InteractiveFly: GeneBrief

Spd-2: Biological Overview | References


Gene name - Spd-2

Synonyms - CG17286

Cytological map position- 73A10-73A11

Function - centriole duplication factor

Keywords - recruitment of pericentriolar material to sperm centrioles after fertilization

Symbol - Spd-2

FlyBase ID: FBgn0027500

Genetic map position - 3L: 16,584,306..16,588,142 [-]

Classification - ASH (ASPM, SPD-2, Hydin) domain

Cellular location - nuclear


Recent literature
Baumbach, J., Novak, Z.A., Raff, J.W. and Wainman, A. (2015). Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo. PLoS Genet 11: e1005261. PubMed ID: 26020779
Summary:
Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. This study shows that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2-the same proteins that appear to drive mitotic centrosome assembly in flies. This finding was used to ablate aMTOC formation in acentriolar cells, and perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. It was shown that although aMTOCs could nucleate microtubules, these microtubules did not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. However, they were found to be required for robust microtubule array assembly in cells without centrioles that also lacked microtubule nucleation from around the chromatin. Importantly, aMTOCs were also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). The study proposes an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

Conduit, P. T. and Raff, J. W. (2015). Different Drosophila cell types exhibit differences in mitotic centrosome assembly dynamics. Curr Biol 25: R650-651. PubMed ID: 26241137
Summary:
Centrosomes are major microtubule organising centres comprising a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, and we previously showed that two key PCM components, Centrosomin (Cnn) and Spd-2, cooperate to form a scaffold structure around the centrioles that recruits the mitotic PCM in Drosophila; the SPD-5 and SPD-2 proteins appear to play a similar function in C. elegans. In fly syncytial embryos, Cnn and Spd-2 are initially recruited into a central region of the PCM and then flux outwards. This centrosomal flux is potentially important, but it has so far not been reported in any other cell type. This study examine the dynamic behaviour of Cnn and Spd-2 in Drosophila larval brain cells. Spd-2 fluxes outwards from the centrioles in both brains and embryos in a microtubule-independent manner. In contrast, although Cnn is initially incorporated into the region of the PCM occupied by Spd-2 in both brains and embryos, Cnn fluxes outwards along microtubules in embryos, but not in brain cells, where it remains concentrated around the centrosomal Spd-2. Thus, the microtubule-independent centrosomal-flux of Spd-2 occurs in multiple fly cell types, while the microtubule-dependent outward flux of Cnn appears to be restricted to the syncytial embryo.

BIOLOGICAL OVERVIEW

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-2z3-5711 and DSpd-2z3-3316 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-2z3-5711 and DSpd-2z3-3316 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-2z3-5711/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-2z3-5711/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-2z3-5711/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-2z3-5711/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-2G20143, 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-2G20143 mutation is not functionally null. Although the DSpd-2 protein is not detectable in western blots from DSpd-2G20143 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 molecular mechanism of mitotic centrosome assembly in Drosophila

Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. This study shows that the centriole protein Asterless (Asl) initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn mitotic PCM assembly is diminished; in the absence of both proteins it appears to be abolished. DSpd-2 helps incorporate Cnn into the PCM and Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies (Conduit, 2014).

Several hundred proteins are recruited to the PCM that expands around the centrioles during centrosome maturation in mitosis, but how so many proteins are organised into a functional mitotic centrosome has remained mysterious. Remarkably, this study shows that the assembly of the mitotic PCM in flies appears to depend on just two proteins, Cnn and DSpd-2. Both proteins appear to form scaffolds that initially assemble around the mother centriole and then spread outward, forming a dynamic platform upon which most, if not all, other PCM proteins ultimately assemble. DSpd-2 and Cnn partially depend on each other for their centrosomal localisation, and both proteins are required to ensure robust centrosome maturation. In the absence of one of these proteins, reduced levels of the other protein still localise around the 2 centrioles and can support the partial assembly of the mitotic PCM. In the absence of both proteins mitotic PCM assembly appears to be abolished (Conduit, 2014).

How are DSpd-2 and Cnn recruited to mother centrioles? The results strongly suggest that in fly embryos Asl initially helps recruit DSpd-2 to centrioles and DSpd-2 then helps to recruit Cnn. Cnn does not appear to be required to recruit either Asl or DSpd-2 to centrosomes, but it is required to properly maintain DSpd-2 within the PCM. It is speculated that this interaction between DSpd-2 and Cnn creates a positive feedback loop that drives the dramatic expansion of the PCM scaffold around mother centrioles during mitosis. Although direct interactions between Asl and DSpd-2 and between DSpd-2 and Cnn have been identified by Y2H, and the endogenous proteins can all co-immunoprecipitate with one another in fly embryo extracts, it is stressed that it is uncertain that these interactions are direct in vivo. The requirement for Asl to initiate the mitotic recruitment of DSpd-2 and Cnn probably explains why these proteins are specifically recruited to mother centrioles. It has been recently shown that although Asl is essential for centriole duplication, it is not incorporated into daughter centrioles until they have passed through mitosis and matured into new mother centrioles, and Asl/Cep152 proteins mainly localise to mother centrioles in several species. The PCM appears to be preferentially associated with mother centrioles in many systems. The current findings provide a potential explanation for why this is so, and raise the intriguing possibility that all the mitotic PCM 0 may be organised exclusively by mother centrioles. Although DSpd-2 seems to be the major recruiter of centrosomal Cnn in embryos, there must be an alternative recruiter, as the centrosomal localisation of Cnn is not abolished in the absence of DSpd-2. Asl is an attractive candidate as anti-Asl antibodies perturb Cnn recruitment to centrioles (although this could be an 7 indirect consequence of their effect on DSpd-2 recruitment), and Asl and Cnn interact in Y2H analysis. Moreover, human Cep152/Asl has a role in the centrosomal recruitment of human Cdk5Rap2/Cnn (Conduit, 2014).

Interestingly, in flies this alternative pathway appears to be stronger in larval brain cells than in eggs/embryos: in the absence of DSpd-2, Cnn levels are reduced by only ~35% in brains but by ~80% in eggs. Thus, the detail of the mitotic PCM assembly pathway may vary between different cell types even in the same species. The data suggest that after DSpd-2 and Cnn have been recruited to centrioles they rapidly assemble into scaffolds that then move slowly away from the centrioles. For Cnn, there is strong data indicating that scaffold assembly is regulated by phosphorylation. Cnn contains a phospho-regulated multimerization (PReM) domain that is phosphorylated by Polo/Plk1 in vitro and at centrosomes during mitosis in vivo. Mimicking phosphorylation allows the PReM domain to multimerise in vitro and Cnn to spontaneously assemble into cytosolic scaffolds in vivo that can organise MTs. Conversely, ablating phosphorylation does not interfere with Cnn recruitment to centrioles, but inhibits Cnn scaffold assembly 06. It is speculated that, like Cnn, DSpd-2 can assemble into a scaffold and that this assembly is regulated in vivo so that it only occurs around mother centrioles. It remains unclear, however, whether DSpd-2 itself can form a scaffold, or whether it requires other proteins to do so (Conduit, 2014).

It is striking that both DSpd-2 and Cnn exhibit an unusual dynamic behaviour at centrosomes. Both proteins incorporate into the PCM from the inside out, and are in constant flux, as the molecules that move slowly outward away from the centrioles are replaced by newly incorporated molecules close to the centriole surface. This inside out assembly is likely to have important consequences, as it means that events close to the centriole surface, rather than at the periphery of the PCM, can ultimately regulate mitotic PCM assembly. This may be particularly important in cells where centrioles organise centrosomes of different sizes, as is the case in certain asymmetrically dividing stem/progenitor cells. Fly neural stem cells, fo example, use centrosome size asymmetry to ensure robust asymmetric division, and there is strong evidence that new and old mother centrioles differentially regulate the rate of Cnn incorporation in these cells. Moreover, mutations in human Cdk5Rap2/Cnn have been implicated in microcephaly, a pathology linked to a failure in neural progenitor cell proliferation, although the precise reason for this is unclear. Although DSpd-2 and Cnn have a major role in centrosome maturation, it is stressed that other PCM components are likely to make important contributions (Conduit, 2014).

Pericentrin, for example, has been implicated in PCM recruitment in several systems, and the fly homologue, D-plp, forms ordered fibrils in cultured S2 cells that extend away from the centriole wall and support PCM assembly in interphas. These centriolar fibrils, however, cannot explain how centrioles organise such a vastly expanded PCM matrix during mitosis, and D-plp appears to have an important, but more minor, role in mitotic PCM 1 assembly in vivo (Martinez-Campos, 2004). Nevertheless, proteins like 2 D-plp will certainly help recruit other PCM proteins and help form structural links within the PCM, thus strengthening the mitotic PCM matrix. The 4 important distinction is that, in flies at least, proteins like D-plp are recruited into the PCM by an underlying PCM scaffold, whereas DSpd-2 and Cnn appear to form this scaffold. Homologues of Asl, DSpd-2 and Cnn have been implicated in PCM assembly in many species suggesting that the mechanism of mitotic PCM recruitment identified in this study may have been conserved in evolution. To date, no PCM component has yet been shown to assemble from the inside out and to flux away from the centrioles in any other system. Nevertheless, although the precise molecular details will likely vary from cell type to cell type and from species to species, it is suspected that this unusual dynamic behaviour of an underlying mitotic PCM scaffold will prove to be a general feature of mitotic centrosome assembly in many systems (Conduit, 2014).

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).

Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. This study, carried out in mammalian cultured cells, showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 (Drosophila homolog: Spd-2) and Cep152 (Drosophila homolog: asterless) serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-alpha-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis (Kim, 2013).


REFERENCES

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. PubMed ID: 16814722

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

Conduit, P. T., Richens, J. H., Wainman, A., Holder, J., Vicente, C. C., Pratt, M. B., Dix, C. I., Novak, Z. A., Dobbie, I. M., Schermelleh, L. and Raff, J. W. (2014). A molecular mechanism of mitotic centrosome assembly in Drosophila. Elife: e03399. PubMed ID: 25149451

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

Delattre, M., Canard, C. and Gonczy, P. (2006). Sequential protein recruitment in C. elegans centriole formation. Curr. Biol. 16(18): 1844-9. PubMed ID: 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. PubMed ID: 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 ID: 18291647

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

Goshima, G., et al. (2007). Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417-421. PubMed ID: 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. PubMed ID: 15068791

Kim, T. S., Park, J. E., Shukla, A., Choi, S., Murugan, R. N., Lee, J. H., Ahn, M., Rhee, K., Bang, J. K., Kim, B. Y., Loncarek, J., Erikson, R. L. and Lee, K. S. (2013). Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152. Proc Natl Acad Sci U S A 110: E4849-4857. PubMed ID: 24277814

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. PubMed ID: 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. PubMed ID: 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. PubMed ID: 17136092

Ponting, C. P. (2006). A novel domain suggests a ciliary function for ASPM, a brain size determining gene. Bioinformatics 22(9): 1031-5. PubMed ID: 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. PubMed ID: 17689959


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date revised: 10 October 2014

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