in asl mutants can be easily explained if one assumes that they correspond to the centrioles (Bonaccorsi, 1998).

In wild-type, each mature primary spermatocyte contains two pairs of duplicated centrioles, with the daughter centriole lying at a right angle with respect to its parent. In preparation of meiosis I, both pairs of centrioles migrate together from the plasma membrane to the nuclear envelope, become associated with centrosomal material, and move to the cell poles while nucleating astral microtubules. Thus, during meiosis I each centrosome contains a pair of duplicated centrioles. However, there is not centriole duplication before the second meiotic division; in secondary spermatocytes each pair of centrioles splits into two single centrioles that migrate to the opposite cell poles. Therefore, each spermatid inherits a single centriole that becomes the basal body of the elongating axoneme (Bonaccorsi, 1998).

Based on centriole behavior in the wild-type, it is proposed that the centrosomin-enriched doublets seen in asl primary spermatocytes correspond to the centrioles. The fact that these doublets are occasionally resolved into four entities further suggests that each element of the doublets does in fact consist of a pair of centrioles. In addition, it is proposed that the two pairs of centrioles, due to the absence of astral microtubules, fail to separate and migrate to the cell poles during both meiotic divisions of asl mutants. Thus, during each meiotic division they are transmitted together to only one of the two daughter cells. This model for centriole behavior in asl mutants is supported by the results obtained by EM. EM analysis has shown that asl cells contain morphologically normal centrioles that in several cases fail to separate properly. Several Nebenkern associated with two instead of a single centriole were observed. Moreover, in some asl spermatids, these two centrioles are lying parallel to each other instead of at a right angle, as do the parent and its daughter centriole in wild-type. This parallel centriole arrangement is consistent with the possibility that the two centrioles in the plane of the section belong to different pairs of centrioles that have been transmitted together to the sectioned spermatid (Bonaccorsi, 1998).

Centrosomin immunostaining and EM analysis clearly indicate that asl meiotic cells contain centrioles of regular morphology that duplicate normally. Thus, the asl¹ function does not appear to be required for either centriole fine structure or duplication. However, the observation that asl centrioles are never associated with γ tubulin and accumulate much less centrosomin than their wild-type counterparts, strongly suggests that asl specifies a function required for the assembly of centrosomal material around the centrioles. The identification of such a function must await the molecular analysis of asl, which, however, may turn out to be particularly difficult (Bonaccorsi, 1998).

This study has shown that despite the absence of asters, asl mutants assemble a peculiar anastral spindle. Meiotic chromosomes appear to play an important role in this process, acting as microtubule-organizing centers and promoting formation of bipolar minispindles. This finding was anticipated by micromanipulation experiments showing that Drosophila male bivalents detached from the spindle can trigger the formation of minispindles (Bonaccorsi, 1998 and references therein).

The aberrant meiosis observed in asl males has many similarities with naturally occurring anastral divisions, such as those accompanying female meiosis in mice, Caenorhabditis, Xenopus, and Drosophila. The asl spindle formation pathway is also reminiscent of the in vitro spindle assembly induced by DNA-coated beads in Xenopus egg extracts. In all these systems, chromatin can induce microtubule nucleation and stabilization. These microtubules are initially randomly oriented; their minus-ends then focus at the spindle poles through the action of minus-end-directed motors and their associated proteins. However, the minispindles associated with the asl bivalents are not always clearly organized into a bipolar array. Moreover, when they do exhibit a bipolar configuration, the poles are broad and are never as focused as those observed in Drosophila female meiosis or in the Xenopus in vitro systems. This result suggests that Drosophila spermatocytes do not have sufficient minus-end motor activity to complete spindle polarization in the absence of centrosomes (Bonaccorsi, 1998 and references therein).

These results on asl mutants indicate that cells in which spindle assembly is normally driven by centrosomes nonetheless have the ability to form anastral spindles. Similar findings have been obtained with crane fly spermatocytes, but not with grasshopper spermatocytes where both the chromosomes and the centrosomes are essential for spindle formation. In addition, a series of studies has clearly shown that spindle assembly during mitotic division of a variety of vertebrate cell types invariably requires the presence of functional centrosomes. Together, these findings raise the question of why the ability to form anastral spindles in cells that normally contain centrosomes is restricted to a few meiotic systems. It is possible that this property reflects different types of interaction between chromosomes and microtubules. In vertebrate mitotic cells and in grasshopper spermatocytes, the chromosomes can only capture and stabilize the microtubules nucleated by the centrosomes, and do not appear to have the ability to stimulate microtubule growth. In contrast, in Drosophila male meiosis and most likely also in meiosis in the crane fly Pales, the chromosomes act as microtubule- organizing centers, even in the absence of centrosomes. Thus, it is suggested that anastral spindles are assembled only in those centrosome-containing systems where the chromosomes can induce formation of a sufficient number of microtubules. In systems where the chromosomes are unable to promote substantial microtubule growth, there would not be enough microtubules to form a bipolar spindle (Bonaccorsi, 1998 and references therein).

One of the most remarkable features of asl male meiosis is the formation of a morphologically normal central spindle in most ana-telophases. This finding challenges the classical view of central spindle assembly through interaction of antiparallel polar microtubules. The results argue for a self-organization of the central spindle using either preexisting or newly formed microtubules. Most likely, central spindle formation during male meiosis is mediated by microtubule cross-linking, plus-end-directed kinesin-like motors. This hypothesis is supported by the finding that mutations in KLP3A, a Drosophila gene encoding a kinesin-like protein that concentrates in the central spindle midzone during male meiosis, disrupts central spindle formation and cytokinesis (Bonaccorsi, 1998 and references therein).

An open question about cell cleavage in animal systems is the source of signals that stimulates contractile ring formation and cytokinesis. It has been suggested that these signals may be provided either by the metaphase chromosomes or the asters or the central spindle. The current results clearly show that the asters are not needed for the cytokinetic signal. Moreover, the fact that asl chromosomes are scattered within the cell and never congress into a metaphase plate strongly suggests that chromosomes cannot dictate the positioning of the cleavage furrow. This conclusion agrees very well with the results of recent micromanipulation experiments showing that cytokinesis can occur in the absence of chromosomes in grasshopper spermatocytes. Thus, of the three components of the anaphase spindle -- the asters, the chromosomes, and the central spindle -- only the latter appears to be required for signaling cytokinesis. In this respect, the current findings rule out the possibility of the central spindle merely accumulating cytokinetic signals originating from the asters (Bonaccorsi, 1998).

During Drosophila male meiosis, there is a cooperative interaction between the central spindle and the contractile ring; when one of these structures is disrupted the other one is also affected. Thus, the central spindle appears to play an essential role during cytokinesis. The asters, however, may be important for symmetrical positioning of the central spindle between the two daughter cells (Bonaccorsi, 1998).

asterless mutant neuroblasts undergo normal division

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. This study analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. Neuroblast division was examined in asterless mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. These results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. The localization of Miranda was examined during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division (Giansanti, 2001; full text of article).

Search PubMed for articles about Drosophila Asterless

Azimzadeh, J. and Bornens, M. (2007). Structure and duplication of the centrosome J. Cell. Sci. 120: 2139-2142. Full text of article

Baker, J. D., Adhikarakunnath, S. and Kernan, M. J. (2004). Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila, Development 131: 3411-3422. Medline abstract: 15226257

Bonaccorsi, S., Giansanti, M. G. and Gatti, M. (1998). Spindle self-organization and cytokinesis during male meiosis in asterless mutants of Drosophila melanogaster. J. Cell Biol. 142: 751-761. Medline abstract: 9700163

Bonaccorsi, S., Giansanti, M. G. and Gatti, M. (2000). Spindle assembly in Drosophila neuroblasts and ganglion mother cells. Nat. Cell Biol. 2: 54-56. Medline abstract: 10620808

Giansanti, M. G., Gatti, M. and Bonaccorsi, S. (2001). The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts. Development 128: 1137-1145. Medline abstract: 11245579

Martinez-Campos, M., Basto, R., Baker, J., Kernan, M. and Raff, J. W. (2004). The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis, J. Cell Biol. 165: 673-683. Medline abstract: 15184400

Varmark, H., et al. (2007). Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila. Curr. Biol. 17(20): 1735-45. Medline abstract: 17935995

date revised: 10 January 2009

Home page: The Interactive Fly © 2007 Thomas Brody, Ph.D.