InteractiveFly: GeneBrief

Spd-2: Biological Overview | References

In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. These proteins are widely believed to comprise a conserved core duplication module. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles (Pelletier, 2004; Kemp, 2004; Pelletier, 2006; Delattre, 2006). Drosophila Spd-2 is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. Spd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in Spd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, Spd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. Spd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. It is speculated that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm (Dix, 2007).

The predicted Drosophila ortholog of C. elegans SPD-2 is encoded by the gene CG17286, which is referred to as Drosophila Spd-2 (Pelletier, 2004). Rabbit Spd-2 antibodies were generated and affinity-purified; these antibodies recognized centrosomes at all stages of the cell cycle in embryos and specifically in mitosis in brain. A Spd-2-GFP (green fluorescent protein) fusion protein was generated, that localizes to centrosomes throughout the cell cycle in both embryos and larval brain cells (Dix, 2007).

In brain cells, Spd-2-GFP was more strongly recruited to mitotic centrosomes, but it also localizes to interphase centrosomes. Because larval brain cells recruit very little pericentriolar material (PCM) during interphase, this localization suggests that Spd-2-GFP is associated with centrioles in interphase and is also recruited to the PCM in mitosis. In addition, Spd-2-GFP localizes to the large centrioles present in the primary spermatocytes. Taken together, these observations indicate that, like its C. elegans counterpart, Spd-2 localizes both to the centrioles and to the PCM (Dix, 2007).

Interestingly, Drosophila Sas-4, Sas-6, and Sak/Plk4 specifically localize to the proximal and distal ends of the centrioles in primary spermatocytes, and it has been proposed that this localization might be common to proteins involved in the duplication process (Peel, 2007). In contrast, Spd-2 equally distributes along the length of the centrioles in these cells as detected using both the GFP fusion protein and antibodies, suggesting that Spd-2 might not contribute to centriole duplication in the same manner as do the other proteins (Dix, 2007).

To test whether Spd-2 is essential for centriole duplication, a stock was obtained carrying a mutation in the Spd-2 gene. The G20143 mutant stock is an enhanced and promoter (EP) line carrying a P element insertion directly after the initiating ATG codon of the Spd-2 gene. On a Western blot, Spd-2 protein was not detected in Spd-2 mutant brains, and quantitative Western-blot analysis revealed that Spd-2 protein levels were reduced by more than 90%. No Spd-2 was detected at centrosomes or centrioles in Spd-2 homozygous mutant brain cells by immunofluorescence. Thus, this P element insertion severely reduces the expression of the Spd-2 protein. All of the phenotypes described below were rescued by the Spd-2-GFP transgene, demonstrating that the lack of Spd-2 is responsible for these defects (Dix, 2007).

Previous studies have shown that flies lacking centrioles are viable but are severely uncoordinated because of a lack of cilia in their mechanosensory neurons. However, Spd-2 mutant flies are viable and are not uncoordinated, suggesting that they possess both centrioles and cilia. Consistent with this observation, centrioles were detectable in both wild-type (WT) and Spd-2 mutant brains expressing the centriolar markers GFP-PACT, Sas-4-GFP, and Asl-GFP. Quantification of centriole numbers in fixed brain preparations revealed that, in contrast to mutants lacking the other centriole-duplication proteins Sas-4 (Basto, 2006), Sas-6 (Rodrigues-Martins, 2006), and Sak/Plk-4 (Bettencourt-Dias, 2005), there was no dramatic decrease in centriole numbers in Spd-2 mutant cells compared to the WT. These results are consistent with the recent finding that depletion of Spd-2 in a genome-wide screen in Drosophila cultured cells did not affect centriole duplication (Dix, 2007).

To determine whether Spd-2 is essential for centriole duplication in other tissues, the large centrioles were examined in the primary spermatocytes of the male germline. Strikingly, centriole numbers actually increased in the primary spermatocytes lacking Spd-2, suggesting that centrioles can over duplicate in the absence of Spd-2 in some tissues. The presence of extra centrioles leads to the formation of multipolar spindles, resulting in severe meiotic defects and male sterility. Taken together, these results suggest that Spd-2 is not essential for centriole duplication in Drosophila somatic cells. Thus, SPD-2 is the first centriole-duplication factor from the C. elegans pathway that does not have an essential role in this process in Drosophila (Dix, 2007).

In the C. elegans embryo, SPD-2 is essential for PCM recruitment to the centrioles, and it appears to be one of the earliest effectors in the recruitment pathway (Pelletier, 2004; Kemp, 2004). To test whether Spd-2 is also required for this process in flies, the recruitment of the PCM markers Cnn and γ-tubulin to mitotic centrosomes was assessed in WT and Spd-2 mutant third-instar larval brain cells. Both Cnn and γ-tubulin were still detectable on the majority of mitotic centrosomes in Spd-2 brains, but they were present at significantly decreased levels compared to the WT. Thus, Spd-2 appears not to be essential for PCM recruitment in mitotic larval brain cells, but is required to ensure the efficiency of this process (Dix, 2007).

It was asked whether the reduced levels of PCM at Spd-2 mutant centrosomes would affect the ability of the centrosomes to nucleate MTs and hence drive spindle formation. It was found that the mitotic index in WT and Spd-2 mutant third-instar larval brain cells was very similar, suggesting that mitosis occurs with normal timing in mutant cells. Moreover, a live analysis of the mitotic spindles in larval neuroblasts expressing GFP-α-tubulin revealed that the centrosomes in Spd-2 mutant neuroblasts nucleated robust astral MT arrays that participated in spindle formation in a manner indistinguishable from those in WT cells. In addition, Spd-2 mutant neuroblasts invariably divided asymmetrically, as was the case in WT neuroblasts. Thus, in Spd-2 mutant third-instar larval brain cells, the reduced efficiency of PCM recruitment does not detectably perturb mitosis. This is in contrast to C. elegans embryos lacking SPD-2, in which PCM recruitment, MT nucleation, and mitosis are dramatically impaired (Dix, 2007).

The data suggest that Spd-2 is not essential for centriole duplication and has only a minor role in PCM recruitment in Drosophila somatic cells. Despite this fact, Spd-2 mutant females produced embryos that did not hatch as larvae. In fixed 0-4 hr collections of Spd-2 embryos mated with WT males (hereafter Spd-2 embryos), most embryos had been fertilized but were arrested very early in development (Dix, 2007).

To determine the nature of the defect in Spd-2 embryos, 0-15 min collections of embryos were fixed and stained to visualize MTs, PCM (Cnn), and centrioles (Asl-GFP). The first mitotic spindle was frequently observed in 0-15 min collections of WT embryos, but it was never observed in Spd-2 embryos, suggesting that mutant embryos arrest before the first mitosis. In many mutant embryos, it was clear that pronuclear migration had failed, and male and female pronuclei were often observed that remained spatially separated within the embryo. This was never observed in WT embryos of a similar stage (as determined by the morphology of the polar bodies; data not shown). Thus, it appears that Spd-2 mutant embryos are unable to develop because of a failure in pronuclear fusion (Dix, 2007).

Next, whether there were defects in female meiosis that might explain the early arrest of the Spd-2 embryos was analyzed. It was found that female meiosis proceeded normally in Spd-2 embryos, generating a female pronucleus that was positioned appropriately for subsequent capture by the sperm aster. Defects, however, were observed in the recruitment of Cnn to the shared central pole of the meiosis II spindles in Spd-2 embryos. This observation suggests that Spd-2 can have a role in recruiting PCM proteins to MT organizing centers (MTOCs) that do not contain centrioles (Dix, 2007).

To test whether the mutant embryos fail in pronuclear fusion because of a defect in sperm aster assembly, the distribution was analyzed of PCM and MTs around the sperm centriole that enters the egg at fertilization. In the majority of WT embryos in metaphase or later stages of meiosis II, large amounts of Cnn and MTs were recruited around the sperm centriole. In contrast, in Spd-2 embryos at the same stage, sperm centrioles were rarely associated with Cnn or MTs. Thus, in contrast to somatic cells, Spd-2 has an essential role in recruiting the PCM to the sperm centriole: In its absence, the sperm centrosome does not organize a robust array of astral MTs, pronuclear fusion fails, and mutant embryos arrest prior to the first mitotic division (Dix, 2007).

These results suggest that Spd-2 has a particularly important role in PCM recruitment to the sperm centriole. It was therefore wondered whether Spd-2 might also be essential for centriole duplication specifically during the first centriole-duplication event after fertilization. It was observed, however, that in all late meiosis II embryos in which the centrioles could be detected, a newly duplicated centriole could be distinguished in both WT and Spd-2 embryos. This suggests that the single sperm-derived centriole is capable of undergoing the first duplication event in the absence of maternally supplied Spd-2 protein, although the interpretation of this result is not straightforward (Dix, 2007).

These data indicate that the Drosophila ortholog of C. elegans SPD-2 is not essential for centriole duplication and has only a minor role in PCM recruitment in somatic cells. This might suggest that the function of SPD-2 has diverged between worms and flies. It is noted, however, that the essential role of C. elegans SPD-2 in centriole duplication and PCM recruitment has, to date, only been demonstrated in the embryo, during the first events after. The data show that Spd-2 plays a particularly important role in recruiting the PCM to the centriole that enters the fly embryo at fertilization. Thus, it is possible that this family of proteins has a conserved function, which is only absolutely essential when the sperm centriole(s) first enter the fertilized egg (Dix, 2007).

Why would SPD-2 proteins be essential for PCM recruitment to the sperm centriole but not to the centrioles in somatic cells? One possibility is that SPD-2 proteins have a specialized role in facilitating the de novo recruitment of PCM proteins from maternal stores to the “naked” centriole(s) that enter the oocyte at fertilisation. This is a unique situation, because in all subsequent rounds of mitotic PCM recruitment, the centrioles are already associated with at least a small amount of PCM that was present on the centriole during interphase. Hence, SPD-2 proteins might not be essential for PCM recruitment once the PCM has initially been loaded onto the sperm centrioles. It remains possible, however, that SPD-2 proteins are also essential for PCM recruitment to the centrioles during subsequent embryonic cycles. Unfortunately, this possibility cannot be tested because Spd-2 embryos arrest prior to the first mitotic division (Dix, 2007).

As is the case for PCM recruitment, there are currently no data suggesting that SPD-2 is essential for centriole duplication in worm somatic cells. Indeed, the results of a recent candidate-based siRNAi screen in human tissue culture cells suggested that human SPD-2 (Cep192) might not be essential for centriole duplication in somatic cells (Kleylein-Sohn, 2007). It therefore remains possible that SPD-2 proteins could be specifically required for the initial duplication of the fertilizing sperm centriole. At a first glance, these data appear to contradict this hypothesis because it was shown that the first round of centriole duplication can occur in Drosophila embryos lacking Spd-2. It remains possible, however, that the initial events of centriole duplication have already occurred in the sperm prior to fertilization, and so are not dependent on the maternally supplied pool of Spd-2. It is not possible to test this hypothesis directly by the fertilization of Spd-2 embryos with Spd-2 mutant sperm because these sperm are immotile (Dix, 2007).

How could SPD-2 proteins coordinate these two important centrosome-cycle events—centriole duplication and centrosome maturation? It is proposed that SPD-2 could act as a general protein-recruitment factor, which is only absolutely essential for the recruitment of centriole-duplication factors and PCM proteins to the sperm centriole(s) after fertilization. More work will elucidate whether SPD-2 proteins are also dispensable for these processes in the somatic cells of other species, or whether the functions of worm and fly SPD-2 proteins have diverged. In either case, these data highlight the importance of studying the roles of proteins implicated in the centrosome cycle in a range of organisms and cellular contexts (Dix, 2007).

Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation

SPD-2 is a C. elegans centriolar protein required for both centriole duplication and pericentriolar material (PCM) recruitment (Pelletier, 2004; Kemp, 2004; Delattre, 2005; Pelletier, 2006). SPD-2 is conserved in Drosophila (DSpd-2) and is a component of the fly centriole (Dix, 2007; Rodrigues-Martins, 2007; Goshima, 2007). The analysis of a P element-induced hypomorphic mutation has shown that DSpd-2 is primarily required for PCM recruitment at the sperm centriole but is dispensable for both centriole duplication and aster formation (Dix, 2007). This study shows that null mutations carrying early stop codons in the DSpd-2 coding sequence suppress astral microtubule (MT) nucleation in both neuroblasts (NBs) and spermatocytes. These mutations also disrupt proper Miranda localization in dividing NBs, as has been seen in mutants lacking astral MTs. Spermatocyte analysis revealed that DSpd-2 is enriched at both the centrioles and the PCM and is required for the maintenance of cohesion between the two centrioles but not for centriole duplication. DSpd-2 localization at the centrosome requires the wild-type activity of Asl but is independent of the function of D-PLP, Cnn, γ-tubulin, DGrip91, and D-TACC. Conversely, DSpd-2 mutants displayed normal centrosomal accumulations of Asl and D-PLP, strongly reduced amounts of Cnn, γ-tubulin, and DGrip91, and diffuse localization of D-TACC. These results indicate that DSpd-2 functions in a very early step of the PCM recruitment pathway (Giansanti, 2008).

The DSpd-2^z3-5711 and DSpd-2^z3-3316 mutations were identified by a cytological screen of the Zuker's collection, which includes more than 2000 male sterile mutants. Males homozygous for either mutant allele showed spermatocytes devoid of asters and aberrant spermatids. Both mutations failed to complement Df(3L)st-j7 and Df(3L)st-g24 for this phenotype but did complement Df(3L)st-7P, indicating that they map to a region that contains only six genes. RNAi was performed for these six genes in S2 cells and it was found that one of them, CG17286, is required for aster formation. CG17286 encodes a polypeptide of 1146 amino acids (aa), which shares a highly conserved 200 aa domain with C. elegans SPD-2. DNA sequence analysis revealed that the DSpd-2^z3-5711 and DSpd-2^z3-3316 mutant alleles carry early stop codons that truncate the DSpd-2 protein to a 56 and a 365 aa polypeptide, respectively. Flies homozygous or hemizygous for either DSpd-2 mutant allele were viable and morphologically normal. However, they were sterile in both sexes and showed an unequivocal uncoordinated phenotype; mutant males also showed nonmotile sperm tails. The uncoordinated phenotype is often associated with defects in the cilia of mechanosensory neurons and has been observed in mutants affecting centriole structure and replication, such as uncoordinated (unc), Drosophila pericentrin-like (d-plp), and DSas-4, but not in mutants in PCM components such as centrosomin (cnn) (Giansanti, 2008).

An antibody was raised against the C-terminal region of DSpd-2. This antibody recognized a band of the predicted molecular weight in blots from both larval brain and adult testis extracts. Neither this band nor the truncated polypeptides were detected in extracts from mutant animals, even after long exposures. The antibody immunostained the centrosomes of both brain cells and spermatocytes. In larval brains, the antibody detected the interphase centrosomes as small dots and strongly stained the mitotic centrosomes, suggesting that DSpd-2 is enriched at both the centrioles and the PCM (Giansanti, 2008).

To obtain further insight into DSpd-2 localization, focus was placed on spermatocytes, which contain centrioles that are approximately 10-fold larger than somatic centrioles. Centrioles duplicate early during spermatocyte development and increase in length during spermatocyte growth. In mature spermatocytes, paired centrioles appear as two rods joined at their proximal ends to form a characteristic V-shaped structure. Immunostaining of testes that express the centriole-specific marker YFP-Asl showed that DSpd-2 colocalizes with Asl throughout spermatocyte development. However, when the centrosomes recruit the PCM and nucleate astral MTs, the DSpd-2 signal increases in intensity at the proximal ends of the paired centrioles, exceeding the Asl signal. In telophase cells, concomitant with a reduction in the length of astral MTs, the Asl and DSpd-2 signals colocalize again. This staining pattern is very similar to that elicited by the Sigma GTU-88 anti-γ-tubulin ascites fluid, which stains both the centrioles and the pericentriolar γ-tubulin. Collectively, these results strongly suggest that DSpd-2 associates with centrioles throughout the cell cycle and becomes part of the PCM during cell division (Giansanti, 2008).

To determine the role of DSpd-2 in neuroblast (NB) mitosis, brain preparations were examined from DSpd-2^z3-5711/Df(3L)st-j7 larvae stained for both tubulin and Cnn. Drosophila brains contain mostly NBs and ganglion mother cells (GMCs). NBs are stem cells that divide asymmetrically, giving rise to another NB and a smaller GMC. Prometaphase and metaphase wild-type NBs display centrosomes and asters of similar sizes at both cell poles. However, with progression through anaphase and telophase, the MTs of the basal aster shorten dramatically and those of the apical aster elongate slightly. Concomitantly, the basal centrosome becomes substantially smaller than the apical one. The analysis of DSpd-2 mutant NBs revealed that they contain centrosomes that recruit less Cnn than their wild-type counterparts. Examination of hundreds of cells from several mutant brains revealed that more than 90% of mutant centrosomes were not associated with astral MTs. In the few cases in which mutant centrosomes appeared to have a residual MT-nucleation ability, the astral MTs were extremely short. In addition, mutant metaphase cells displayed variable numbers of centrosomes, ranging from zero to four. This finding is thought to reflect a failure of mutant centrosomes to properly localize at the opposite spindle poles, leading to an abnormal centrosome distribution to the daughter cells. Consistent with previous results, the absence of asters did not substantially affect the asymmetry of NB division, and most DSpd-2 mutant NBs displayed asymmetric cytokinesis (Giansanti, 2008).

It was also asked whether DSpd-2 mutations affect the mitotic parameters of larval brain cells. In DSpd-2^z3-5711/Df(3L)st-j7 brains stained for tubulin and DNA, the mitotic index (MI) was higher than in controls, indicating a slight increase in the duration of mitosis. In addition, the frequency of anaphases and telophases (relative to all mitotic figures) was lower in mutant brains than in wild-type, suggesting a specific increase in the length of prometaphase and/or metaphase. However, chromosome preparations from mutant brains showed only 1.9% hyperploid metaphases versus 1% in wild-type, and 2.7% polyploid metaphases versus 0.2% in wild-type, indicating that most mutant NBs successfully complete mitosis (Giansanti, 2008). DSpd-2 larval NBs expressing the tubulin marker Jupiter were examined. In vivo imaging showed that wild-type NBs display prominent asters, but asters are absent or drastically reduced in mutant NBs. Mutant NBs showed MT nucleation around the chromosomes, followed by progressive spindle focusing. However, despite these problems in spindle assembly, NBs divided asymmetrically. These results are consistent with observations on fixed brains and confirm that DSpd-2 is required for astral MT nucleation (Giansanti, 2008).

Astral MTs mediate spindle rotation during NB division. If the spindle does not rotate properly, the cell-fate determinants fail to localize at the poles of the dividing NBs and are not correctly partitioned between the daughter cells. For example, Miranda (Mira) forms a crescent at the basal side of dividing NBs. In wild-type NBs, this crescent remains at the basal pole throughout mitotic division and segregates to the GMC upon completion of cytokinesis. In contrast, in asl, cnn, and DSas-4 NBs, which lack astral MTs, approximately 50% of metaphase figures exhibit Mira mislocalization. Likewise, whereas 96% of wild-type NBs showed a basal Mira crescent, 16% of DSpd-2 mutant NBs showed a diffuse Mira localization and 39% displayed Mira crescents whose midpoint formed an angle > 30° with the spindle axis. However, only 15% of DSpd-2 mutant telophases showed Mira mislocalization. This phenomenon, named telophase rescue, has been observed in inscuteable (insc) and bazooka (baz) mutants. Collectively, these results indicate that DSpd-2 plays an essential role in NB aster formation and that NBs can assemble a functional spindle in the absence of centrosome-driven MT nucleation. However, a severe reduction of astral MTs results in cell-fate-determinant mislocalization (Giansanti, 2008).

Centrosome assembly and spindle formation were analyzed in DSpd-2^z3-5711/Df(3L)st-j7 spermatocytes stained for both tubulin and Cnn. In wild-type primary spermatocytes, Cnn was strongly enriched at the centrosomes, which nucleated prominent asters. In contrast, mutant spermatocytes showed tiny Cnn signals associated with a variable number of centrosomes. In late-prophase spermatocytes, the number of centrosomes per cell ranged from zero to eight. Importantly, mutant centrosomes did not appear to have MT-nucleating ability, because 95% of mutant prometaphases (n = 480) were devoid of asters whereas the remaining 5% displayed extremely reduced asters. Nonetheless, mutant spermatocytes were able to assemble anastral spindles from MTs nucleated around the chromosomes, to undergo anaphase and telophase, and to form regular central spindles and contractile rings. However, most mutant telophases displayed unequal chromosome segregation and/or multiple nuclei at one or both cell poles. Collectively, these results indicate that the abnormalities seen in DSpd-2 spermatocytes are identical to those previously observed in meiotic cells of asl males (Giansanti, 2008).

Living spermatocytes expressing EGFP-tagged β-tubulin were imaged. Wild-type spermatocytes displayed prominent asters and underwent anaphase approximately 10 min after nuclear-envelope disassembly. In contrast, in DSpd-2^z3-5711/Df(3L)st-j7 spermatocytes, asters were either absent or extremely reduced, and the time elapsed between nuclear-envelope breakdown and anaphase onset was always longer than 25 min. However, mutant cells managed to form a central spindle comparable to that seen in wild-type cells. These observations are consistent with those on fixed material; they clearly show that the centrosomes of DSpd-2 mutants are unable to nucleate astral MTs (Giansanti, 2008).

Next centriole structure was analyzed in DSpd-2 primary spermatocytes that express the centriole-specific marker YFP-Asl. Examination of cysts in late prophase showed that mutant and wild-type centrioles have comparable lengths. However, whereas wild-type spermatocytes contained two pairs of centrioles, mutant spermatocytes were associated with a variable number of centrioles ranging from zero to 15. Nonetheless, the average number of centrioles per spermatocyte observed in mutants and wild-type testes were similar. Finally, in mutant cysts, 11% of centrioles were unpaired and appeared as single barrels and not as V-shaped structures. These results indicate that DSpd-2 is not required for centriole elongation during spermatocyte growth but is necessary to maintain centriole cohesion within each pair. These findings also suggest that DSpd-2 is not strictly required for centriole duplication. The variable number of centrioles in mutant spermatocytes can be attributed to errors in gonial divisions leading to daughter cells with either two or no centrosomes. However, the presence of spermatocytes containing a single unduplicated centriole raises the possibility that unpaired centrioles replicate less efficiently than those that are regularly paired (Giansanti, 2008).

To determine the role of DSpd-2 in centrosome assembly, DSpd-2 localization was examined in brains from mutants with defective centrosomes. As expected, DSpd-2 showed a diffuse staining in DSas-4 mutant brain cells, which do not contain centrioles. A diffuse staining was also found in asl mutant brain cells, which do contain (defective) centrioles. However, normal DSpd-2 signals in brain cells from mutants in the d-plp gene, which specifies a centriolar protein essential for ciliogenesis but with a minor role in PCM recruitment. DSpd-2 showed a normal centrosomal localization also in mutants in the γ-tubulin 23C, dd4 (Dgrip91), and d-tacc genes, which encode components of the PCM. The localization of centrosomal proteins was examined in DSpd-2 mutant brain cells. The Asl (YFP-Asl) and D-PLP proteins were normally associated with the centrioles of DSpd-2 mutant cells. However, the centrosomes of the same cells showed substantially reduced concentrations of Cnn. These results on brain cells are strongly supported by similar findings obtained with spermatocytes. Together, these studies indicate that in the centrosome assembly pathway, DSpd-2 acts downstream of Asl but upstream of Cnn, γ-tubulin, Dd4/Dgrip91, and D-TACC (Giansanti, 2008).

DSpd-2 mutant flies display an uncoordinated phenotype, and their centrosomes are unable to mediate aster formation in both NBs and spermatocytes. In addition, mutant NBs contain variable numbers of centrosomes and exhibit frequent Mira mislocalization. These phenotypic traits were not observed in flies bearing the P element-induced mutation DSpd-2^G20143, leading to the conclusion that DSpd-2 is mostly required for the recruitment of PCM to the sperm centriole. The results suggest that the DSpd-2^G20143 mutation is not functionally null. Although the DSpd-2 protein is not detectable in western blots from DSpd-2^G20143 mutant flies, it is likely that these animals express very low levels of wild-type protein that are sufficient to support astral MT nucleation (Giansanti, 2008).

The results indicate that DSpd-2 associates with the centrioles throughout the cell cycle but is not directly required for centriole duplication. During cell division, DSpd-2 also accumulates around the centrioles, mediating the recruitment of additional PCM components such as γ-tubulin and Cnn. This latter result is consistent with earlier studies in C. elegans and recent work showing that Cep192, the human homolog of Spd-2, is required for PCM recruitment and aster formation (Gomez-Ferreria, 2007. However, the precise role of DSpd-2 in aster formation is unclear. Although the centrosomes of DSpd-2 mutants contain residual amounts of Cnn and γ-tubulin, they are unable to form asters. This finding suggests that in addition to its role in PCM recruitment, the DSpd-2 protein might play a direct role in either astral MT nucleation or stabilization (Giansanti, 2008).

A novel domain suggests a ciliary function for ASPM, a brain size determining gene

The N-terminal domain of human abnormal spindle-like microcephaly-associated protein (ASPM) is identified as a member of a novel family of ASH (ASPM, SPD-2, Hydin) domains. These domains are present in proteins associated with cilia, flagella, the centrosome and the Golgi complex, and in Hydin and OCRL whose deficiencies are associated with hydrocephalus and Lowe oculocerebrorenal syndrome, respectively. Genes encoding ASH domains thus represent good candidates for primary ciliary dyskinesias. ASPM has been proposed to function in neurogenesis and to be a major determinant of cerebral cortical size in humans. Support for this hypothesis stems from associations between mutations in ASPM and primary microcephaly, and from the rapid evolution of ASPM during recent hominid evolution. The identification of the ASH domain family instead indicates possible roles for ASPM in sperm flagellar or in ependymal cells' cilia. ASPM's rapid evolution may thus reflect selective pressures on ciliary function, rather than pressures on mitosis during neurogenesis (Ponting, 2006).

Search PubMed for articles about Drosophila Spd-2

Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C. G., Khodjakov, A. and Raff, J. W. (2006). Flies without centrioles. Cell 125: 1375-1386. Medline abstract: 16814722

Bettencourt-Dias, M. et al. (2005). SAK/PLK4 is required for centriole duplication and flagella development, Curr. Biol. 15: 2199-2207. Medline abstract: 16326102

Delattre, M., et al. (2004). Centriolar SAS-5 is required for centrosome duplication in C. elegans. Nat. Cell Biol. 6: 656-664. Medline abstract: 15232593

Delattre, M., Canard, C. and Gonczy, P. (2006). Sequential protein recruitment in C. elegans centriole formation. Curr. Biol. 16(18): 1844-9. Medline abstract: 16979563

Dix, C. I. and Raff, J. W. (2007). Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication. Curr. Biol. 17(20): 1759-64. Medline abstract: 17919907

Giansanti, M. G., Bucciarelli, E., Bonaccorsi, S. and Gatti. M. (2008). Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation. Curr. Biol. 18(4): 303-9. PubMed citation: 18291647

Gomez-Ferreria, M. A. et al. (2007). Human Cep192 is required for mitotic centrosome and spindle assembly. Curr. Biol. 17: 1960-1966. PubMed citation: 17980596

Goshima, G., et al. (2007). Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417-421. PubMed citation: 17412918

Kemp, C. A., Kopish, K. R., Zipperlen, P., Ahringer, J. and O'Connell, K. F. (2004). Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2. Dev. Cell 6: 511-523. Medline abstract: 15068791

Kleylein-Sohn J., Westendorf J., Le Clech M., Habedanck R., Stierhof Y. D. and Nigg E. A. (2007). Plk4-induced centriole biogenesis in human cells. Dev. Cell. 13: 190-202. Medline abstract: 17681131

Peel, N., Stevens, N. R., Basto, R. and Raff, J. W. (2007). Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation. Curr. Biol. 17: 834-843. Medline abstract: 17475495

Pelletier L., Ozlu N., Hannak E., Cowan C., Habermann B., Ruer M., Muller-Reichert T. and Hyman A.A. (2004). The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication. Curr. Biol. 14:863-873

Pelletier, L., et al. (2006). Centriole assembly in Caenorhabditis elegans. Nature 444(7119): 619-23. Medline abstract: 17136092

Ponting, C. P. (2006). A novel domain suggests a ciliary function for ASPM, a brain size determining gene. Bioinformatics 22(9): 1031-5. Medline abstract: 16443634

Rodrigues-Martins, A., et al. (2007). DSAS-6 organizes a tube-like centriole precursor, and its absence suggests modularity in centriole assembly. Curr. Biol. 17: 1465-1472. Medline abstract: 17689959

date revised: 6 January 2007

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