InteractiveFly: GeneBrief

Spd-2: Biological Overview | References

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-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 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).

Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics

The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. This study reports a novel role for the multifunctional adaptor protein centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of gamma-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, these results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division (Malerod, 2018).

During cell division, the mitotic spindle (MS) that forms between the two centrosomes ensures faithful segregation of the chromosomes between the two daughter cells, positions the cleavage furrow, and is anchored to the cell cortex to ensure proper spindle orientation. Different subpopulations of microtubules (MTs); the kinetochore, interpolar/astral, and astral MTs, are involved in controlling each process, respectively. Correct orientation of the MS ensures proper segregation of molecules defining cell fate and is important during asymmetric stem cell division to generate one daughter cell which self-renews and one which undergoes differentiation. The orientation of the MS further defines the cleavage plane of the cell and thereby its position within the tissue, exemplified by the planar division of epithelial cells to generate a monolayered epithelium. The precise orientation of the MS can be influenced by internal cues (cell polarity determinants) or external cues (neighboring cells or extracellular matrix) and is cell type-dependent (Malerod, 2018).

Regardless of the molecular mechanisms setting the orientation, the MS is anchored to the cell cortex by the astral MTs radiating from the centrosomes. The centrosome is the major MT-organizing center in most cell types and nucleates astral MTs and the other MT subpopulations of the MS. The centrosome is composed of a centriole pair and the surrounding pericentriolar material (PCM), generated by dynamic assembly of proteins found to stabilize each other via positive feedback loops. During mitosis, the centrosome matures when the PCM expands extensively due to recruitment of scaffold and MT nucleating proteins, which promote MS formation. The γ-tubulin ring complexes (γTuRCs) of the PCM, composed of γ-tubulin and associated proteins (γ-tubulin complex proteins, GCPs), nucleate MT filaments at the centrosome. The ring of γ-tubulin within γTuRC resembles the MT geometry and serves as a template for assembly of α/β-tubulin-dimers, which polymerize into long filaments, MTs. Although the centrosomes represent the major centers for MT nucleation, MTs may alternatively be formed at the Golgi, chromosomes, nuclear envelope, plasma membrane, and pre-existing MTs. Importantly, γ-tubulin seems to be implicated in the nucleation process regardless of the intracellular localization (Malerod, 2018).

Microtubules of the MS, including the astral MTs, are dynamic and their timely assembly and disassembly is tightly controlled by proteins regulating nucleation, severing, and stability of the filaments. MT stability is regulated by MT-associated proteins. These proteins stabilize MTs by binding to the growing plus-end of the filaments to prevent catastrophe, or alternatively, by decorating the MTs to prevent interaction with severing proteins. Furthermore, the γTuRC itself has also been reported to modulate the stability of MTs by interacting with motor proteins such as dynein, kinesin-5, and kinesin-14 as well as the plus-end tracking protein EB1 (Malerod, 2018).

Astral MT regulation occurs at several levels to achieve proper MS orientation: (1) astral MT nucleation at the centrosomes, (2) astral MT dynamics and stability, and (3) astral MT anchoring and behavior at the cell cortex. Aberrant regulation of astral MTs has been shown to correlate with spindle misorientation. For example, centrosomal proteins regulating γTuRC-mediated nucleation of MTs and MAPs controlling MT stability have been shown to regulate spindle orientation in their capacity of modulating MT dynamics. Despite the emerging insight into how astral MT formation is controlled to ensure proper MS orientation, the molecular mechanisms are incompletely understood (Malerod, 2018).

The multifunctional adaptor protein ALG-2-interacting protein X (ALIX) has been shown to localize to centrosomes in interphase and during cell division. However, the biological roles of centrosomal ALIX are not known. Extensive research has implicated ALIX in a diversity of cellular processes, such as apoptosis, endocytosis and endosome biogenesis, cell adhesion, virus release, plasma membrane repair, and cytokinesis. Specifically, ALIX controls cytokinesis by participating in recruiting abscission-promoting proteins of the endosomal sorting complex required for transport (ESCRT) to the midbody. The current study has investigated the role of centrosomal ALIX during cell division. ALIX is shown to localize to the PCM, where it interacts with and stabilizes γTuRC, thus promoting efficient nucleation of astral MTs. In addition, centrosomal ALIX recruits MAP1S, which stabilizes the newly formed MTs radiating from the centrosomes. It is concluded that ALIX facilitates efficient formation of astral MTs by stimulating their nucleation and stabilization, which promotes correct MS orientation during both asymmetric and symmetric cell division (Malerod, 2018).

This study has unraveled a novel role of ALIX located at the centrosomes during cell division in regulation of MS orientation by modulating the formation of astral MTs. ALIX is recruited to the PCM via DSpd-2/Cep192, which recruit PCM components (including Cnn/Cep215, γ-tubulin, and Dgrip91/GCP3), to promote nucleation of astral MTs. Notably, even though DSpd-2/Cep192 appears to be a major recruiter of ALIX to centrosomes, the fact that ALIX was still partially detected at centrosomes in the absence of DSpd-2/Cep192 indicates that additional recruitment mechanisms exist. Centrosomal protein of 55 kDa (CEP55), which localizes to centrosomes during early phases of cell division and moves to and recruits ALIX to the midbody during cytokinesis, represents a possible additional recruiter of ALIX to centrosomes in human cells. However, because CEP55 orthologues lack in lower eukaryotes, such as D. melanogaster (and C. elegans), other proteins could also participate in recruiting ALIX to centrosomes. Interestingly, a direct interaction between DSpd-2/Cep192 and γ-tubulin has not been elucidated. Based on the current results, it is therefore tempting to speculate that ALIX serves a scaffolding role at the interface between DSpd-2/Cep192 and γTuRC, since it was found that ALIX binds DSpd-2, γ-tubulin, and Dgrip91 in vitro. Thus, the results provide mechanistic insight into DSpd-2/Cep192-mediated regulation of astral MT formation and proper orientation of the MS during metaphase in Drosophila and human cells (Malerod, 2018).

The current model shows the MS orientation in cells with or without ALIX. The PCM protein DSpd-2/Cep192 recruits ALIX to the PCM, where ALIX recruits γ-tubulin of the γTuRC at the centrosomes, thus facilitating nucleation of astral MTs. Furthermore, ALIX recruits MAP1S to the centrosomes, in close proximity to the newly formed MTs which are then stabilized by MAP1S. Thus, ALIX facilitates both nucleation of and stabilization of astral MTs emanating from the centrosomes, thus promoting efficient formation of stable astral MTs which mediate anchoring to the cell cortex and thus correct positioning of the MS. By this mechanism, ALIX is one of several molecules controlling MS orientation, providing robustness to correctly orient the MS during cell division (Malerod, 2018).

Astral MTs seem to be equally essential for correct spindle orientation in diverse cell types, in difference to internal polarity cues or external signals provided by neighboring cells. Interestingly, that loss of ALIX induced spindle misorientation in a variety of cell types, including stem cells (NBs and mGSCs) and epithelial cells (SOPs, FECs, and Caco-2 cells), corresponds well with the current data showing that ALIX controls spindle orientation by facilitating the formation of astral MTs and indicates a general role of ALIX in this process. Furthermore, the defective localization of Miranda and aPKC in alix mutant NBs reflects the compromised formation of astral MTs, rather than aberrant cell polarity, since astral MTs have previously been shown to stabilize these determinants at the basal and apical membranes, respectively (Malerod, 2018).

ALIX was shown to maintain the epithelial blood-cerebrospinal fluid barrier by facilitating assembly of tight junctions, which were recently reported to control spindle orientation in Caco-2 cyst cells. In general, cell-cell contacts such as tight junctions seem to control MS orientation in epithelial cells by F-actin, an essential component of the cell cortex facilitating capture of astral MTs. In human epithelial cells, ALIX might thus affect both the formation of astral MTs, as has been shown in this study, and their anchoring to the cell cortex. Also in Drosophila SOPs, septate junctions, resembling tight junctions, regulate the MS orientation. Whether ALIX regulates septate junctions in Drosophila epithelial cells remains to be elucidated, but the current data showing that cold-induced depolymerization of MTs potentiated the spindle misorientation only in wild-type FECs, and not in alix1 FECs, suggest that ALIX regulates the MS orientation by MT-dependent mechanisms (Malerod, 2018).

The current study suggests a dual role for ALIX during astral MT formation: (1) by promoting nucleation via γ-tubulin recruitment and (2) by stabilization of MTs via stabilizing MAP1S at the centrosomes. Although MAP1S is predominantly associated along MTs, it has also been shown to concentrate at the centrosomes. Here, MAP1S has been suggested to stabilize newly formed MT filaments, which likely explains the reduced regrowth of MTs observed at early time points after cold-induced depolymerization in MAP1S-depleted cells. Accordingly, this study found that ectopically expressed MAP1S was unable to rescue the reduced number of astral MTs observed in ALIX-deficient cells, thus arguing against that MAP1S influences the nucleation of MTs as such. Rather, MAP1S significantly increased the length of astral MTs in ALIX knockdown cells, supporting the hypothesis that ALIX facilitates MT stability via MAP1S. It is envisioned that ALIX stabilizes MAP1S adjacent to the PCM, close to the ends of the newly formed MTs emanating from the centrosomes. A simultaneous interaction of MAP1S with both MTs and ALIX seems plausible since the MT-interacting domain is located in the light chain of MAP1S, whereas ALIX seems to bind the heavy chain (Malerod, 2018).

In summary, the current study identifies a novel evolutionarily conserved role of centrosomal ALIX in promoting astral MT formation to orient the MS. The reduced, rather than absent, recruitment of γ-tubulin, MAP1S and consequently appearance of astral MTs in ALIX-deficient cells, clearly suggests that ALIX represents one of several mechanisms to ensure formation of astral MTs. Thus, ALIX provides robustness to correctly orient the MS during asymmetric and planar cell division (Malerod, 2018).

Evidence that a positive feedback loop drives centrosome maturation in fly embryos

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. This study shows that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. These findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).

Centrosomes play an important part in many aspects of cell organisation, and they form when a mother centriole recruits pericentriolar material (PCM) around itself. The PCM contains several hundred proteins, allowing the centrosome to function as a major microtubule (MT) organising centre, and also as an important coordination centre and signalling hub. Centrosome dysfunction has been linked to several human diseases and developmental disorders, including cancer, microcephaly and dwarfism (Alvarez-Rodrigo, 2019).

During interphase, the mother centriole recruits a small amount of PCM that is highly organised. As cells prepare to enter mitosis, however, the PCM expands dramatically around the mother centriole in a process termed centrosome maturation. Electron microscopy (EM) studies suggest that centrioles organise an extensive 'scaffold' structure during mitosis that surrounds the mother centriole and recruits other PCM components such as the γ-tubulin ring complex (γ-TuRC) (Alvarez-Rodrigo, 2019).

In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, a relatively simple pathway seems to govern the assembly of this mitotic PCM scaffold. The conserved centriole/centrosome protein Spd-2/SPD-2 (fly/worm nomenclature) cooperates with a large, predominantly predicted-coiled-coil, protein (Cnn in flies, SPD-5 in worms) to form a scaffold whose assembly is stimulated by the phosphorylation of Cnn/SPD-5 by the mitotic protein kinase Polo/PLK-1. Mitotic centrosome maturation is abolished in the absence of this pathway, and some aspects of Cnn and SPD-5 scaffold assembly have recently been reconstituted in vitro. Vertebrate homologues of Spd-2 (Cep192), Cnn (Cdk5Rap2/Cep215) and Polo (Plk1) also have important roles in mitotic centrosome assembly, indicating that elements of this pathway are likely to be conserved in higher metazoans. In vertebrate cells another centriole and PCM protein, Pericentrin, also has an important role in mitotic centrosome assembly that is dependent upon its phosphorylation by Plk1. Pericentrin can interact with Cep215/Cnn, but in flies the Pericentrin-like-protein (Plp) has a clear, but relatively minor, role in mitotic PCM assembly when compared to Spd-2 and Cnn (Alvarez-Rodrigo, 2019).

Although most of the main players in mitotic centrosome-scaffold assembly appear to have been identified, several fundamental aspects of the assembly process remain mysterious. Cells entering mitosis, for example, contain two mother centrioles that assemble two mitotic centrosomes of equal size. It is unclear how this is achieved, as even a slight difference in the initial size of the two growing centrosomes would be expected to lead to asymmetric centrosome growth-as the larger centrosome would more efficiently compete for scaffolding subunits. The centrioles in fly embryos appear to overcome this problem by constructing the PCM scaffold from the 'inside-out': Spd-2 and Cnn are only incorporated into the scaffold close to the mother centriole, and they then flux outwards to form an expanded scaffold around the mother centriole. In this way, the growing PCM scaffold could ultimately attain a consistent steady-state size-where incorporation around the mother centriole is balanced by loss of the scaffold at the centrosome periphery-irrespective of any initial size difference in the PCM prior to mitosis (Alvarez-Rodrigo, 2019).

A potential problem with this 'inside-out' mode of assembly is that the rate of centrosome growth is limited by the very small size of the centriole. Mathematical modelling indicates that the incorporation of a crucial PCM scaffolding component only around the mother centriole cannot easily account for the high rates of mitotic centrosome growth observed experimentally. To overcome this problem, it has been proposed that centrosome growth is 'autocatalytic', with the centriole initially recruiting a key scaffolding component that can subsequently promote its own recruitment. It has been proposed that Spd-2 and Cnn could form a positive feedback loop that might serve such an autocatalytic function: Spd-2 helps recruit Cnn into the scaffold, and Cnn then helps to maintain Spd-2 within the scaffold, thus allowing higher levels of Spd-2 to accumulate around the mother centriole, which in turn drives higher rates of Cnn incorporation (Alvarez-Rodrigo, 2019).

In worms and vertebrates, SPD-2/Cep192 can help recruit PLK1/Plk1 to centrosomes and Cep192 also activates Plk1 in vertebrates, in part through recruiting and activating Aurora A, another mitotic protein kinase implicated in centrosome maturation. It is suspected, therefore, that in flies Spd-2 might recruit Polo into the centrosome-scaffold to phosphorylate Cnn and so help to generate a positive feedback loop that drives the expansion of the mitotic PCM. In flies, however, no interaction between Polo and Spd-2 has been reported. Indeed, an extensive Y2H screen for interactions between key centriole and centrosome proteins identified interactions between Spd-2 and the mitotic kinases Aurora A and Nek2, and between Polo and the centriole proteins Sas-4, Ana1 and Ana2, but not between Polo and Spd-2. A possible explanation for this result is that Polo/Plk1 is believed to be largely recruited to its many different locations in the cell, including centrosomes, through its Polo-Box-Domain (PBD), which binds to phosphorylated S-S/T(p) motifs. Perhaps any such Polo binding sites in fly Spd-2 were simply not phosphorylated in the Y2H experiments. In support of this possibility, phosphorylated S-S/T(p) motifs in SPD-2/Cep192 have previously been shown to help recruit PLK1/Plk1 to centrosomes in worms, frogs and humans (Alvarez-Rodrigo, 2019).

This study examined the potential role of Spd-2 in recruiting Polo to centrosomes in Drosophila embryos. Like Cnn, Spd-2 is largely unphosphorylated in the cytosol, but is highly phosphorylated at centrosomes, where Spd-2 and Polo extensively co-localise within the pericentriolar scaffold. A Spd-2 fragment containing 19 S-S/T motifs exhibits enhanced binding to the PBD in vitro when it has been phosphorylated by Plk1, but no enhancement is seen if these S-S/T motifs are mutated to T-S/T-a mutation that strongly perturbs PBD binding. This study expressed forms of Spd-2 in vivo in which either all 34 S-S/T motifs, or the 16 most conserved S-S/T motifs, have been mutated to T-S/T to perturb PBD-binding. These mutant Spd-2 proteins are still recruited to mother centrioles, as are Polo and Cnn, and these proteins assemble a PCM scaffold around the mother centriole. Strikingly, however, this PCM scaffold can no longer expand outwards, and centrosome maturation fails. These observations provide strong support for the hypothesis that Spd-2, Polo and Cnn cooperate to form a positive feedback loop that is required to drive the rapid expansion of the mitotic PCM in fly embryos (Alvarez-Rodrigo, 2019).

It was previously proposed that three proteins -- Spd-2, Polo and Cnn -- together form a scaffold that expands around the mother centriole to recruit other PCM components to the mitotic centrosome. The data presented in this study suggests that these three proteins cooperate to form a positive feedback loop that drives the dramatic expansion of the mitotic PCM scaffold in fly embryos (Alvarez-Rodrigo, 2019).

The following model is proposed (see Polo and Cnn appear to form a positive feedback loop that drives the expansion of the mitotic PCM scaffold). In interphase cells, Spd-2, Polo and Cnn are recruited around the surface of the mother centriole, but Polo is inactive and Spd-2 and Cnn are not phosphorylated-so no scaffold is assembled. As cells prepare to enter mitosis, centrosomal Spd-2 becomes phosphorylated. In vitro data suggests that Polo is involved in this phosphorylation (via a 'self-priming and binding' mechanism), but other mitotic kinases may also be involved. Phosphorylation allows Spd-2 to form a scaffold that fluxes outwards and that can recruit both Polo (via phosphorylated S-S/T(p) motifs) and Cnn . The active Polo phosphorylates Cnn, allowing it to also form a scaffold. The Spd-2 scaffold is inherently unstable, so it can only accumulate around the mother centriole if it is stabilised by the Cnn scaffold. The Cnn scaffold therefore allows the Spd-2 scaffold to expand outward, increasing Spd-2 levels within the PCM scaffold and allowing Spd-2 to recruit more Cnn and more Polo into the scaffold. This is a classical positive feedback loop in which the Output (the PCM scaffold in toto) directly increases the Input (the Spd-2 scaffold) (Alvarez-Rodrigo, 2019).

If Spd-2 cannot efficiently recruit Polo, as appears to be the case with the Spd-2-ALL and Spd-2-CONS mutants, it can still recruit Cnn, and this is, at least initially, phosphorylated by the pool of Polo that is still present around the mother centriole. The data suggests that this centriolar pool of Polo is not recruited by Spd-2 (at least not via the PBD), and it is suspected that S-S/T(p) motifs in other centriole proteins, such as Sas-4, normally recruit Polo to centrioles. As a result, mutant Spd-2 proteins can still support the assembly of a 'mini-scaffold' around the mother centriole, and this can recruit some PCM and organise some MTs. The mutant Spd-2 scaffold that fluxes outwards from the mother centriole, however, cannot recruit Polo. Therefore the Cnn recruited by the expanding Spd-2 network cannot be phosphorylated, and it cannot form a scaffold to support the expanding Spd-2 network. As a result, the expanding mitotic PCM scaffold rapidly dissipates into the cytosol (Alvarez-Rodrigo, 2019).

Although this mechanism is autocatalytic-as the expanding Spd-2 scaffold allows Polo and Cnn to be recruited into the PCM at an increasing rate-crucially, the mother centriole remains the only source of Spd-2. This potentially explains the conundrum of how mitotic PCM growth is autocatalytic, but at the same time requires the mother centriole. This requirement for centrioles can also potentially explain how two spatially separated centrosomes usually grow their mitotic PCM to the same size, as PCM size may ultimately be determined by how much Spd-2 can be provided by the centrioles, rather than how much PCM was present in the centrosome when maturation was initiated (Alvarez-Rodrigo, 2019).

A key feature of this proposed mechanism is that Cnn cannot recruit itself or Spd-2 or Polo into the scaffold (although it helps to maintain the Spd-2 scaffold recruited by the centriole). If it could do so, mitotic PCM growth would no longer be constrained by the centriole as Cnn could catalyse its own recruitment. Interestingly, although Spd-2 and Cnn are of similar size in flies (1146aa and 1148aa, respectively) Spd-2 has >5X more conserved potential PBD-binding S-S/T motifs than Cnn. Moreover, a similar ratio of conserved sites is found when comparing human Cep192 (1941aa) to human Cep215/Cdk5Rap2 (1893aa), even though the human and fly homologues of both proteins share only limited amino acid identity. Perhaps, these two protein families have evolved to ensure that phosphorylated Spd-2/Cep192 can efficiently recruit Polo/Plk1, whereas phosphorylated Cnn/Cep215 cannot (Alvarez-Rodrigo, 2019).

The data indicates that multiple S-S/T(p) motifs in Spd-2 may be involved in Polo recruitment to the PCM. When only the most conserved motifs are mutated, other motifs in Spd-2 appear to be able to help recruit Polo, as evidenced by the additive effect of the Spd-2-ALL mutant compared to the Spd-2-CONS mutant. This mechanism of multi-site phosphorylation and recruitment could help amplify the maturation process (as the additional Polo recruited would allow Cnn to be phosphorylated at a higher rate) and so contribute to the establishment of the positive feedback loop (Alvarez-Rodrigo, 2019).

Another important feature of this proposed mechanism is that Spd-2 is incorporated into the mitotic PCM at the centriole surface and then fluxes outwards. This Spd-2-flux has so far only been observed in Drosophila embryos and mitotic brain cells. In fly embryos, Cnn also fluxes outwards but, unlike Spd-2, this flux requires MTs and is only observed in embryos. In C. elegans embryos, SPD-5 behaves like Cnn in somatic cells: it does not flux outwards and is incorporated isotropically throughout the volume of the PCM. Moreover, a very recent study found no evidence for an outward centrosomal flux of SPD-2 in worm embryos. Clearly, it will be important to determine whether Spd-2/Cep192 homologues flux outwards in other species and, if so, whether this flux provides the primary mechanism by which the mother centriole influences the growth of the expanding mitotic PCM (Alvarez-Rodrigo, 2019).

In vertebrates, Cep192 serves as a scaffold for Plk1 and also Aurora A-another mitotic protein kinase that plays an important part in centrosome maturation in many species. There appears to be a complex interplay between Cep192, Plk-1 and Aurora A in vertebrates, with Cep192 acting as a scaffold that allows these two important regulators of mitosis to influence each other's activity and centrosomal localisation. Spd-2 clearly plays an important part in recruiting Aurora A to centrosomes in fly cells-although it is unclear if this is direct, as fly and worm Spd-2/SPD-2 both lack the N-terminal region in vertebrate Cep192 that recruits Aurora A. How Aurora A might influence the assembly of the Spd-2, Polo/PLK-1 and Cnn/SPD-5 scaffold remains to be determined, although in worms AIR-1 (the Aurora A homologue) is required to initiate centrosome maturation, but is not required for subsequent PCM growth (Alvarez-Rodrigo, 2019).

Finally, there has been great interest recently in the idea that many non-membrane bound organelles like the centrosome may assemble as 'condensates' formed by liquid-liquid phase separation. In support of this possibility for the centrosome, purified recombinant SPD-5 can assemble into condensates in vitro that have transient liquid-like properties, although they rapidly harden into a more viscous gel- or solid-like phase. Moreover, a mathematical model that describes centrosome maturation in the early worm embryo treats the centrosome as a liquid, and it is from this model that the importance of autocatalysis was first recognised. In vivo, however, the Cnn and SPD-5 scaffolds do not appear to be very liquid-like and fragments of Cnn can assemble into micron-scale assemblies in vitro that are clearly solid- or very viscous-gel-like. The current data suggests that the incorporation of Spd-2 into the PCM only at the surface of the centriole, coupled to an amplifying Spd-2/Polo/Cnn positive feedback loop, could provide an 'autocatalytic' mechanism that functions within the conceptual framework of a non-liquid-like scaffold that emanates from the mother centriole (Alvarez-Rodrigo, 2019).

Ana1 helps recruit Polo to centrioles to promote mitotic PCM assembly and centriole elongation

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and it has been shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. This study shows that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. It is concluded that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly (Alvarez-Rodrigo, 2021).

Polo kinase (PLK1 in mammals) is an important cell cycle regulator. During mitosis, it is recruited to several locations in the cell -- such as centrosomes, kinetochores and the cytokinesis apparatus -- where it performs multiple functions. PLK1 is usually recruited to these locations by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs in target proteins. Mutating the first serine in the PBD-binding motif to threonine strongly reduces PBD binding in vitro and in vivo (Alvarez-Rodrigo, 2021).

PLK1 has several key functions at centrosomes. These organelles are important microtubule (MT) organising centres that form around a pair of centrioles (comprising a mother and daughter centriole) when the mother recruits a matrix of pericentriolar material (PCM) around itself. During interphase, centrosomes organise relatively little PCM, but as cells prepare to enter mitosis the PCM expands dramatically in a process termed centrosome maturation. PLK1 is an essential driver of this process, and several PCM proteins have been identified as PLK1 targets. In vertebrate cells, PLK1 phosphorylates pericentrin, which cooperates with CDK5RAP2 (also known as Cep215) to promote mitotic PCM assembly, whereas in flies and worms Polo/PLK1 kinases phosphorylate Cnn and SPD-5 (functional homologues of CDK5RAP2), respectively, which allows these proteins to assemble into a PCM scaffold around the mother centriole that recruits other PCM proteins (Alvarez-Rodrigo, 2021).

Towards the end of mitosis, the mother and daughter centrioles disengage from each other. PLK1 is essential for disengagement and also for the subsequent maturation of the daughter centriole into a new mother centriole that is itself capable of duplicating and organising PCM. The old mother (OM) and new mother (NM) centrioles then both duplicate during S phase by nucleating the assembly of a daughter centriole on their side. PLK1 is not essential for centriole duplication per se, but it is required for the growth of the centriole MTs that occurs during G2, at least in human cells, and for the subsequent maturation of the daughter centriole into a new mother centriole. After duplication in S phase, the two centrosomes (each now comprising a duplicated centriole pair) are held together by a linker, and PLK1 also helps disassemble this linker to promote centrosome separation as cells prepare to enter mitosis (Alvarez-Rodrigo, 2021).

How PLK1 is recruited to centrosomes to execute its multiple functions is largely unclear, although this recruitment appears to be dependent on the PBD. In vertebrate systems, Cep192 is required for centrosome maturation and it is phosphorylated by Aurora A (also known as AURKA) to create PBD-binding sites that recruit PLK1; this promotes the activation of both kinases at the centrosome. The fly and worm homologues of Cep192, Spd-2 and SPD-2, respectively, are concentrated at centrioles and centrosomes, and their phosphorylation also helps recruit Polo/PLK1 kinases to the mitotic PCM to phosphorylate Cnn in flies and SPD-5 in worms. In fly embryos, Spd-2, Polo and Cnn have been proposed to form a positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. In this scenario, Spd-2 starts to be phosphorylated at centrioles as cells prepare to enter mitosis, and this allows Spd-2 to form a scaffold that can recruit other PCM proteins and that fluxes outwards from the mother centriole. The Spd-2 scaffold itself is weak, but it can recruit Polo and Cnn; the recruited Polo phosphorylates Cnn, which then forms a Cnn scaffold that recruits other PCM components and strengthens the Spd-2 scaffold. This allows more Spd-2 to accumulate around the centriole, which in turn drives the recruitment of more Polo and Cnn - so forming a positive feedback loop. In this way, Spd-2 recruits Polo and Cnn to the PCM to help drive centrosome maturation in flies (Alvarez-Rodrigo, 2021).

If Drosophila Spd-2 cannot efficiently recruit Polo (because all its S-S/T motifs have been mutated to T-S/T) Polo recruitment to the PCM is dramatically reduced, but Polo is still strongly recruited to the mother centriole, indicating that other proteins must help recruit Polo to centrioles. The centriole protein Sas-4 is phosphorylated by Cdk1 during mitosis on threonine 200 (T200), creating a PBD-binding site that recruits Polo to newly formed daughter centrioles. This allows the daughter to recruit Asl (Cep152 in humans), which allows the daughter to mature into a new mother that can duplicate and organise PCM - since Asl is required for both of these processes. Although the single PBD-binding site in Sas-4 recruits Polo to mother centrioles, it is suspected that other proteins must also be required. This study attempted to identify such proteins by mutating all the S-S/T motifs to T-S/T in several candidates. The centriole protein Ana1 (Cep295 in humans) was found to normally help recruit Polo to mother centrioles. Ana1 and Cep295 are required for centriole maturation, and in flies Ana1 helps recruit and/or maintain Asl at new mother centrioles. Thus, flies lacking Ana1 lack centrioles, centrosomes and cilia, presumably because the centrioles cannot duplicate without Ana1 as they cannot recruit Asl. This study shows that centrioles that do not efficiently recruit Polo via Ana1 can still recruit Sas-4, Cep135 and Asl, and can still duplicate, disengage and organise cilia, but they cannot efficiently recruit mitotic PCM or elongate during G2. It is proposed that Ana1 recruits Polo to centrioles specifically to promote centriole elongation in G2 and mitotic PCM assembly (Alvarez-Rodrigo, 2021).

Polo has many important functions at centrioles and centrosomes, and previous work has shown that it is initially recruited to newborn centrioles in flies when Cdk1 phosphorylates the Sas-4 T200 S-T motif during mitosis. This initial recruitment of Polo is important to allow the newborn centrioles to subsequently mature into mothers that can recruit Asl and so duplicate and recruit mitotic PCM. This study showed that the centriole protein Ana1 also plays an important part in recruiting Polo to mother centrioles. The data suggests that Ana1 can recruit Polo directly and that Polo itself can phosphorylate Ana1 at several S-S/T motifs to 'self-prime' its own recruitment. It cannot be excluded that other protein kinases may prime these S-S/T motifs, or that Ana1 could recruit Polo to centrioles indirectly in ways that are disrupted when the S-S/T motifs are mutated to T-S/T. Regardless of mechanism, the Ana1-dependent centriolar pool of Polo appears to be required to drive efficient mitotic PCM expansion and centriole elongation in G2 (Alvarez-Rodrigo, 2021).

Although Ana1 helps recruit and/or maintain Asl at centrioles, and therefore is essential for both mitotic PCM recruitment and centriole duplication, this function of Ana1 does not appear to require the ability to recruit Polo. Thus, Ana1-S34T centrioles recruit and maintain normal levels of Asl (and of Cep135, as well as slightly increased levels of Sas-4) and can duplicate normally. This is in contrast to the situation with Sas-4, where T200 phosphorylation is required for proper Asl recruitment and so for both centriole duplication and mitotic PCM assembly. Presumably, the Polo recruited by Sas-4 is either sufficient for Asl recruitment, or it phosphorylates centriole substrates other than Ana1 to promote Asl recruitment. Interestingly, PLK1 is also essential for efficient centriole disengagement, but neither the Ana1-S34T nor Sas-4-T200 mutations appear to perturb this process, indicating that a separate pathway must recruit Polo to centrioles to drive centriole disengagement. Centrosome separation in G2 is also normally dependent on PLK1, and centrosomes/duplicated centriole pairs were often observed that failed to separate properly in embryos expressing Ana1-S34T. As these centriole pairs almost always organised very little PCM, however, it is suspected that this defect may be an indirect consequence of the failure to properly recruit PCM, rather than a direct consequence of the inability of Ana1 to recruit Polo (Alvarez-Rodrigo, 2021).

These new findings further support the hypothesis that centrioles activate a Spd-2-Polo-Cnn positive feedback loop that drives the expansion of the mitotic PCM around the mother centriole. A key feature of this model is that Spd-2 can only be phosphorylated to initiate scaffold assembly at the surface of the mother centriole, and the phosphorylated Spd-2 then fluxes outwards away from the centriole: the Spd-2-Polo-Cnn scaffold itself cannot phosphorylate and/or recruit new Spd-2 into the scaffold. This is important, as it can explain why the mother centriole is required to drive efficient mitotic PCM assembly, why the size of the centriole influences the size of the mitotic PCM and why centrioles are constantly required to drive the growth of the mitotic PCM. All of these findings can be explained if the mother centriole is the only source of the phosphorylated Spd-2 scaffold. The observation that the pool of Polo recruited by Ana1, which unlike Spd-2, is not a PCM component and is restricted to the centriole - is required for the efficient expansion of the PCM demonstrates that the PCM-associated pool of Polo (recruited by Spd-2) is not sufficient to drive efficient PCM expansion on its own. It is important to stress, however, that so far an outward flux of Spd-2 from the centriole has only been observed in fly embryos and cells and has not been detected for SPD-2 in C. elegans embryos\. Clearly it will be important to establish whether such a Spd-2 or Cep192 flux exists in other species (Alvarez-Rodrigo, 2021).

The ability of Ana1 to recruit Polo also appears to be required for centriole elongation during G2. In human cells, PLK1 is required for this process, although a previous study did not report any change in centriole length after long-term Polo-inhibition in fly spermatocytes. Clearly more work is required to establish whether Polo recruitment by Ana1 has a role in G2 centriole elongation in flies, as the current work suggests, and, if so, what Polo's relevant substrates are at the centriole distal end (Alvarez-Rodrigo, 2021).

Finally, it is noted that both the Ana1/Cep295 and Spd-2/Cep192 protein families have a relatively high density of potential PBD-binding sites (S-S/T motifs) when compared to several other centriole and centrosome proteins. This suggests that these proteins might have evolved to function as scaffolds that amplify Polo levels at specific locations within the cell during mitosis. It will be interesting to examine whether other proteins with a high density of potential PBD-binding domains serve a similar function at other locations within the mitotic cell. The strategy of mutating all S-S/T motifs to T-S/T in candidate proteins may be a good way of testing this possibility as, for both Ana1 and Spd-2 at least, the S-to-T substitutions seem to specifically impair Polo-recruitment without more generally perturbing the function of the proteins or centriole/centrosome structure (Alvarez-Rodrigo, 2021).

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).

Search PubMed for articles about Drosophila Spd-2

Alvarez-Rodrigo, I., Steinacker, T. L., Saurya, S., Conduit, P. T., Baumbach, J., Novak, Z. A., Aydogan, M. G., Wainman, A. and Raff, J. W. (2019). Evidence that a positive feedback loop drives centrosome maturation in fly embryos. Elife 8. PubMed ID: 31498081

Alvarez-Rodrigo, I., Wainman, A., Saurya, S. and Raff, J. W. (2021). Ana1 helps recruit Polo to centrioles to promote mitotic PCM assembly and centriole elongation. J Cell Sci 134(14). PubMed ID: 34156068

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

Malerod, L., Le Borgne, R., Lie-Jensen, A., Eikenes, A. H., Brech, A., Liestol, K., Stenmark, H. and Haglund, K. (2018). Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics. EMBO J. 37(13): PubMed ID: 29858227

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

date revised: 5 December 2023

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