InteractiveFly: GeneBrief

SAK: Biological Overview | References

BIOLOGICAL OVERVIEW

SAK/PLK4 is a distinct member of the polo-like kinase family. SAK^−/− mice die during embryogenesis, whereas SAK^+/− mice develop liver and lung tumors and SAK^+/− MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. This study shows that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, this study shows that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. Thus SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role (Bettencourt-Dias, 2005).

Polo-like kinases (Plks) belong to a conserved family of mitotic serine-threonine protein kinases that play key roles in centrosome function and are misregulated in many human tumors. Two branches of the family have emerged in metazoans, and they are represented in Drosophila by Polo and SAK (also called Plk4) (Lowery, 2005). Whereas much is now known about the mitotic functions of the founder member of the family, Drosophila Polo, and its related mammalian counterparts (Plk1-3), the precise mitotic roles of SAK remain obscure. Conservation of the structures of the two types of Plks throughout evolution suggests that different roles may have been preserved. Both branches of the family have an amino-terminus kinase domain and a regulatory C-terminal domain that contains conserved polo boxes (PBs). However, whereas the two PB domains of mammalian Plk1 interact with each other to create a positively charged cleft able to bind phosphopeptides, the SAK PB forms an intermolecular homodimer in which different sequences are exposed (Lowery, 2005; Elia, 2003. Moreover, SAK has a second very divergent PB (cryptic PB) (Leung, 2002; Swallow, 2005) that does not bind to its conserved PB. The Plk1-3 group are more akin to the single Plks found in the yeasts, such as Cdc5 in S. cerevisiae, and expression of either Plk1 or Plk3, but not SAK, rescues the mitotic defects of temperature-sensitive cdc5-1 mutant cells (Bettencourt-Dias, 2005).

Drosophila Polo and its closest mammalian counterpart, Plk1, are associated with centrosomes, kinetochores, and the late-mitotic central spindle, reflecting their functions in centrosome maturation, in the metaphase-anaphase transition, and in cytokinesis. SAK also localizes to the centrosome, and SAK^−/− mice die shortly after gastrulation, showing a 20-fold increase in cell death (Leung, 2002; Hudson, 2001). It was recently reported that elderly SAK^+/− mice display a 15-times-higher incidence of spontaneous liver and lung cancers than their wild-type littermates (Ko, 2005). Similarly, the human SAK gene maps to a chromosome region, 4q28, that is frequently rearranged in hepatocellular carcinomas. Multipolar mitotic spindles were reported in livers and MEFS of SAK^+/− mice, suggesting that haploinsufficiency for tumor suppression may result from chromosome instability in the oncogenic pathway. This study addresses the underlying cause of these mitotic defects, showing that in both Drosophila and human cells, SAK is required for centriole duplication. It is therefore essential for centrosome integrity and thereby fidelity of the mitotic apparatus. Moreover, SAK is also required for development of axonemal structures, reflecting the dual nature of centrioles and basal bodies (Bettencourt-Dias, 2005).

To examine SAK function, more than 70% of SAK mRNA was depleted in cultured Drosophila cells. A 12-fold increase was observed in the percentage of mitotic cells that had no γ-tubulin at the poles (1.6%, controls versus 24.7%, SAK RNAi). Other proteins typically recruited to Drosophila centrosomes in mitosis were absent: CP190 and Cnn. In a recent survey of the cell-cycle function of all the protein kinases in Drosophila, loss of function of only one other kinase, Polo, led to loss of γ-tubulin from the centrosome, consistent with the known function of Polo/Plk1 in centrosome maturation. However, in contrast to the striking metaphase arrest following polo RNAi, there was no change in mitotic index or in the flow-cytometry profile of DNA content after SAK RNAi. Thus, in S2 cells, SAK is required for centrosome integrity but not for progression through the cell cycle or cell survival (Bettencourt-Dias, 2005).

Dividing cells were examined in the CNS of SAK mutant larvae. Such cells showed an extremely similar phenotype to SAK RNAi cultured cells: a notable absence of γ-tubulin from the poles of spindles that were often disorganized and splayed. Meiotic spindles in testes of such mutants also often lacked γ-tubulin and Cnn at the poles. It was found that Drosophila SAK localized to centrosomes in both interphase and mitosis of wild-type cells, consistent with its having a function in ensuring centrosome integrity (Bettencourt-Dias, 2005).

In contrast to Polo-depleted cells, the poles of SAK-depleted cells were broad, similar to the mitotic figures of a Drosophila acentriolar cell line. This led to a determination of whether the innermost centrosomal structures, the centrioles, might be disrupted by depletion of SAK. It was found that the majority of mitotic cells that showed no γ-tubulin at the poles following SAK RNAi had no detectable pericentrin-like protein, normally present in both centrioles and the pericentriolar material (PCM). This was in contrast to polo or cnn RNAi cells, where D-PLP was still present in more than 75% of cells lacking γ-tubulin at both poles, reflecting the roles of Polo and Cnn in centrosome maturation but not centriolar integrity. In interphase, the majority of SAK RNAi cells had either zero or a single centrosome, whereas interphase polo or cnn RNAi cells did not show a significant change in centrosome number. The absence of D-PLP in a large proportion of interphase SAK RNAi cells suggested that they might be missing both centrioles and PCM. To confirm loss of centrioles after SAK RNAi, such cells were stained for the centriole marker centrin and then embedded and serially sectioned them for transmission electron microscopy (TEM). In SAK-depleted cells in which centrin staining was absent at the spindle poles, no centrioles were detected by TEM. Thus, the downregulation of SAK leads to loss of centrioles from affected cells, with no effect on the ability of those cells to proliferate (Bettencourt-Dias, 2005).

To determine whether loss of centrosomes also occurred in SAK mutants and still permit proliferation of diploid cells, the central nervous system of mutant larvae was examined. This revealed cells with two, one, or zero centrosomes, a phenotype identical to SAK RNAi. Moreover, the brains of mutant larvae were of normal size and the proportion of brain cells in mitosis was comparable to the wild-type, indicating no obvious defects in cell-cycle progression. SAK mutants were able to pupate, and adults eclosed from the pupae. However, the majority of adults were uncoordinated and died after getting stuck in the food. This phenotype is similar to D-PLP mutations that cause defects in basal bodies (Martinez-Campos, 2004), the centriolar-derived structures required for formation of cilia in neurons of type-I sensory organs that function in transduction of sensory stimuli. The lack of centriole markers in cells of the larval central nervous system of SAK mutants suggests that the uncoordinated adult phenotype is likely to reflect absence of basal bodies in sensory neurons (Bettencourt-Dias, 2005).

Whether the above defects could reflect a failure to assemble new centrosomes was examoned. This could account for the increased frequency of cells with only one centrosome in SAK mutants, if new centrosomes are not formed but cells continue to cycle. The prediction was that if cultured cells were allowed to continue dividing while being exposed to SAK dsRNA, the average number of centrosomes per cell would decrease with time. This was tested by repeatedly transfecting cells with SAK dsRNA at 4-day intervals. Successive transfections led to a progressive increase in the proportion of cells with no centrosomes, rising to greater than 93% of cells after 16 days. This dilution is consistent with SAK's having a role in centrosome assembly. Such a role was further substantiated by the finding that transfection of S2 cells with the active, but not inactive, kinase led to an increase in cells with more than two D-PLP foci. These foci are unlikely to result from aborted cell division because the majority of those cells had a single nuclei. These foci behaved as microtubule organizing centers clustering at the poles of mitotic spindles. Although additional experiments will be needed to verify the origin of the multiple foci, these results suggest that overexpression of SAK may result in multiple centrosomes (Bettencourt-Dias, 2005).

To determine whether the phenotypes observed in SAK mutant flies were associated with defective centrosome separation, abnormal centrosome inheritance, or problems in centriole duplication, spermatogenesis was examined in SAK mutant males. The germline has a stereotyped pattern of mitotic and meiotic divisions, a pattern that allows the cellular history of centrosomes and centrioles to be deduced. Additionally, their centrioles are approximately 10-fold longer than those found in other Drosophila cells and can be easily visualized by fluorescence of a GFP-PACT fusion protein harboring the centriole-targeting domain of D-PLP protein (Bettencourt-Dias, 2005).

A very high proportion of primary spermatocytes from SAK mutants had no centrioles at one or both spindle poles in meiosis I. When centrioles were absent, the spindle poles were broad and there were no astral microtubules, or very disorganized spindles were formed. Transverse TEM sections of wild-type sperm tails revealed the classic '9 + 2' axonemal microtubules that were missing from the majority of elongating SAK spermatids. Accordingly, the majority of sperm from SAK mutant testes were nonmotile, and males were sterile. Frequently, cells that had no axonemes had irregular size and numbers of mitochondrial derivatives per cell. This is usually associated with defective chromosome segregation and cytokinesis during meiosis. The lack of a strong spindle-assembly checkpoint in meiosis makes cells more likely to progress all the way through both meiotic divisions even in the presence of abnormalities. When axonemes were present, they appeared to be normal in structure (Bettencourt-Dias, 2005).

Wild-type primary spermatocytes enter meiosis I (MI) with a pair of centrioles at each spindle pole. Daughter cells inherit a pair of centrioles that are not duplicated in Drosophila. These centrioles separate, each generating a centrosome at the pole of the second meiotic spindle. Thus, each daughter spermatid inherits a single centriole. Failure to observe more than a single centriole in the products of meiosis II in the SAK mutant led to discarding of the possibility of defects in centriole separation (Bettencourt-Dias, 2005).

The cysts of 16 primary spermatocytes encapsulate the history of centriole duplication in the four preceding cell divisions. It was therefore asked whether the number of centrioles per cell in SAK mutant cysts could reflect abnormal centriole duplication. Mature cysts of primary spermatocytes in both wild-type and SAK mutant testes always contained 16 spermatocytes of comparable size, indicating success in the four rounds of premeiotic mitosis. However, whereas wild-type primary spermatocytes contained four centrioles (64 per cyst), the majority of spermatocytes from SAK mutants had no centrioles, and a smaller proportion of cells had intermediate numbers between one and four. The absence of cells having more than two centrosomes/four centrioles provided a second indication of the lack of defects in centrosome segregation to daughter cells. To test whether the distribution of centrioles observed resulted from a defect in centriole duplication, a mathematical model was generated to describe the centriole-duplication cycle and adjusted it for the four mitotic divisions of germ cells in a cyst. Multitype branching-process theory was used to evaluate the distribution of cells with a given number of centrioles. The model has some analogies with previous analytical work on plasmid copy number in bacteria. It assumes that centrosome separation and segregation is perfect, and that the variable number of centrioles in G2 after the four germline divisions is due to partially defective centriole duplication having a probability θ. Generating functions were built to follow the dynamics of the mean number of cells with a given number of centrioles. The proportion of cells with a given number of centrioles in G2 was evaluated after four cell divisions. Finally, a value of θ was found that best fit the empirical data. The function is very peaked around 0.55, fitting the empirical data very well and giving high confidence in the estimated duplication rate. Thus, it was possible to model the reduction in centriole number during the premeiotic divisions of SAK hypomorphic mutants solely by assuming reduced success of centriole duplication (Bettencourt-Dias, 2005).

Finally, it was asked whether the role of SAK in centriole duplication was conserved in human cells and SAK siRNA conditions were developed that reduced levels of SAK transcripts by more than 70%. Such depletion resulted in a 10-fold increase of HeLa cells with just one centriole. This was associated with an increased mitotic index and a higher-than-2-fold increase in apoptosis, leading to a decrease in cell number. A similar reduction was observed in centriole number after SAK RNAi in U2OS cells. It is known that centrioles continue to replicate when U2OS cells are blocked in S phase by treatment with the DNA polymerase α-inhibitor aphidicolin (AF) or the ribonucleotide reductase inhibitor hydoxyurea (HU). Following SAK RNAi, it was found that the number of cells accumulating supernumerary centrosomes in an S phase block is less than half that of control cells. Thus, downregulation of SAK reduces centriole duplication in cells blocked in S phase. There was no increase in the proportion of cells with a single centriole after SAK RNAi when cells were inhibited from dividing by treatment with AF or HU, a result consistent with inhibition of centriole duplication. It was therefore conclude that the human SAK kinase is required for centriole duplication in both HeLa and U2OS cells and for centriole reduplication in AF- or HU-treated U2OS cells (Bettencourt-Dias, 2005).

Reduced centriolar number was associated with a 6-fold increase in abnormal mitotic spindles 72 hr after SAK SiRNA in HeLa cells. These included monopolar and multipolar spindles. Similar defects were observed in U2OS cells. Curiously, in a few cases, recruitment of γ-tubulin to the acentriolar poles of multipolar spindles was seen, suggesting that acentriolar poles may organize some PCM in mammalian cells. No such cases were observed in Drosophila. In summary, spindle organization is affected in the absence of two canonical centrosomes having two centrioles, and this is likely to contribute to the reduced chromosome-transmission fidelity that has been suggested from observations of SAK-deficient mouse cells (Bettencourt-Dias, 2005).

This study has shown that depletion of SAK, but not Polo, leads to cells with a reduced number of centrioles. Centriole loss in a cycling population of cells can arise through defective centrosome duplication, abnormal separation of centrosomes at entry to mitosis, or abnormal centrosome segregation to the daughter cells in cytokinesis. In both human and Drosophila cells that there is no abnormal centrosome segregation in the absence of SAK because there is always loss but never gain of centrosomes/centrioles after cell division. Moreover, centriole reduplication in S phase is reduced after SAK knockdown in the absence of cell division in human cells. This analysis of centriole distribution in Drosophila SAK spermatids revealed no defects in centrosome separation. Moreover, the distribution of centrioles in cysts of 16 primary spermatocytes was consistent with a mathematical model assuming defects in centriole duplication. Together, these observations point to a conserved role of this member of the PLK family in centriole duplication. However, it cannot be entirely excluded that there is an additional function for SAK in mitosis until a complete loss-of-function mutant is isolated (Bettencourt-Dias, 2005).

Experiments in C. elegans have suggested five proteins to be important for centrosome duplication in embryogenesis, and these include one protein kinase, ZYG1 (O'Connell, 2001). Although ZYG-1 has only low sequence similarity to Drosophila SAK, it is the closest homolog in a BLAST search, and SAK may thus represent the ortholog of ZYG-1 in flies and vertebrates (Bettencourt-Dias, 2005).

What could be the role of SAK in centriole duplication? Structurally compromised centrioles have never been observed either in SAK mutants or after SAK depletion in either Drosophila or human tissue-culture cells. Moreover, overexpression of SAK, but not of the inactive kinase, leads to the formation of multiple D-PLP foci, suggestive of overduplication of centrioles. Together, these data suggest that SAK has a regulatory role in centriole duplication. ZYG-1 has been found to be high in the hierarchy of molecules necessary for centriole assembly and essential for the recruitment of SAS-6/SAS-5 to the centriole (Leidel, 2005), but it remains to be discovered whether this role has been conserved in Drosophila and vertebrates (Bettencourt-Dias, 2005).

The perdurance of centrosomal structures in SAK mutants studied here may result from some residual SAK function due to the hypomorphic nature of this allele and/or from remaining wild-type maternal SAK protein provided by the heterozygous mother. Such perdurance of maternal protein to the third larval-instar stage is a common feature of mitotic mutants in Drosophila. Nonetheless, SAK mutants provide the first opportunity to assess the consequences of the absence of centrioles and centrosomes upon the development of an organism. This examination of the larval central nervous system suggests that as many as 72% of dividing cells have fewer centrosomes than expected, with 28% possessing no centrosomes at all. In cysts of primary spermatocytes, only 8% of the cells have the correct number of centrioles, whereas 68% have none. It has been proposed that centrosomes are not required for the formation of mitotic spindles, but do provide speed and fidelity to this process and so may be necessary for proper cell-cycle progression. it is conclude that in cultured Drosophila cells and in the whole organism, centrosomes are not essential for mitotic progression or cell survival. However, the absence of centrioles, and hence basal bodies, compromised both meiotic divisions and the formation of sperm axonemes. The only cilia and flagella known in the fly are found in the peripheral nervous system and in the male germline. Accordingly, it was found SAK mutants to be both uncoordinated and sterile (Bettencourt-Dias, 2005).

What could be the consequences of the lack of SAK in vertebrates? Mammalian cells appear to be more sensitive to depletion of SAK and the lack of centrosomes than Drosophila cells. There was a significant increase in abnormal mitoses, in mitotic index, and in apoptosis after depletion of SAK in human cells. Mitotic abnormalities and an increase in cell death are also observed after depletion of SAS-6, a protein involved in centriole replication, in U2OS cells and may thus be a general consequence of centriole loss in vertebrates. Mice homozygotes for SAK die very early during embryogenesis (Hudson, 2001), hindering study of the effects on cilia formation and development. However, these embryos showed increased cell death and a higher mitotic index. SAK^+/− MEFs also show abnormal spindles and chromosome segregation, and, significantly, SAK^+/− mice are more prone to develop cancer (Ko, 2005). Human Plk4 has been shown to be a key regulator of centriole duplication. Both gain- and loss-of-function experiments demonstrate that Plk4 is required - in cooperation with Cdk2, CP110 (see Drosophila CP110) and Hs-SAS6 - for the precise reproduction of centrosomes during the cell cycle (Habedanck, 2005). The link between centrosome duplication, embryonic survival, and haploinsufficiency for tumor suppression is also seen with nucleophosmin, a regulator of centrosome duplication. Together, these studies reinforce the link between centrosome defects and oncogenesis. The sensitivity with which two tumor cell lines undergo aberrant mitosis and cell death after downregulation of the SAK kinase suggests that it may be a valuable target for cancer therapy (Bettencourt-Dias, 2005).

Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation

Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. This study investigated the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila -- DSas-6, DSas-4, and Sak. By directly observing centriole replication in living Drosophila embryos, this study shows that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. These data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg (Peel, 2007).

This study shows that DSas-6, like DSas-4 and Sak, is required for centriole duplication in Drosophila. Studying the effects of overexpressing each of the three proteins, the following is shown: first, the overexpression of DSas-6 in vivo can drive extra rounds of templated centriole replication within a single cell cycle. Second, the overexpression of these proteins induces the formation of extra centriole-like structures to varying extents in different tissues. Third, the overexpression of any of these proteins at high levels can drive the de novo formation of centriole-like structures in unfertilized eggs. The implications of each of these findings is discussed in turn (Peel, 2007).

It has previously been shown that the overexpression of Plk4/Sak in human cells leads to an accumulation of extra centrioles and HsSAS-6 appears to have a similar effect. Because these experiments were performed with fixed cultured cells, it was unclear how the extra centrioles formed and whether these proteins could drive centriole accumulation in vivo. In the current experiments, extra rounds of templated centriole replication driven by the overexpression of DSas-6 were directly visualized in vivo. Moreover, these extra centrioles appear to be fully functional because they organize PCM and MTs and, most importantly, they can undergo further rounds of replication in synchrony with the other centrioles in the embryo (Peel, 2007).

Recent studies in C. elegans have revealed that centriole replication requires the ordered activity of SPD-2, ZYG-1, SAS-5, and SAS-6, and finally SAS-4. The current findings demonstrate that DSas-6 levels are critical in determining the number of centrioles formed during centriole replication in Drosophila embryos. How might DSas-6 regulate centriole number during replication? One possibility is that, when overexpressed, DSas-6 is recruited normally to the mother centriole but is then inappropriately recruited to the newly formed daughter centriole, thereby inducing the formation of a 'granddaughter' centriole. Another possibility is that excessive recruitment of DSas-6 to the mother centriole expands the area where centrioles can form, thereby resulting in the generation of multiple daughter centrioles. Neither mechanism is mutually exclusive, and both of these configurations of centrioles have been observed in Drosophila somatic cells in which the inactivation of Cdk1 led to centriole overduplication (Vidwans, 2003; Peel, 2007 and references therein).

Extra rounds of templated centriole replication were not directly observed in Ubq-GFP-Sak embryos, but it is suspected that this is because the protein was expressed at very low levels in embryos. In larval brain cells and ovarian nurse cells, Sak was the most potent of the three replication proteins at inducing the formation of extra centriole-like structures. The formation of these extra structures required DSas-4, and the structures contained several centriole markers and could organize PCM markers and MTs. Nevertheless, EM studies will be required to confirm that these structures are true centrioles (Peel, 2007).

A priori, it is perhaps surprising that two different proteins can drive centriole overduplication, because only one protein would be expected to be rate limiting in any given system. The data suggest that it is the amount of DSas-6 at the centriole that determines the number of daughter centrioles formed during each round of replication (the 'litter' size), and it is suspected that overexpressed Sak can recruit extra DSas-6 to the centrioles even when DSas-6 is not overexpressed. The configuration of the extra centrioles in human cells overexpressing Plk4/Sak is consistent with this proposal, and the formation of these extra centrioles requires HsSAS-6. The observation that DSas-4 overexpression does not induce templated-centriole overduplication in any of the cell types examined is consistent with this hypothesis, because SAS-4 is recruited to centrioles only after ZYG-1 and SAS-6 in C. elegans. Overexpressed DSas-4 is presumably unable to recruit extra DSas-6 to the centrioles (Peel, 2007).

The results demonstrate that the overexpression of centriole duplication proteins can have different effects in different tissues. The overexpression of GFP-Sak or GFP-DSas-6 leads to an accumulation of extra centrioles in larval brain cells but not in larval spermatocytes. It seems unlikely that these differences result only from differing expression levels in the different tissues, because the Ubq promoter appears to drive higher levels of GFP-DSas-6 and DSas-4-GFP in the testes than in the brain. It is speculated, therefore, that additional mechanisms may regulate the activities of these proteins, and these mechanisms may differ between tissues (Peel, 2007).

Perhaps the most surprising of the observations is that the expression of any of the three fusion proteins at high levels can trigger the de novo formation of many hundreds of centriole-like structures in unfertilized eggs. EM studies will be required to see whether these structures are normal centrioles, but they all incorporate endogenous centriole markers and organize PCM and astral MTs. Nevertheless, there are clear morphological differences between the structures formed by the overexpression of GFP-DSas-6 and those formed by the overexpression of DSas-4-GFP and GFP-Sak. Interestingly, it has previously been shown that the expression of a dominant mutant form of dynein heavy chain, Laborc^D, can lead to the rapid de novo formation of centriole-like structures in a manner very similar to that reported in this study. An EM analysis revealed that these structures were 'rudimentary centrioles' that consisted of hollow tubes that lacked any associated MTs. The de novo formation of centrioles in cultured cells also leads to the formation of centriole-like structures that, initially, do not have the normal appearance of centrioles at the EM level (Peel, 2007).

Whereas the de novo formation of centrioles in cultured cells is a slow process that occurs over several hours, the centriole-like structures that was observed in unfertilized eggs appear very rapidly upon egg deposition. Even in 30 min collections of both UAS-GFP-Sak and UAS-GFP-DSas-6 unfertilized eggs, it was found that >95% of the eggs had at least ~50 of these structures and most had several hundred structures that had already recruited PCM components and were nucleating MTs. Because the expression of these replication proteins does not lead to the abnormal persistence of centrioles during oogenesis, it is concluded that the centriolar components in these unfertilized eggs must be organized in such a way that they can be very rapidly assembled into centriole-like structures when the egg is deposited (Peel, 2007).

This is further supported by the observation that even DSas-4-GFP can induce the formation of centriole-like structures in unfertilized eggs. SAS-4 functions at a late step in centriole duplication, so it is unlikely that it could induce the de novo formation of centriole-like structures unless the centriolar components were already partially assembled. It is speculated that centriolar components normally have a tendency to transiently self-assemble into 'centriole precursors' in these eggs. The overexpression of any of the replication proteins can stabilize these precursors, allowing them to mature into centriole-like structures upon egg deposition (Peel, 2007).

These observations are consistent with the hypothesis that normal templated centriole replication may depend upon the presence of centriole-precursors in the cytoplasm. In this model, cells normally contain centriole precursors, but during replication only one of these becomes stabilized when it contacts the mother centriole, thereby allowing it to mature into a daughter centriole. In unfertilized Drosophila eggs, the overexpression of replication proteins may stabilize these centriole precursors throughout the egg, thereby circumventing the normal requirement that the centriole precursors contact the mother centriole to become stabilized (Peel, 2007).

Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation

Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms. Although Plk4's centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. This study shows that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent (Dzhindzhev, 2014).

SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication

Centrioles are 9-fold symmetric structures duplicating once per cell cycle. Duplication involves self-oligomerization of the centriolar protein SAS-6, but how the 9-fold symmetry is invariantly established remains unclear. This study found that SAS-6 assembly can be shaped by preexisting (or mother) centrioles. During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. The removal or release of luminal SAS-6 requires Plk4 and the cartwheel protein STIL. Abolishing either the recruitment or the removal of luminal SAS-6 hinders SAS-6 (or centriole) assembly at the outside wall of mother centrioles. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number (Fong, 2014).

Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos

Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with human disease. Centrioles duplicate once per cell cycle, when a mother centriole assembles an adjacent daughter during S phase. Daughter centrioles cannot support the assembly of another daughter until they mature into mothers during the next cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. Pioneering studies in C. elegans identified a set of core proteins essential for centriole duplication, and a similar set have now been identified in other species. The protein kinase ZYG-1/Sak/Plk4 recruits the inner centriole cartwheel components SAS-6 and SAS-5/Ana2/STIL, which then recruit SAS-4/CPAP, which in turn helps assemble the outer centriole microtubules. In flies and humans, the Asterless/Cep152 protein interacts with Sak/Plk4 and Sas-4/CPAP and is required for centriole duplication, although its precise role in the assembly pathway is unclear. This study shows that Asl is not incorporated into daughter centrioles as they assemble during S phase but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, requires DSas-4, and, crucially, is essential for daughter centrioles to mature into mothers that can support centriole duplication. Therefore a 'dual-licensing' model of centriole duplication is proposed, in which Asl incorporation provides a permanent primary license to allow new centrioles to duplicate for the first time, while centriole disengagement provides a reduplication license to allow mother centrioles to duplicate again (Novak, 2014).

This study demonstrates that Asl recruitment to disengaged new centrioles has a critical role in allowing these centrioles to mature into mothers that can duplicate for the first time. During all subsequent duplication cycles, however, mother centrioles already contain a pool of immobile Asl, and this appears to be sufficient to allow subsequent rounds of duplication, because anti-Asl antibodies block the recruitment of the mobile fraction of Asl to mother centrioles but do not block their duplication. For an old centriole to duplicate again, therefore, disengagement of the daughter centriole appears to be the crucial licensing event that allows reduplication, because immobile Asl incorporation has already occurred. Taken together, these findings suggest a dual-licensing model in which the recruitment of the immobile fraction of Asl by DSas-4 provides an irreversible primary license to allow newly formed centrioles to duplicate for the first time, while centriole disengagement provides a reduplication license to allow older centrioles to duplicate again (Novak, 2014).

How might Asl perform this primary licensing function? In flies, Asl localizes Sak to centrioles, probably explaining why Asl incorporation is a crucial step in converting a disengaged daughter centriole into a mother centriole that can duplicate. Cep152 (human Asl) is also required for the efficient loading of Plk4 (human Sak) onto centrioles in verte- brate cells, although it appears to share this function with Cep192 (human SPD-2). This model is consistent with superresolution microscopy studies on fixed cells, which show that Asl/Cep152 is associated with the mother centriole in an engaged centriole pair, suggesting that a similar model may operate in vertebrates. Although the primary and reduplication licensing steps are mechanistically different, it is suspected that they share a common purpose: to provide an Asl platform that is competent to recruit Sak to initiate daughter centriole assembly (Novak, 2014).

The model can explain why only mother centrioles can support certain types of experimentally induced centriole reduplication, including that induced by Sak overexpression or by ablation of one of the engaged centrioles during an arrested S phase. It can also explain why daughter centrioles appear to have to be 'modified' before they can support any duplication; the results strongly suggest that this modification, at least in flies, is Asl incorporation. How is Asl recruited to centrioles? It is speculated that DSas-4 initially recruits the immobile fraction of Asl, which then recruits the mobile fraction. This would explain the 50:50 ratio of immobile to mobile Asl. The finding that anti-Asl antibodies strongly block the recruitment of the mobile fraction of Asl to mother centrosomes also supports this possibility. It is tempting to speculate that the mobile fraction of Asl may be important for the previously described role of Asl in mitotic PCM recruitment. It is also interesting to note that only very low levels of Asl seem to be required at new mother centrioles to allow duplication (Novak, 2014).

It remains to be determined what regulates the interaction between DSas-4 and Asl such that Asl is only recruited to daughter centrioles at about the time they separate from their mothers. It is speculated that the phosphorylation state of either or both proteins could be altered at the end of mitosis, perhaps increasing the affinity of their interaction. Polo/Plk1 seems to play a crucial part in resetting the reduplication license at old centrioles through the regulation of centriole disengagement; perhaps it also has an important role in the primary licensing of new centrioles by regulating the interaction between DSas-4 and Asl (Novak, 2014).

A homeostatic clock sets daughter centriole size in flies

Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing 'mother' centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. This study shows that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size (Aydogan, 2018).

How organelles grow to the right size is a fundamental problem in cell biology. For many organelles, however, this question is difficult to address: the number and distribution of an organelle within a cell can vary, and it can also be difficult to determine whether an organelle's surface area, volume, or perhaps the amount of a limiting component, best defines its size. Centrioles are highly structured organelles that form centrosomes and cilia. Their length can vary by an order of magnitude between different species and tissues but is very consistent within a given cell type. Centrioles are potentially an attractive system with which to study organelle size control, as their numbers are precisely regulated: most cells are born with a single centriole pair that is duplicated once per cell cycle, when a single daughter centriole grows outwards from each mother centriole during S-phase. Moreover, the highly ordered structure of the centriole means that the complex 3D question of organelle size control can be simplified to a 1D question of daughter centriole length control (Aydogan, 2018).

Much progress has been made recently in understanding the molecular mechanisms of centriole duplication. Polo-like kinase 4 (Plk4) initiates duplication and is first recruited in a ring surrounding the mother centriole; this ring ultimately resolves into a single 'dot' that marks the site of daughter centriole assembly. Plk4 recruits and phosphorylates Ana2/STIL, which helps recruit Sas-6 to initiate the assembly of the ninefold-symmetric cartwheel that forms the structural backbone of the growing daughter centriole. How Plk4 is ultimately localized to a single site on the side of the mother is unclear, but Plk4 can dimerize and autophosphorylate itself in trans to trigger its own destruction. In addition, binding to Ana2/STIL activates Plk4's kinase activity and also appears to stabilize Plk4. Thus, the binding of Plk4 to Ana2/STIL at a single site on the side of the mother could activate and protect the kinase at this site, whereas the remaining Plk4 around the mother centriole is degraded (Aydogan, 2018).

Although studies have provided important insight into how mother centrioles grow only a single daughter, the question of how daughter centrioles subsequently grow to the correct length has been difficult to address. This is in part because centrioles are small structures (usually 100-500 nm in length), making it hard to directly monitor the kinetics of centriole growth. Also, cells usually only assemble two daughter centrioles per cell cycle, and this makes it difficult to measure centriole growth in a quantitative manner. The early Drosophila melanogaster embryo is an established model for studying centriole and centrosome assembly, and it is potentially an attractive system for measuring the kinetics of daughter centriole growth. First, it is a multinucleated single cell (a syncytium) that undergoes 13 rounds of nearly synchronous, rapid nuclear divisions. During nuclear cycles 10-14, the majority of nuclei (and their associated centrioles) form a monolayer at the cortex, allowing the simultaneous observation of many centrioles as they rapidly and synchronously progress through repeated rounds of S-phase and mitoses without intervening gap phases. Second, centrioles in flies are structurally simpler than those in vertebrates. All centrioles start to assemble around the cartwheel in S-phase, but vertebrate centrioles often exhibit a second phase of growth during G2/M, when the centriolar microtubules (MTs) extend past the cartwheel. Fly centrioles usually do not exhibit this second phase of growth, so the centrioles are relatively short, and the cartwheel extends throughout the length of the daughter centriole. It was reasoned, therefore, that the fluorescence incorporation of the cartwheel components Sas-6-GFP or Ana2-GFP could potentially be used as a proxy to measure daughter centriole length in D. melanogaster embryos(Aydogan, 2018).

This study shows that this is the case, and the first quantitative description is provided of the kinetics of daughter centriole growth in a living cell. The findings reveal an unexpected inverse relationship between the centriole growth rate and growth period: in embryos where daughter centrioles tend to grow slowly, they tend to grow for a longer period. Surprisingly, Plk4 influences both the centriole growth rate and growth period and helps coordinate the inverse relationship between them. Thus, Plk4 functions as a homeostatic clock that helps to ensure daughter centrioles grow to the correct size in fly embryos (Aydogan, 2018).

Several models have been proposed to explain how daughter centrioles might grow to the correct size, but none of these have been tested, primarily because of the lack of a quantitative description of centriole growth kinetics. The observations suggest an unexpected, yet relatively simple, model by which centriolar Plk4 might determine daughter centriole length in flies (Aydogan, 2018).

It is proposed that a small fraction of centriolar Plk4, perhaps the fraction bound to both Asl and Ana2, influences both the rate of cartwheel growth (by determining the rate of Sas-6 and Ana2 recruitment to the centriole) and the period of cartwheel growth (by determining the rate of Plk4 recruitment to the centriole, and so how quickly centriolar Plk4 accumulates to trigger its own destruction). This model is consistent with the observation that daughter centrioles grow at a relatively constant rate even as centriolar levels of Plk4 fluctuate (indicating that the majority of Plk4 located at the centriole during S-phase is not directly promoting daughter centriole growth) and that centriolar Plk4 levels appear to influence the rate at which Plk4 is accumulated at centrioles (suggesting that Plk4 can recruit itself, either directly or indirectly, to centrioles) (Aydogan, 2018).

In this model, Plk4 functions as a homeostatic clock (see Schematic illustration of how a Plk4-dependent homeostatic clock might set daughter centriole length in flies), regulating both the rate and period of daughter centriole growth, and ensuring an inverse relationship between them: the more 'active' the Plk4, the faster the daughters grow, but the faster Plk4 is recruited and so inactivated. The activity of this Plk4 fraction is probably a function of both the total amount of Plk4 in this fraction and its kinase activity. It is speculate that this activity is determined before the start of S-phase by a complex web of interactions between Plk4, Ana2, Sas-6, and Asl that influence each other's recruitment and stability and also, directly or indirectly, Plk4's kinase activity. These interactions are likely to be regulated by external factors (such as the basic cell cycle machinery), allowing cells to set centriole growth parameters according to their needs. In cells with a G1 period, for example, Plk4 could be activated as cells progress from mitosis into G1, allowing the mother centriole to recruit an appropriate amount of Sas-6 and Ana2/STIL at this stage, which could then be incorporated into the cartwheel when cells enter S-phase. This could explain why in some somatic cells Plk4 levels appear to be higher during mitosis/G1 than in S-phase, and why Plk4 kinase activity appears to be required primarily during G1, rather than S-phase (Aydogan, 2018).

This model can explain why halving the dose of Plk4 leads to a decrease in the growth rate and an increase in the growth period: halving the dose of Plk4 would be predicted to lower both the kinase activity of centriolar Plk4 (so slowing the growth rate) and the amount of centriolar Plk4 (so increasing the growth period). It can also potentially explain why doubling the dose of Plk4 might change the growth period without changing the growth rate: increasing the dose could lead to an increased rate of Plk4 recruitment (because of its increased cytoplasmic concentration), without increasing the amount or kinase activity of the Plk4 fraction bound to Asl or Ana2 (if these were already near saturation). Finally, it could explain why decreasing the kinase activity of Plk4 decreases the rate of growth without changing the growth period: the decrease in Plk4 kinase activity might affect the rate at which it recruits Ana2/Sas-6 without affecting the amount of centriolar Plk4, and so the rate at which Plk4 recruits itself to centrioles (Aydogan, 2018).

Importantly, although the cartwheel extends throughout the entire length of the daughter centriole in worms and flies, this is not the case in vertebrates, where centrioles exhibit a second phase of growth during G2/M and the centriolar MTs grow to extend beyond the cartwheel. It is suspected that the homeostatic clock mechanism described in this study may regulate the initial phase of centriole/cartwheel growth in all species, but the subsequent extension of the daughter centriole beyond the cartwheel that occurs in vertebrates will likely require a separate regulatory network (Aydogan, 2018).

The concept of a homeostatic clock regulating organelle size has not been proposed previously. This mechanism is plausible for Plk4, because it can behave as a 'suicide' kinase: the more active it is, the faster it will trigger its own inactivation. This mechanism relies on delayed negative feedback, a principle that helps set both the circadian clock and the somite segmentation clock. A similar mechanism might operate with other kinases that influence organelle biogenesis and whose activity accelerates their own inactivation, such as PKC, which regulates lysosome biogenesis. It will be interesting to determine whether homeostatic clock mechanisms that rely on delayed negative feedback could regulate organelle size more generally (Aydogan, 2018).

Centriole distal-end proteins CP110 and Cep97 influence centriole cartwheel growth at the proximal-end

Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. Previous work showed that an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. This study showed that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, in a manner that appears to be entrained by the core Cdk/Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110/Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, changing the levels of CP110/Cep97 appears to alter the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected potential crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which may help to coordinate centriole growth (Aydogan, 2022).

This study shows that fluorescent fusions of CP110 and Cep97 are recruited to the distal-end of daughter centriole MTs in a cyclical manner as they grow during S-phase, with levels peaking, and then starting to decline at about mid-S-phase, which is normally when the centrioles appear to stop growing in these embryos. These recruitment dynamics, however, do not appear to play a major part in determining the period of daughter centriole MT growth, and the findings strongly suggest that centriole MTs do not stop growing when a threshold level of CP110 and Cep97 accumulates at the centriole distal end. Thus, although in many systems the centriole MTs are dramatically elongated in the absence of CP110 or Cep97, it is speculated that they have finished growing, rather than because the centriole MTs grow too quickly as the new daughter centriole is being assembled. It remains a formal, though unlikely, p that this cyclical recruitment of CP110 and Cep97 is an artefact of their fluorescent tagging (Aydogan, 2022).

CP110 and Cep97 levels do not peak at centrioles because the proteins reach saturating levels on the centriole MTs, as the amount of CP110 and Cep97 recruited to centrioles is increased when either protein is overexpressed. It is unclear how these proteins interact specifically with the distal-ends of the centriole MTs, but it is concluded that their binding sites are normally far from saturated, at least in the rapidly cycling Drosophila embryo. Importantly, the phase of CP110 and Cep97 recruitment appears to be influenced by the activity of the core cell-cycle oscillator (CCO). It is suspected, therefore, that the cyclical recruitment dynamics of CP110 and Cep97 in these embryos might simply reflect the ability of these proteins to bind to centrioles when Cdk-Cyclin activity is low, but not when it is high. CP110 was originally identified as a Cdk substrate, and presumably the CCO modifies (perhaps by phosphorylating) CP110 and/or Cep97 and/or their centriolar recruiting factor(s) to inhibit recruitment as cells prepare to enter mitosis. It is presently unclear why it might be important to prevent CP110 and Cep97 binding to centrioles during mitosis (Aydogan, 2022).

Perhaps surprisingly, this study showed that CP110 and Cep97 levels appear to influence the growth of the centriole cartwheel, at least in part, by altering the parameters of the Plk4 oscillation at the base of the growing daughter centrioles. This reveals an unexpected crosstalk between proteins that are usually thought to influence events at the proximal end of the cartwheel (Plk4) and at the distal end of the centriole MTs (CP110 and Cep97). It is currently not understood how CP110 and Cep97 might influence the behaviour of Plk4, but the data suggests they do not alter the abundance of each other in the cytoplasm. Nevertheless, it might be that Plk4 and CP110 and/or Cep97 interact in the cytoplasm, and this interaction influences the amount of Plk4 available for recruitment to the centriole (explaining why less Plk4 is recruited when these proteins are overexpressed and more is recruited with they are absent). Alternatively, perhaps these proteins interact at the centriole during the very early stages of daughter centriole assembly, when they are all present at the nascent site of assembly but have not yet been spatially separated by the growth of the daughter centriole. Clearly it will be important to test whether Plk4 and CP110 and/or Cep97 interact in Drosophila embryos and, if so, how this interaction is regulated in space and time (Aydogan, 2022).

CP110 and Cep97 are not essential for centriole duplication in mice or flies, but CP110 (also known as CCP110 in mammals) is required for Plk4-induced centriole overduplication in cultured human cells, and Plk4 can interact with and phosphorylate CP110 to promote centriole duplication in these cells. Thus, although the physiological significance and molecular mechanism of Plk4 and CP110 and Cep97 crosstalk is currently unclear, this crosstalk might be conserved in other species (Aydogan, 2022).

Finally, it is important to note that changing the levels of CP110 and Cep97 influences the Plk4 oscillation in a surprising way. In the absence of CP110 and Cep97, the cartwheel seems to grow faster and for a shorter period, but the Plk4 oscillation has a higher amplitude and a longer period. Previous observations would suggest that faster centriole growth for a shorter period would be associated with Plk4 oscillation that has a higher amplitude but a shorter period. One way to potentially explain this conundrum is if Plk4 is more active in the absence of CP110 and Cep97 - so the cartwheel would be built faster but for a shorter period, as was observed - but the inactivated Plk4 is not efficiently released from its centriolar receptors (so Plk4 would accumulate at centrioles to a higher level and for a longer period). Clearly further work is required to understand how the Plk4 oscillation drives cartwheel assembly, and how this process is influenced by CP110 and Cep97 (Aydogan, 2022).

The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/PLK4

Centrioles are essential for the formation of microtubule-derived structures, including cilia and centrosomes. Abnormalities in centrosome number and structure occur in many cancers and are associated with genomic instability. In most dividing animal cells, centriole formation is coordinated with DNA replication and is highly regulated such that only one daughter centriole forms close to each mother centriole. Centriole formation is triggered and dependent on a conserved kinase, SAK/PLK4. Downregulation and overexpression of SAK/PLK4 is associated with cancer in humans, mice, and flies. This study shows, in Drosophila cultured cells, that centrosome amplification is normally inhibited by degradation of SAK/PLK4, mediated by the SCF/Slimb ubiquitin ligase. This complex physically interacts with SAK/PLK4, and in its absence, SAK/PLK4 accumulates, leading to the striking formation of multiple daughter centrioles surrounding each mother. This interaction is mediated via a conserved Slimb binding motif in SAK/PLK4, mutations of which lead to centrosome amplification. This regulation is likely to be conserved, because knockout of the ortholog of Slimb, beta-Trcp1 in mice, also leads to centrosome amplification. Because the SCF/beta-Trcp complex plays an important role in cell-cycle progression, these results lead to new understanding of the control of centrosome number and how it may go awry in human disease (Cunha-Ferreira, 2009).

Substrates of the SCF/Slimb complex show a conserved degron that is recognized by Slimb. SAK/PLK4 protein has a modified Slimb recognition site in Drosophila (DSGIIT; position 293) and humans (DSGHAT; position 285). In previous studies, human SAK/PLK4 was shown to be ubiquitinated and removal of a region encompassing amino acids 272–311 led to stabilization of the human SAK/PLK4, although no analysis was performed on centrosome number. The conserved Slimb recognition site was mutated to DAGIIA in Drosophila SAK/PLK4, which is called SAK-ND (nondegradable). This mutation abolished SAK's ability to interact with Slimb and led to a decrease in SAK's ubiquitinated species. Moreover, SAK-ND is more stable in comparison to its WT counterpart. Thus, this mutation allowed testing of the biological significance of SAK/PLK4 degradation by the SCF/Slimb complex. Upon expression of the mutated SAK/PLK4 construct, a very similar phenotype was observed to the one registered after Slimb RNAi, i.e, rosette-like structures. These structures contained centriole percursors or elongating centrioles. Those results suggest a common centriole-amplification phenotype observed after Slimb RNAi and after expression of the SAK-ND mutant. The observation of at least five centrioles clustered at spindle poles in mitosis suggests that some of the supranumerary centrioles formed in one cycle after impairment of SAK/PLK4 degradation by the SCF/Slimb complex become proper microtubule-organizing centers. The formation of supranumerary centrosomes was quantified at the light microscope level. Upon transient low-level expression of the SAK-ND construct fused to GFP, a statistical significant increase in centrosome number was systematically observed in transfected cells compared to cells expressing low levels of the WT SAK fused to GFP. Indeed, expression of low levels of SAK-ND led to a 2-fold increase in centrosome amplification and after expression of a dominant-negative form of Slimb, suggesting that high levels of SAK/PLK4 underlie those phenotypes (Cunha-Ferreira, 2009).

Together, these results show that degradation of SAK/PLK4 by the SCF/Slimb complex is critical to restrict its function, preventing centrosome amplification. Although other mechanisms for regulation of SAK/PLK4 activity may exist, proteolytic degradation is likely to be conserved across species and be relevant in the animal. The Slimb-binding phosphodegron in SAK/PLK4 is conserved in vertebrates. Moreover, knockout of the ortholog of Slimb in mice, β-Trcp1, and both SkpA and Slimb Drosophila mutants show an increase in centrosome number, as do flies that overexpress SAK/PLK4 (Cunha-Ferreira, 2009).

It has been suggested that centriole overduplication is normally prevented by a 'licensing event,' the disengagement of centrioles that occurs at the exit of mitosis, required for the next duplication cycle. Requirement for this event ensures that centrioles duplicate only once in every cell cycle. However, it was recently shown that centriole amplification can also occur through the simultaneous formation of many daughters from a single mother, as it is after overexpression of SAK/PLK4 and SAS-6. These data suggest that proteolysis mediated by SCF/Slimb plays an important role in limiting the amount of SAK/PLK4, but not SAS-6, available to trigger multiple daughter formation. When SAK/PLK4 is not degraded, multiple daughter centrioles may be generated, which led to centrosome amplification. The regulation of centriole number thus emerges as a multistep mechanism, where proteolysis controls the activity of key players, SAK/PLK4 and SAS-6 (Cunha-Ferreira, 2009).

The mammalian F-box counterpart of Slimb, β-Trcp, plays a role in the DNA damage response by halting the cycle in response to genotoxic inputs. Misregulation of β-Trcp has been observed in cancer cells in which centrosome number is often increased after stress. It is thus possible that Slimb/β-Trcp coordinates checkpoints that monitor the status of DNA replication with centrosome number. The misregulation of this F-box protein would therefore result in changes in levels of SAK/PLK4, which are associated with mitotic abnormalities and oncogenesis. Given that other substrates of the Slimb/β-Trcp F-box protein require to be phosphorylated and the current results with the SAK-ND mutant, it is likely that SAK/PLK4 also requires such a modification at the serine and threonine residues in its DSGIIT degron to mark it for degradation. Future research on the identity and regulation of the kinase that phosphorylates SAK/PLK4 and on the localization of Slimb and SAK/PLK4 phosphorylated at the DSGIIT degron should indicate how different signaling events are coordinated in the cell to prevent centrosome amplification. These results open new avenues for understanding the mechanisms underlying centrosome misregulation that are of direct relevance to human disease (Cunha-Ferreira, 2009).

Regulation of autophosphorylation controls PLK4 self-destruction and centriole number

Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/betaTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/betaTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. This study shows that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/betaTrCP binding. A multisite trans-autophosphorylation mechanism is demonstrated that is likely to ensure that both a threshold of PLK4 concentration is attained, and a sequence of events is observed before PLK4 can autodestruct. First, it was shown that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, the importance was shown of PLK4-Slimb/betaTrCP regulation as it operates in both soma and germline. As betaTrCP, PLK4, and centriole number are deregulated in several cancers, this work provides novel links between centriole number control and tumorigenesis (Cunha-Ferreira, 2014).

Polo-like Kinase 4 autodestructs by generating its Slimb-binding phosphodegron

Polo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly. Previous study has found that Drosophila Plk4 protein levels are actively suppressed during interphase. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCFSlimb/betaTrCP. Since Plk4 stability depends on its activity, the consequences were examined of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Plk3 was shown to be directly responsible for extensively autophosphorylating and for generating its Slimb-binding phosphodegron (the residues that direct the starting place of degradation). Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. These data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCFSlimb. Taken together, these findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s) (Klebba, 2013).

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. This paper shows that PP2A [Protein Phosphatase 2A(Twins)] counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification (Brownlee, 2011).

Plk4 protein is maintained at near-undetectable levels for the majority of the cell cycle by ubiquitin-mediated proteolysis. The ubiquitin ligase SCF^Slimb is responsible for Plk4 degradation and recognizes an extensively phosphorylated degron situated immediately downstream of the kinase domain (KD; ~50 amino acids containing the Slimb-binding domain)KD. Slimb is appropriately positioned on centrioles throughout the cell cycle to promote rapid Plk4 destruction, but centrioles are not required for its activity. In any case, Plk4 degradation is critical in blocking all pathways of centriole amplification. Unlike other Polo kinase members, Plk4 is a homodimer capable of autophosphorylating its downstream regulatory element (DRE), a serine-rich region surrounding its SBD, in trans to promote Slimb binding. Autoregulation is a conserved feature of Plk4. Moreover, a RNAi screen of the fly kinome suggests that no other kinase is required for Plk4 degradation. The continuous and efficient degradation of Plk4 indicates that Plk4 is immediately active when expressed and that control of Plk4’s protein level is key to regulating its activity (Brownlee, 2011).

However, surprisingly little is known about the converse event: how Plk4 is activated. The results reveal the existence of a previously unknown facet of the regulation of centriole duplication, a process which transiently stabilizes and activates Plk4 specifically during mitosis. Serine/threonine phosphatases were investigated as possible effectors to counteract Plk4 autophosphorylation. PP2A is an excellent candidate to fulfill this role as it has important functions in mitosis and localizes to mitotic centrioles in cultured fly cells and centrosomes in dividing Caenorhabditis elegans embryos. A previous study found that the number of γ-tubulin foci in mitotic S2 cells was diminished after PP2A RNAi, but whether this resulted from a bona fide loss of centrioles or instead reflects a requirement for PP2A for centrosome maturation was not determined. Subsequently, a role for PP2A in centrosome maturation was identified in a genome-wide RNAi screen. The current results indicate that PP2A and the regulatory subunit Tws are required for centriole duplication by dephosphorylating and stabilizing Plk4. Without PP2A^Tws, Plk4 cannot be stabilized, and centrioles fail to duplicate. PP2A is also required for centriole assembly in C. elegans embryos but functions downstream in the centriole assembly process (Kitagawa, 2011; Song, 2011). Although the catalytic and structural PP2A subunits are abundant, regulatory subunits are needed for intracellular targeting and recognition of a myriad of substrates. Tws overexpression is sufficient to stabilize Plk4 in a dose-dependent manner, causing centriole amplification and multipolar spindle formation. Like Plk4, Tws protein levels are low during interphase but rise and peak during mitosis. Accordingly, the results suggest that PP2A^Tws stabilizes mitotic Plk4 by counteracting Plk4 autophosphorylation, enabling cells to switch Plk4 activity (and thus centriole duplication) on and off. This mechanism is inherently highly sensitive to the presence of Tws, a rate-limiting component. Moreover, this is likely a conserved mechanism because overexpression of human Tws also stabilizes fly Plk4 in S2 cells. Clearly, an important goal for future studies is to establish whether the regulation of Tws levels and cell cycle control are linked. In addition, the results suggest that up-regulation of Tws could be a means to amplify centrioles in multiciliated cells and that increased Tws activity could be a condition found in cancerous cells (Brownlee, 2011).

Centrosome amplification is a hallmark of cancer and is also observed upon expression of DNA tumor virus proteins, which include SV40 ST, human papillomavirus E7, human T cell leukemia virus type-1 Tax, hepatitis B virus oncoprotein X, and human adenovirus E1A. However, mechanisms for centrosome amplification by viral oncoproteins are not known. SV40 ST has been found to directly bind the highly conserved Drosophila catalytic and structural PP2A subunits and to induce centrosome overduplication in cultured fly cells (Kotadia, 2008). Notably, ST is a well-established PP2A inhibitor and is known to bind structural PP2A subunits, forcing endogenous PP2A regulatory subunits to be displaced and inhibiting PP2A activity. However, the current results demonstrate that ST expression does not inhibit all PP2A activities but, instead, stimulates PP2A stabilization of Plk4. This represents the first evidence that ST mimics the function of a PP2A regulatory subunit in cells. It will be important to determine whether ST targets additional PP2A substrates during tumorigenesis and whether other tumorigenic viruses (e.g., human papillomavirus and hepatitis B) known to promote centrosome amplification exploit this same mechanism. Intriguingly, human papillomavirus E7 oncoprotein binds PP2A catalytic and structural subunits and prevents PP2A from dephosphorylating Akt. Although a previous study has suggested that PP2A may function as a tumor suppressor, these findings indicate that unregulated PP2A activity leads to centriole amplification and chromosomal instability and should therefore be considered as a potential oncogenic factor (Brownlee, 2011).

Identification of a Polo-like Kinase 4-dependent pathway for de novo centriole formation

Supernumerary centrosomes are a key cause of genomic instability in cancer cells. New centrioles can be generated by duplication with a mother centriole as a platform or, in the absence of preexisting centrioles, by formation de novo. Polo-like kinase 4 (Plk4) regulates both modes of centriole biogenesis, and Plk4 deregulation has been linked to tumor development. This study shows that Plx4, the Xenopus homolog of mammalian Plk4 and Drosophila Sak, induces de novo centriole formation in vivo in activated oocytes and in egg extracts, but not in immature or in vitro matured oocytes. Both kinase activity and the polo-box domain of Plx4 are required for de novo centriole biogenesis. Polarization microscopy in 'cycling' egg extracts demonstrates that de novo centriole formation is independent of Cdk2 activity, a major difference compared to template-driven centrosome duplication that is linked to the nuclear cycle and requires cyclinA/E/Cdk2. Moreover, it was shown that the Mos-MAPK pathway blocks Plx4-dependent de novo centriole formation before fertilization, thereby ensuring paternal inheritance of the centrosome. The results define a new system for studying the biochemical and molecular basis of de novo centriole formation and centriole biogenesis in general (Eckerdt, 2011).

Revisiting the role of the mother centriole in centriole biogenesis

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. Overexpression of SAK/PLK4 can induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two proteins required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly (Rodrigues-Martins, 2007).

Centrioles are essential for the formation of cilia and flagella and for the organization of the centrosome. Normally, centrioles duplicate in coordination with the cell cycle. A new centriole, the daughter, arises orthogonally to each old one, the mother, in S phase. This led to the idea that the mother centriole templates the formation of the daughter. However, daughter centrioles do not incorporate a substantial proportion of the mother, and centrioles can also form de novo when existing centrioles are naturally lost during development or are physically removed, questioning the idea of the mother centriole as a template (Rodrigues-Martins, 2007).

SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical centriole duplication and is necessary for centriole formation. The Caenorhabditis elegans ZYG-1 kinase, a homolog of SAK, is part of a conserved module of proteins, which also includes SAS-6 and SAS-4, necessary for the normal centriole duplication cycle. ZYG-1 is an upstream regulator in that process, a role consistent with formation of multiple centrioles in cultured cells following overexpression of active SAK kinase. The generation of multiple centrioles associated with high SAK expression also occurs physiologically in the olfactory mucosa. The Drosophila egg contains all the proteins necessary to make 213 centriole pairs (centrosomes). Centrioles are naturally eliminated from the oocyte cytoplasm in the course of development and provided to the egg in the form of the basal body of the sperm. Thus, the consequences of overexpressing SAK was studied in a cytoplasm that either contained centrioles (the embryo) or lacked them (the unfertilized egg) (Rodrigues-Martins, 2007).

Embryos overexpressing SAK did not develop and were filled with free asters of microtubules not associated with spindles. Those asters were focused around Drosophila pericentrin-like protein (D-PLP)-containing structures, a centriolar and pericentriolar material (PCM) marker. These centrosomes first appeared in 15- to 30-min-old embryos and spread to fill the entire embryo after 2 to 3 hours. The observed supernumerary centrosomes led to abnormal mitotic progression and impaired embryonic development, as id observed upon microtubule depolymerization by colchicine treatment. To address the origins of those centrosomes, the very early stages of embryonic development were examined in embryos overexpressing SAK. Both the sperm aster around the incoming basal body and the first mitotic spindle were normal. However, at anaphase or telophase of the first mitosis, more than two centrosomes were observed at each pole, an indication of the onset of centrosome amplification. No other centrosomes were seen in the embryo at this stage. Moreover, it is estimated that a minimum of 3700 centrosomes (equivalent to 12 duplication cycles) were present after 60 min in embryos overexpressing SAK. After 60 min, a wild-type embryo only showed 128 centrosomes. Duplicating centrioles were observed in groups, suggesting they originated by duplication of a progenitor. Thus, upon fertilization of eggs overexpressing SAK, the basal body of the sperm enters an environment that promotes accelerated canonical duplication, overriding any existing controls that would normally couple the centrosome and chromosome cycles (Rodrigues-Martins, 2007).

Uncoupling between centrosome and chromosome cycles occurs when embryos are arrested in S-phase-like conditions. However, this did not seem to be so in this case, because proliferating cell nuclear antigen (PCNA), which appears early in S phase, was not detected in DNA of SAK-overexpressing embryos (Rodrigues-Martins, 2007).

It was next asked whether SAK could promote centriolar assembly in the absence of centrioles. Centrioles were lost normally in oocytes overexpressing SAK. Yet observations of unfertilized eggs at varying developmental intervals revealed free centrosomes in eggs overexpressing SAK that had exited meiosis II but never in wild-type eggs. Thus, in the absence of a basal body provided by the sperm, SAK can induce de novo formation of centrosomes. Whereas in embryos centrosomes appeared in a single cluster in the first mitotic spindle and spread throughout the cytoplasm, in unfertilized eggs they appeared scattered at random positions, including at the anterior and posterior poles. The formation of the first centrioles started later in eggs than in embryos [at 30 min, 0 amplification in eggs versus 51% amplification in embryos; after 1 hour, the amounts were 18% versus 89%, respectively], suggesting that centrioles take longer to be made in the absence of a template. However, once the first centrosomes had formed in eggs, their spreading in space and time was very similar to that seen in embryos, indicative of canonical biogenesis. Thus, once the first centrioles are formed de novo, they probably duplicate through the canonical pathway (Rodrigues-Martins, 2007).

There is precedent for defects in de novo-formed centrioles. The presence of SAK and two other molecules required for centriole duplication was confirmed: DSAS-6 and DSAS-4. PCM components were also detected, including γ-tubulin, centrosomin (CNN), and centrosomal protein 190 (CP190). Moreover, electron microscopy showed that centrioles in both embryos and eggs overexpressing SAK were structurally normal. It also showed the presence of procentrioles next to the completed ones in both embryos and eggs, a result suggesting that SAK-induced centrioles can duplicate (Rodrigues-Martins, 2007).

These results show that SAK is sufficient to induce both canonical and de novo centriole biogenesis. If both rely on self-assembly of the structure, the use of the same regulatory molecules would be predicted. The dependency of SAK-promoted centriole biogenesis on DSAS-4 and DSAS-6 was examined. Advantage was taken of the fact that centrioles can be eliminated from Drosophila tissue culture cells. After depletion of SAK in four rounds of RNA interference (RNAi) over a period of 16 days, more than 80% of the cells lacked centrioles, presumably because the remainder are diluted in each division cycle. Subsequent overexpression of SAK led to a clear increase in the number of cells with several centrosomes (from 4% to 48%). Depletion of DSAS-6 or DSAS-4 prevented SAK-induced centrosome biogenesis in cells with and without centrioles (Rodrigues-Martins, 2007).

These results suggest that centriole biogenesis is a template-free self-assembly process that is locally triggered and regulated by molecules such as SAK, DSAS-6, and DSAS-4. What could be the role of the mother centriole? The presence of SAK at the centriole and the fact that assembly is faster in the presence of centrioles suggest that the mother centriole is not a bona fide template but a platform for regulatory molecules, hence catalyzing and regulating daughter centriole assembly. The establishment of that platform is probably less efficient in the absence of centrioles. The mother centriole could in principle establish a temporally and spatially regulated gradient of SAK activity, as demonstrated for RanGTP, a small guanosine triphosphatase involved in spindle assembly, perhaps counteracted in the cytoplasm by other molecules. These data also point to a role for centrioles in regulating total centriole number, because their presence precludes de novo formation. This is true even in a large embryo (~800 µm) containing very large amounts of SAK. Whether this indicates sequestering of active SAK or its substrates in existing centriolar structures or an active inhibitory effect of centrioles upon de novo assembly requires further study (Rodrigues-Martins, 2007).

The regulation of SAK activity is essential in the control of centriole number and may be a parameter that is regulated according to cellular needs, because multiciliated cells of the respiratory tract have high SAK levels. The activity of SAK may be inhibited in the acentriolar female meiosis, as de novo centrosome formation only occurs after meiosis exit in eggs overexpressing SAK. Drosophila eggs and embryos should provide an ideal experimental system for further analyses of the control of centriole biogenesis and how it may go awry in cancer (Rodrigues-Martins, 2007).

Centrosome amplification can initiate tumorigenesis in flies

Centrosome amplification is a common feature of many cancer cells, and it has been proposed that centrosome amplification can drive genetic instability and so tumorigenesis. To test this hypothesis, Drosophila lines were generated that have extra centrosomes in ~60% of their somatic cells. Overexpression of the centriole replication protein SAK/PLK4 leads to the formation of extra centrosomes in cells. To analyze the consequences of centrosome amplification within the context of a developing multicellular organism, stable transformed Drosophila lines that expressed a GFP-SAK fusion protein under the control of the Ubiquitin promoter were analyzed (SAKOE cells). Many cells with extra centrosomes initially form multipolar spindles, but these spindles ultimately become bipolar. This requires a delay in mitosis that is mediated by the spindle assembly checkpoint (SAC). As a result of this delay, there is no dramatic increase in genetic instability in flies with extra centrosomes, and these flies maintain a stable diploid genome over many generations. The asymmetric division of the larval neural stem cells, however, is compromised in the presence of extra centrosomes, and larval brain cells with extra centrosomes can generate metastatic tumors when transplanted into the abdomens of wild-type hosts. Thus, centrosome amplification can initiate tumorigenesis in flies (Basto, 2008).

These observations reveal that, in vivo, the presence of extra centrosomes does not lead to large-scale genetic instability in somatic cells. As cells with extra centrosomes enter mitosis, most of the centrosomes are active and nucleate robust asters of MTs. As mitosis proceeds, however, many of the extra centrosomes become clustered together to form two dominant poles that assemble a bipolar mitotic spindle. This phenomenon of centrosome clustering has been described in several systems. In addition, however, many extra centrosomes do not become clustered at the spindle poles. Instead, these extra centrosomes appear to be gradually inactivated, and they organize less PCM and nucleate fewer MTs as mitosis proceeds. The mechanism of this inactivation is not understood, but it could result from a competition for limiting supplies of PCM components. Perhaps the centrosomes that cluster together can communally organize more PCM and so nucleate more MTs than isolated centrosomes. This would then provide a negative feedback loop as mitosis progresses so that, eventually, the isolated centrosomes are inactivated. Whatever its mechanism, the inactivation of isolated centrosomes ensures that they do not form extra spindle poles efficiently (Basto, 2008).

Cells with extra centrosomes are delayed in mitosis, and this delay is maintained by the SAC. It is possible that the presence of extra centrosomes somehow directly maintains the activity of the SAC, although previous reports suggest that this is not the case. It is suspected, therefore, that sister chromatids may be inefficiently aligned on multipolar spindles, and these improperly attached kinetochores ensure the maintenance of SAC activity until a bipolar spindle has formed (Basto, 2008).

The SAC is not normally essential for fly development, as mad2 mutant flies lack the SAC but are viable and fertile. Flies with too many centrosomes, however, completely depend on the SAC as mad2,SAKOE flies exhibit high levels of spindle multipolarity and genetic instability and do not survive to adulthood. Thus, the SAC is essential to allow enough time for cells with extra centrosomes to organize bipolar spindles. Interestingly, while SAKOE flies are severely delayed in development, the mad2,SAKOE flies develop faster than WT flies, indicating that the SAC-dependent delay in mitosis also slows the development of SAKOE flies (Basto, 2008).

It has previously been shown that centrosome clustering in cells with extra centrosomes is dependent on the activity of dynein. This study shows that another minus-end-directed motor, Ncd (HSET in vertebrates), also plays a role in this process. Ncd is not essential for Drosophila development, and ncd mutants develop at normal rates with few mitotic defects. It was found that ncd,SAKOE flies were severely delayed in development, exhibited elevated levels of spindle multipolarity during early mitosis, had a dramatically increased mitotic index, but ultimately divided in a bipolar fashion. It is concluded that Ncd enhances the efficiency of bipolar spindle formation in cells with extra centrosomes, but it is not absolutely essential, and the SAC ensures that these cells do not exit mitosis until they have formed a bipolar spindle (Basto, 2008).

The development of flies with extra centrosomes in their somatic tissues is delayed but otherwise appears to proceed normally. It is known, however, that the role of the centrosome differs between embryonic and somatic tissues in Drosophila. While somatic fly cells can tolerate the absence of centrosomes, these organelles are essential for early embryonic development in flies. Although SAKOE flies are viable and fertile, ~60% of SAKOE embryos accumulate mitotic defects and die during early embryonic development. Moreover, the mechanisms that ensure bipolar spindle formation in the presence of extra centrosomes may be absent in male germ cells; the presence of extra centrioles or centriole fragments in these cells leads to the formation of multipolar spindles and to male sterility. The overexpression of SAK does not lead to centrosome amplification in male germ cells, presumably explaining why SAKOE male flies are fertile. Interestingly, no uncoordinated behavior could be detected in flies with extra centrosomes, suggesting that cilia assembly and function are unaffected by the presence of extra centrioles (Basto, 2008).

Brain cells with extra centrosomes can form tumors when injected into WT adult flies. The pathways that lead to tumor formation are complex, and the events that initiate this process remain controversial. This work shows, however, that centrosome amplification is sufficient to initiate tumorigenesis in the fly (Basto, 2008).

It is not clear how centrosome amplification initiates tumor formation. It was originally hypothesized that extra centrosomes might promote tumorigenesis by promoting genetic instability. The nature of the link between aneuploidy and cancer, however, remains controversial. Recently, it has been shown that increased levels of aneuploidy in mouse models can promote tumor formation in certain tissues at later stages in life but suppress tumor formation upon exposure to certain carcinogens or upon the loss of particular tumor suppressor genes. Thus, aneuploidy does not invariably lead to cancer formation. In SAKOE brains the rate of aneuploidy is low, although it is higher than that observed in WT brains (1.75% compared to 0.7%, respectively). It is possible that this modest increase in aneuploidy could allow cells with extra centrosomes to initiate tumor formation in flies (Basto, 2008).

Alternatively, previous studies in flies have shown that there is a correlation between defects in the asymmetric divisions of larval neural stem cells (neuroblasts) and the ability of injected mutant brain tissue to form tumors when transplanted into WT hosts. Defective asymmetric divisions can result in the expansion of the neuroblast population, which ultimately leads to overproliferation. The asymmetric division of neuroblasts is perturbed in SAKOE brains, and this leads to an expansion of the neuroblast population -- a defect that could allow SAKOE-injected brains to overproliferate and form tumors. Indeed, there is much interest in the idea that mutations in stem cells could be central to the generation of cancer. Importantly, although the increase in aneuploidy and the defects in asymmetric division are only seen in SAKOE cells that have extra centrosomes, the possibility cannot be ruled out that SAK overexpression induces tumors via some other mechanism that is unrelated to centrosome amplification. Indeed, mice that are heterozygous for SAK have a variety of cell-cycle defects and have an increased incidence of spontaneous tumor formation (Basto, 2008).

These observations have important implications for understanding the potential link between centrosomes and cancer. In the literature it is often stated as fact that the presence of extra centrosomes in cells generates genetic instability. This assumption is based on the observation that extra centrosomes clearly lead to spindle multipolarity and genetic instability in some systems and the strong association between these two phenotypes in many cancer cells. The data demonstrate, however, that the presence of extra centrosomes does not inevitably lead to genetic instability in vivo, at least in a relatively simple organism like Drosophila. Instead, extra centrosomes are reasonably well tolerated in flies because several pathways cooperate to ensure that these cells ultimately divide in a bipolar fashion. Only when one or more of these pathways is compromised is large-scale genetic instability generated (Basto, 2008).

These findings highlight the possibility that the presence of extra centrosomes could prove to be an 'Achilles heel' for many different cancers. Fly cells with too many centrosomes are viable, but they are much more reliant on certain pathways (such as the SAC) or proteins (such as Ncd) for their survival than normal cells. It seems plausible that inhibiting these pathways in cancer patients could effectively kill the cancer cells, while leaving normal cells relatively unharmed (Basto, 2008).

Sequential protein recruitment in C. elegans centriole formation

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells. Five proteins have been identified as being essential for centriole formation in C. elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2. The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. This study established the order in which these five proteins are recruited to centrioles, and molecular epistasis experiments were conducted. SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. These results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and it is expected that the assembly pathway that stems from this work is conserved (Delattre, 2006).

Centrioles are minute cylindrical structures that contain nine sets of microtubules arranged in a radial fashion. At the onset of the centrosome duplication cycle, the two tightly apposed centrioles split slightly from one another. Each of these mother centrioles then seeds formation of a daughter centriole. Centriole formation has been described by ultrastructural analysis in vertebrate cells that primarily monitored the growth of the microtubules, which constitute the defining feature of centrioles. By contrast, the molecular tenets of this assembly process have remained elusive (Delattre, 2006).

The C. elegans embryo has proven well suited for investigating centrosome duplication. The five proteins known to be essential for centriole formation in this organism are enriched at centrioles and present at lower levels in the cytoplasm of early embryos. ZYG-1 is found at centrioles primarily during anaphase, whereas the four other proteins are centriolar throughout the cell cycle. Furthermore, SPD-2 is enriched in the PCM compared to the cytoplasm, possibly reflecting its additional role in PCM assembly (Delattre, 2006).

The order in which ZYG-1, SAS-4, SAS-5, SAS-6, and SPD-2 are recruited to centrioles was investigated. Experiments were designed that distinguish de novo centriolar recruitment from the prior presence of proteins at centrioles. Such experiments are rendered possible because the sperm contributes the sole pair of centrioles to the newly fertilized embryo. These two centrioles split slightly from one another, and each seeds the formation of a daughter centriole. Because the initial pair of centrioles is of paternal origin, one can assay specifically centriolar recruitment or exchange that occurs in the one-cell-stage embryo, provided the centrioles contributed by the sperm do not harbor the protein under scrutiny (Delattre, 2006).

Initially, SPD-2 was analyzed. Because GFP-SPD-2 is not present in sperm, in contrast to the endogenous protein, the time at which GFP-SPD-2 is first detected at centrioles was determined in one-cell-stage embryos. Double labeling was used with antibodies against SAS-4 to mark all centrioles and against GFP to detect GFP-SPD-2 recruitment. GFP-SPD-2 was first detected at centrioles during meiosis I. Even though endogenous SPD-2 is present in sperm, it is lost rapidly after fertilization in embryos depleted of SPD-2. Therefore, recruitment of the endogenous protein was studied and it was found that, as for GFP-SPD-2, SPD-2 is first detected at centrioles during meiosis I. Endogenous ZYG-1 is not present in sperm, which enabled assay of its recruitment after fertilization. ZYG-1 was also first detected at centrioles during meiosis I. In conducting these experiments, it was noted that the paternally contributed centrioles can be first distinguished a single entities during meiosis II, indicating that splitting has occurred by that time. Overall, it is concluded that SPD-2 and ZYG-1 centriolar recruitment initiates as early as meiosis I, prior to when splitting of the centriole pair can be observed by light microscopy (Delattre, 2006).

Next, SAS-5 was examined. In this case, both the endogenous protein and GFP-SAS-5 are present in sperm centrioles. Therefore, marked mating experiments were performed by crossing hermaphrodites expressing GFP-SAS-5 to wild-type males, the sperm of which provide centrioles not carrying the fusion protein. By contrast to the situation with SPD-2 and ZYG-1, it was found that GFP-SAS-5 is not present at centrioles during meiosis I. Instead, GFP-SAS-5 is first detected weakly at the end of meiosis II, with the centriolar signal becoming more robust thereafter. Because GFP-SAS-6 is not present in sperm, in contrast to the endogenous protein, it was possible to assess when the fusion protein is first recruited to centrioles after fertilization. It was found that centriolar GFP-SAS-6 is first detected shortly after meiosis II and more robustly soon thereafter, much like GFP-SAS-5. This is in line with the fact that SAS-5 and SAS-6 physically interact and are mutually dependent for their centriolar localization. Overall, it is concluded that SAS-5 and SAS-6 are recruited after SPD-2 and ZYG-1 (Delattre, 2006).

Because both SAS-4 and GFP-SAS-4 are present in sperm centrioles 3 and 4, marked mating experiments were conducted to investigate GFP-SAS-4 centriolar recruitment in one-cell-stage embryos. GFP-SAS-4 was incorporated progressively to centrioles during the first cell cycle, starting at the time of pronuclear formation. Taken together, these observations establish the following temporal sequence of recruitment to centrioles: first, SPD-2 and ZYG-1; second, SAS-5 and SAS-6; and third, SAS-4 (Delattre, 2006).

Next, whether this temporal sequence corresponds to related episatic interactions was assessed. In one extreme scenario, the five proteins could be recruited independently of one another. Alternatively, the proteins that are recruited early in the sequence may be needed for the presence of some that are recruited later. Whether SPD-2 is required for the centriolar recruitment of the other four proteins was investigated.ZYG-1, GFP-SAS-5, GFP-SAS-6, and GFP-SAS-4 all fail to be recruited to centrioles in spd-2(RNAi) embryos. Moreover, levels of SAS-4 on paternally contributed centrioles are diminished in spd-2(RNAi) embryos compared to the wild-type, as suggested by previous observations. Because SAS-4 is stably associated with the centriole in the wild-type, this indicates that SPD-2 also plays a role in maintaining SAS-4 after its incorporation into centrioles. In a converse set of experiments, it was found that SPD-2 distribution is not altered in zyg-1(RNAi), sas-5(RNAi), sas-6(RNAi), or sas-4(RNAi) embryos. Overall, it is concluded that SPD-2 controls centriolar recruitment of the four other proteins (Delattre, 2006).

ZYG-1, which is required for the presence of centriolar SAS-5 and SAS-6, which are themselves needed for GFP-SAS-4 recruitment, was investigated. In a converse set of experiments, ZYG-1 distribution was examined in embryos compromised for SAS-5, SAS-6, or SAS-4 function. In the wild-type, levels of ZYG-1 at centrioles are regulated across the cell cycle, with the signal being minimal during interphase and maximal during anaphase. ZYG-1 still localizes to centrioles in sas-5(RNAi) embryos as well as in sas-5(t2079) mutant embryos, in which SAS-5 and SAS-6 are not present at centrioles. ZYG-1 also localizes to centrioles in sas-6(RNAi) and sas-4(RNAi) embryos. Together, these observations establish that ZYG-1 acts upstream of SAS-5 and SAS-6 centriolar recruitment, which themselves act upstream of SAS-4 centriolar recruitment (Delattre, 2006).

In the course of these experiments it was discovered that ZYG-1 levels at centrioles remain high throughout the cell cycle in sas-5(t2079) mutant embryos and sas-6(RNAi) embryos. By contrast, levels of centriolar ZYG-1 still oscillate across the cell cycle in sas-4(RNAi) embryos, with the signal being minimal during interphase and maximal during anaphase. Together, these results indicate that SAS-5 and SAS-6 are required for the diminution of centriolar ZYG-1 during interphase. Because SAS-5 and SAS-6 are present in the cytoplasm but absent from centrioles in sas-5(t2079) mutant embryos, these results suggest in addition that this requirement reflects the presence or activity of centriolar SAS-5 and SAS-6 (Delattre, 2006).

It was of interest to place the recruitment of centriolar microtubules in the pathway that emerges from this study. However, their recruitment could not be assayed using GFP-β-tubulin, because the fusion protein is also incorporated in the remainder of the microtubule cytoskeleton, masking the specific centriolar signal. Therefore, the timing of centriolar microtubule recruitment relative to the five proteins discussed above is not known. Nevertheless, attempts were made to test whether astral microtubules are required for the recruitment of these proteins using RNAi against the alpha-tubulin gene tba-2 (Delattre, 2006).

In severely affected tba-2(RNAi) embryos, tubulin is detected only in paternally contributed centrioles and their immediate vicinity, as expected from the fact that RNAi does not target sperm under these experimental conditions. Interestingly, it was observed that the two paternally contributed centrioles split from one another in one-cell-stage tba-2(RNAi) embryos. Therefore, astral microtubules do not appear to be needed for centriole splitting at the onset of the duplication cycle in C. elegans embryos, as in vertebrate somatic cells. It was noted also that there are only two centrosomes in tba-2(RNAi) embryos, even after several cell cycles. In principle, these two centrosomes could each contain a pair of centrioles if just one round of centriole formation had occurred. However, since centrioles can split from one another in tba-2(RNAi) embryos, four centrosomes, each containing one centriole, would be expected in this scenario. As only two centrosomes are present, it appears instead that completion of daughter centriole formation is impaired and that each centrosome contains only one paternally contributed centriole in tba-2(RNAi) embryos. Therefore, centriole formation does not seem to occur in tba-2(RNAi) embryos. Similarly, centriole formation fails in vertebrate somatic cells treated with high doses of colcemid (Delattre, 2006).

It has been reported that GFP-SAS-5 and GFP-SAS-6 are recruited to centrioles in tba-2(RNAi) embryos. The same is true for GFP-SPD-2, as well as SPD-2, ZYG-1, and GFP-SAS-4. Although the possibility that residual tubulin contributes to the recruitment of these proteins, these results strongly suggest that SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 can all be recruited independently of astral microtubules (Delattre, 2006).

This study has provided evidence for an emerging pathway for centriole formation in C. elegans. Together with earlier work, these findings lead a proposed sequence of events. First, SPD-2 is recruited to each mother centriole or to a closely associated structure. SPD-2 is needed for the centriolar recruitment of ZYG-1, which in turn is required for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for SAS-4 to be incorporated thereafter. Furthermore, these results suggest that assembly of centriolar microtubules occurs downstream of SAS-4 incorporation or in parallel to the entire pathway. In addition, the PCM components SPD-5 and gamma-tubulin play a partially redundant role in centriole formation, and it will be interesting to investigate their placement in this sequence. Overall, it is concluded that, like other assembly processes, centriole formation can be described as a series of consecutive steps that entails the sequential recruitment of at least five proteins that ensure formation of a daughter centriole next to each mother centriole once per cell cycle (Delattre, 2006).

SPD-2 is unique among the five proteins investigated in being also required for PCM assembly. In embryos depleted of SPD-2, the coiled-coil protein SPD-5 is not recruited to centrosomes, resulting in the absence of other PCM components, including the Aurora kinase AIR-1 and gamma-tubulin. Thus, SPD-2 lies upstream in the pathway for PCM assembly. Similarly, it was found that SPD-2 is the upstream-most component among the five proteins essential for centriole formation. Therefore, SPD-2 plays a pivotal role in coordinating assembly of the two principal constituents of centrosomes. Perhaps SPD-2 acts in a manner analogous to scaffold proteins in signaling networks, which serve to localize and modulate kinases and their substrates. In this scenario, ZYG-1 and its substrates may be brought together by SPD-2 during centriole formation. spd-2 and zyg-1 exhibit a strong genetic interaction, compatible with the two components having a close relationship. Interestingly, it was discovered that SAS-5 and SAS-are needed for the diminution of centriolar ZYG-1 during interphase. Whereas it remains to be determined whether diminution of centriolar ZYG-1 is important, it is tempting to speculate that this serves as a signal ensuring that SAS-5 and SAS-6 have been recruited before further steps can take place (Delattre, 2006).

The conserved protein SZY-20 in C. elegans opposes the Plk4-related kinase ZYG-1 to limit centrosome size

Microtubules are organized by the centrosome, a dynamic organelle that exhibits changes in both size and number during the cell cycle. This study shows that SZY-20, a putative RNA-binding protein, plays a critical role in limiting centrosome size in C. elegans. SZY-20 localizes in part to centrosomes and in its absence centrosomes possess increased levels of centriolar and pericentriolar components includingγ-tubulin and the centriole duplication factors Plk4-related kinase ZYG-1 and SPD-2. These enlarged centrosomes possess normal centrioles, nucleate more microtubules, and fail to properly direct a number of microtubule-dependent processes. Depletion of ZYG-1 restores normal centrosome size and function to szy-20 mutants, whereas loss of szy-20 suppresses the centrosome duplication defects in both zyg-1 and spd-2 mutants. These results describe a pathway that determines centrosome size and implicate centriole duplication factors in this process (Song, 2008).

A key finding of this work is that loss of SZY-20 results in both expansion of the PCM and elevated levels of centrosome-associated ZYG-1. This result suggests two possible models for how SZY-20 suppresses centrosome duplication defects. First, suppression may result from concentrating ZYG-1 at centrosomes, thereby facilitating the ability of the mutant protein to execute its function. Since SPD-2 is required to recruit ZYG-1 to the site of centriole assembly, the enhanced localization of ZYG-1 may also compensate for a partial loss of SPD-2 activity. Second, suppression could be due to the expansion of PCM, which could enhance the ability of the centrosome to concentrate the depleted factors around mother centrioles. Although both models are possible, the finding that ZYG-1 is required for the expansion of PCM in szy-20 mutants argues in favor of the first model. Most likely, loss of SZY-20 results in an elevated level of centrosome-associated ZYG-1, which then enhances recruitment of downstream factors such as SAS-6 and ultimately PCM components. These results highlight the attractive interactions between centrioles and PCM; not only does the PCM attract centriole duplication factors, but as the results show, the reverse is also true (Song, 2008).

Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos

Despite being essential for proper cell division, the mechanisms governing centrosome duplication are incompletely understood and represent an important open question in cell biology. Formation of a new centriole next to each existing one is critical for centrosome duplication. In C. elegans embryos, the proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but the mechanisms underlying their requirement remain unclear. This study demonstrates that the kinase ZYG-1 phosphorylates the coiled-coil protein SAS-6 at serine 123 in vitro. This phosphorylation event is shown to be crucial for centriole formation in vivo. Furthermore, it was established that such phosphorylation ensures the maintenance of SAS-6 at the emerging centriole. Overall, these findings establish that phosphorylation of the evolutionarily conserved protein SAS-6 is critical for centriole formation and thus for faithful cell division (Kitagawa, 2009).

The centrosome is the major microtubule organizing center (MTOC) of animal cells and comprises two centrioles surrounded by pericentriolar material (PCM). Centrioles and the related basal bodies are microtubule-based structures that comprise nine microtubule blades arranged in a radial symmetric fashion. Centrioles recruit and organize the PCM from which most microtubules are nucleated. Duplication of the centrosome occurs once per cell cycle and the two resulting centrosomes assemble a bipolar spindle during mitosis. Formation of a new centriole next to each existing one is essential for centrosome duplication, but the mechanisms governing this process are incompletely understood (Kitagawa, 2009).

Time-resolved electron tomography in C. elegans embryos revealed that centriole formation begins with the assembly of a central tube, onto which microtubules are then added. The central tube is thought to be related to the cartwheel that is apparent at the onset of centriole or basal body formation in other species and that appears to impart the 9-fold radial symmetry. Forward genetic and functional genomic screens identified five proteins essential for centriole formation in C. elegans: the kinase ZYG-1 and the coiled-coil proteins SPD-2, SAS-6, SAS-5, and SAS-4. Molecular epistatic experiments indicate that SPD-2 is required for the centriolar localization of the four other proteins, whereas ZYG-1 is needed for centriolar SAS-6 and SAS-5. SAS-6 and SAS-5 physically interact and are themselves essential for SAS-4 loading onto centrioles and subsequent microtubule addition (Kitagawa, 2009 and references therein).

The ZYG-1 related Polo-like kinase 4 (Plk4, also known as SAK) is necessary for centriole formation in human cells and Drosophila. Moreover, Plk4 overexpression induces formation of multiple new centrioles in human cells, as well as amplification and de novo formation of centrioles in D. melanogaster. Although these studies established that ZYG-1/Plk4 is crucial for regulating centriole formation, the underlying mechanisms have not been identified to date (Kitagawa, 2009 and references therein).

Proteins of the SAS-6 family are invariably present in organisms with centrioles or basal bodies and are essential for their formation. Chlamydomonas reinhardtii and Tetrahymena thermophila SAS-6 homologs localize to the cartwheel, and the human protein likewise localizes to the proximal part of the new centriole. Suggestively, in addition, SAS-6 centriolar recruitment proceeds in parallel with elongation of the central tube in C. elegans. Together, these observations indicate that proteins of the SAS-6 family play a fundamental role in an early stage of centriole formation across evolution, but how they are regulated to perform this function is not known (Kitagawa, 2009 and references therein).

This work refines the working model of centriole formation in C. elegans, in particular regarding the contribution of ZYG-1 and SAS-6. An initial step leading to centriole formation entails centriolar recruitment of SAS-6, which occurs in an SAS-5-dependent manner. These findings indicate that this recruitment can occur in most embryos despite ZYG-1 depletion or the absence of SAS-6 phosphorylation at S123. Next, ZYG-1 phosphorylates SAS-6 at S123, which ensures that SAS-6 is maintained in the central tube. Thereafter, SAS-4 promotes the recruitment of microtubules to the fully formed central tube, thus completing the process of centriole formation (Kitagawa, 2009).

Since ZYG-1 localizes to centrioles just before the initial recruitment of SAS-6, a model is favored in which ZYG-1 phosphorylates SAS-6 in the vicinity of the existing centriole. However, the possibility cannot be excluded that this phosphorylation event takes place in the cytoplasm. Although ZYG-1 has been postulated to be essential for the presence of SAS-6 at centrioles, this conclusion was based primarily on examining embryos during mitosis. The present analysis reveals in addition that the recruitment of SAS-6 can occur upon ZYG-1 inactivation. Even though it cannot be formally rule out that such initial recruitment reflects residual ZYG-1 function, this is viewed as unlikely, notably because recruitment is observed not only for SAS-6 and GFP-SAS-6 upon ZYG-1 depletion, but also for GFP-SAS-6RR[S123A] upon depletion of endogenous SAS-6. Interestingly, the relationship between ZYG-1 and SAS-6 uncovered in this study in C. elegans embryos mirrors that observed between the related proteins in human cells, where HsSAS-6 is recruited, but not maintained, at centrioles in cells depleted of Plk4. This analogous relationship raises the possibility that phosphorylation of SAS-6 proteins by ZYG-1/Plk4-related kinases is an evolutionarily conserved mechanism that promotes centriole formation (Kitagawa, 2009).

Plk4-induced centriole biogenesis in human cells

Overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This has provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 (see Drosophila CP110) are required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associates only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 is recruited early and then associates with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells (Kleylein-Sohn, 2007)

Centriole biogenesis in mammalian cells remains poorly understood, but substantial progress has recently been made in invertebrate organisms. In Caenorhabditis elegans, a protein kinase, Zyg-1, and four putative structural proteins, termed SPD-2, Sas-4, Sas-5, and Sas-6, are required for centriole duplication. Moreover, through elegant epistasis experiments and electron tomography the five proteins could be shown to assemble sequentially on nascent procentrioles. Independently, the protein kinase Plk4 (also known as Sak) has been identified as a key regulator of centriole duplication in both Drosophila and human cells. Although the two kinases lack obvious sequence homology, it is plausible that Plk4 represents a functional homolog of C. elegans Zyg-1. When overexpressed in unfertilized eggs of Drosophila, Plk4 (Sak) induces the de novo formation of centrioles, demonstrating that this kinase is able to induce centriole biogenesis even in the absence of pre-existing centrioles. Homologs of nematode Sas-4 and Sas-6 were also required for centriole biogenesis in Drosophila (see Drosophila Sas-4), and a requirement for Sas-6 was demonstrated for human cells, suggesting that fundamental aspects of centriole biogenesis have most likely been conserved during evolution (Kleylein-Sohn, 2007).

Overexpression of Plk4 in human cells causes the recruitment of electron-dense material onto the proximal walls of parental centrioles, suggesting that Plk4 is able to trigger procentriole formation. This study used a cell line allowing the temporally controlled expression of Plk4 to study the formation of centrioles in human cells. Plk4 is shown to trigger the simultaneous formation of multiple procentrioles around each pre-existing centriole. These multiple centrioles form during S phase and persist as flower-like structures throughout G2 and M phase before they disperse in response to disengagement during mitotic exit, giving rise to a typical centriole amplification phenotype. Through siRNA-mediated depletion of individual centrosomal proteins, several gene products important for Plk4-controlled centriole biogenesis have been identified and assigned individual proteins to distinct steps in the assembly pathway. Finally, these functional data have been correlated with morphological analyses using immunoelectron microscopy. Taken together, these results provide a first molecular analysis of centriole formation in human cells (Kleylein-Sohn, 2007).

Stepwise evolution of the centriole-assembly pathway

The centriole and basal body (CBB) structure nucleates cilia and flagella, and is an essential component of the centrosome, underlying eukaryotic microtubule-based motility, cell division and polarity. In recent years, components of the CBB-assembly machinery have been identified, but little is known about their regulation and evolution. Given the diversity of cellular contexts encountered in eukaryotes, but the remarkable conservation of CBB morphology, it was asked whether general mechanistic principles could explain CBB assembly. The distribution of each component of the human CBB-assembly machinery was analyzed across eukaryotes as a strategy to generate testable hypotheses. It was found an evolutionarily cohesive and ancestral module, which was termed UNIMOD, is defined by three components (SAS6, SAS4/CPAP and BLD10/CEP135), that correlates with the occurrence of CBBs. Unexpectedly, other players (SAK/PLK4, SPD2/CEP192 and CP110) emerged in a taxon-specific manner. Gene duplication plays an important role in the evolution of CBB components, and, in the case of BLD10/CEP135, this is a source of tissue specificity in CBB and flagella biogenesis. Moreover, extreme protein divergence was observed among CBB components, and it was shown experimentally that there is loss of cross-species complementation among SAK/PLK4 family members, suggesting species-specific adaptations in CBB assembly. It is proposed that the UNIMOD theory explains the conservation of CBB architecture and that taxon- and tissue-specific molecular innovations, gained through emergence, duplication and divergence, play important roles in coordinating CBB biogenesis and function in different cellular contexts (Carvalho-Santos, 2010).

The conservation of the morphology of the CBB structure contrasts with the diversity of contexts in which it assembles and operates in eukaryotic life. Focusing on the phylogenetic distribution of six proteins essential for centriole assembly in humans, it was found that, in contrast to the previously observed conservation of ciliary and flagella components, CBB-assembly mechanisms evolved in a stepwise fashion. It is proposed that a subset of these proteins, which belong to what is called the universal module (UNIMOD), are necessary to define the CBB structure: the ninefold symmetry and the recruitment and tethering of centriolar microtubules. These proteins have a similar phylogenetic distribution to that previously observed for ciliary and flagella components, and it is likely that new centriole components, such as POC1, will also fall into this subset. Furthermore, the set of proteins needed to form a centriole is likely to be larger than the UNIMOD, including proteins that also have non-centriolar functions and are present in organisms that do not have CBBs, such as α- and β-tubulins and centrin. Mechanisms such as duplication with subfunctionalization of ancestral components (e.g. PLK and the BLD10/CEP135 families), divergence (e.g. SAK/PLK4) and the emergence of new genes (e.g. SPD2/CEP192 and CP110) play important roles in the evolution of CBB biogenesis. It was have shown experimentally that subfunctionalization might have played a role in CBB evolution at least twice. In the case of BLD10/CEP135, duplication and subfunctionalization with the generation of TSGA10 is likely to be important in the development of tissue-specific mechanisms of CBB assembly and flagella formation. In the case of the PLK family, the appearance of SAK/PLK4 with subfunctionalization is likely to play a role in uncoupling the regulation of CBB biogenesis from other cell-cycle events performed by PLKs. It was also shown experimentally that divergence in the PLK4 family leads to loss of cross-species complementation, which might create conditions for further development of species-specific regulation of CBB-assembly mechanisms. Finally, the emergence of novel molecules might have allowed adaptation to new contexts of assembly and new functions of the structure. The appearance in unikonts of SPD2/CEP192, a molecule whose ancestral function is thought to be in PCM recruitment, might have permitted, in animals, CBB biogenesis in contexts in which there is less PCM, such as duplication of the basal body upon fertilization. In animals, CP110 might have coupled the assembly of CBBs to the acquisition of new functions, such as cilia assembly and cytokinesis. Overall, these results strongly support the notion that the molecular machinery that defines the CBB structure is an innovation that emerged in the last eukaryotic common ancestor. This structure evolved through the emergence and divergence of new components that adapted CBB biogenesis and function to the diversification of subcellular contexts and tissue types in which they assemble and function (Carvalho-Santos, 2010).

In its evolutionary mechanisms, the CBB machinery is similar to multiprotein complexes and protein-trafficking pathways. In the former, a conserved core that presumably defines the basic function of the complex can acquire tissue- and organism-specific functions by duplication and specialization of specific components, as well as recruitment of novel interactions. The observation of heterogeneous phylogenetic distributions revealed extensive species-specific adaptations, which suggests that this study has uncovered an approach to identify novel CBB biogenesis players and functions using phylogenetic profiling. It was shown, for example, that both CP110 and CEP97, which are biochemical partners, appeared in animals. This study reveals that it is possible to extend the predictive power of evolutionary-based approaches by considering phylogenetic distributions of genes together with biological structures, and that this will be helpful in predicting both protein functions and interactions. In the future, it will be important to increase the repertoire of species whose genome is sequenced and to thoroughly describe the morphology and function of their CBBs (Carvalho-Santos, 2010).

It was surprising to observe species in which CBBs have not been described, but whose genomes contain SAS6 and SAS4: the algae Ostreococcus and the microsporidiae Encephalitozoon cuniculi and Enterocytozoon bienusi. The Ostreococcus genome also encodes orthologs of axonemal dyneins and other centriolar proteins, such as POC1. However, many flagella components are missing from the Ostreococcus genome. It is proposed that this organism might have an elusive CBB remnant, with no associated flagella, such as that described in the non-flagellated, non-sequenced green algae Kirchneriella. The significance of the presence of these proteins, although severely truncated, in the highly reduced genomes of microsporidial intracellular parasites remains to be determined. Further cell biology research in these enigmatic organisms should reveal mechanisms coupling the loss of cellular structures to the evolution of their molecular assembly machinery or alternatively unveil other functions exhibited by these proteins (Carvalho-Santos, 2010).

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).

Spindle formation in the mouse embryo requires plk4 in the absence of centrioles

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. This study demonstrates that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles (Coelho, 2013).

Structure of the C. elegans ZYG-1 cryptic polo box suggests a conserved mechanism for centriolar docking of Plk4 kinases

Plk4 family kinases control centriole assembly. Plk4s target mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or Asterless/Cep152. This study reports a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular beta sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a different crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over Asterless-derived acidic regions. Overall, these findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly (Shimanovskaya, 2014).

Promotion and suppression of centriole duplication are catalytically coupled through PLK4 to ensure centriole homeostasis

PLK4 (see Drosophila Sak kinase) is the major kinase driving centriole duplication. Duplication occurs only once per cell cycle, forming one new (or daughter) centriole that is tightly engaged to the preexisting (or mother) centriole. Centriole engagement is known to block the reduplication of mother centrioles, but the molecular identity responsible for the block remains unclear. This study shows, using mammalian retinal pigment epithelial cell lines, that the centriolar cartwheel, the geometric scaffold for centriole assembly, forms the identity of daughter centrioles essential for the block, ceasing further duplication of the mother centriole to which it is engaged. To ensure a steady block, the cartwheel was shown to require constant maintenance by PLK4 through phosphorylation of the same substrate that drives centriole assembly. These results support a model that the cartwheel-bound PLK4 directly suppresses centriole reduplication (Kim, 2016).

Protein phosphatase 1 down regulates ZYG-1 levels to limit centriole duplication

In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1 (see Drosophila SAK), is required for centriole duplication, but the understanding of how ZYG-1 levels are regulated remains incomplete. This study identified the two PP1 orthologs, GSP-1 (see Drosophila flw) and GSP-2 (see Drosophila Pp1-87B), and their regulators I-2SZY-2 (see Drosophila I-2) and SDS-22 (see Drosophila sds22) as key regulators of ZYG-1 protein levels. Down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication (see Drosophila centrioles) associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. These results thus define a new pathway that limits the number of daughter centrioles produced each cycle (Peel, 2017).

Search PubMed for articles about Drosophila Sak

Aydogan, M. G., Wainman, A., Saurya, S., Steinacker, T. L., Caballe, A., Novak, Z. A., Baumbach, J., Muschalik, N. and Raff, J. W. (2018). A homeostatic clock sets daughter centriole size in flies. J Cell Biol. PubMed ID: 29500190

Aydogan, M. G., Hankins, L. E., Steinacker, T. L., Mofatteh, M., Saurya, S., Wainman, A., Wong, S. S., Lu, X., Zhou, F. Y. and Raff, J. W. (2022). Centriole distal-end proteins CP110 and Cep97 influence centriole cartwheel growth at the proximal-end. J Cell Sci. PubMed ID: 35707992

Basto, R., et al. (2008). Centrosome amplification can initiate tumorigenesis in flies. Cell 133: 1032-1042. PubMed ID: 18555779

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

Brownlee, C. W., Klebba, J. E., Buster, D. W. and Rogers, G. C. (2011). The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification. J. Cell Biol. 195(2): 231-43. PubMed ID: 21987638

Carvalho-Santos, Z., et al. (2010). Stepwise evolution of the centriole-assembly pathway. J. Cell Sci. 123(Pt 9): 1414-26. PubMed ID: 20392737

Coelho, P. A., Bury, L., Sharif, B., Riparbelli, M. G., Fu, J., Callaini, G., Glover, D. M. and Zernicka-Goetz, M. (2013). Spindle formation in the mouse embryo requires plk4 in the absence of centrioles. Dev Cell 27: 586-597. PubMed ID: 24268700

Cunha-Ferreira, I., et al. (2009). The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/PLK4. Curr. Biol. 19(1): 43-9. PubMed ID: 19084407

Cunha-Ferreira, I., Bento, I., Pimenta-Marques, A., Jana, S. C., Lince-Faria, M., Duarte, P., Borrego-Pinto, J., Gilberto, S., Amado, T., Brito, D., Rodrigues-Martins, A., Debski, J., Dzhindzhev, N. and Bettencourt-Dias, M. (2013). Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number. Curr Biol 23: 2245-2254. PubMed ID: 24184099

Cunha-Ferreira, I., Bento, I., Pimenta-Marques, A., Jana, S. C., Lince-Faria, M., Duarte, P., Borrego-Pinto, J., Gilberto, S., Amado, T., Brito, D., Rodrigues-Martins, A., Debski, J., Dzhindzhev, N. and Bettencourt-Dias, M. (2013). Regulation of autophosphorylation controls PLK4 self-destruction and centriole number. Curr Biol 23: 2245-2254. PubMed ID: 24184099

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

Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Schneider, S., Lattao, R., Fu, J., Debski, J., Dadlez, M. and Glover, D. M. (2014). Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation. Curr Biol 24(21):2526-32. PubMed ID: 25264260

Eckerdt, F., et al. (2011). Identification of a Polo-like Kinase 4-dependent pathway for de novo centriole formation. Curr. Biol. 21: 428-432. PubMed ID: 21353560

Elia, A. E., et al. (2003). The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 115(1): 83-95. PubMed ID: 14532005

Fong, C. S., Kim, M., Yang, T. T., Liao, J. C. and Tsou, M. F. (2014). SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication. Dev Cell 30: 238-245. PubMed ID: 25017693

Habedanck, R., et al. (2005). The Polo kinase Plk4 functions in centriole duplication. Nat. Cell Biol. 7: 1140-1146. PubMed ID: 16244668

Hudson, J. W., et al. (2001). Late mitotic failure in mice lacking Sak, a polo-like kinase. Curr. Biol. 11(6): 441-6. PubMed ID: 11301255

Kim, M., O'Rourke, B. P., Soni, R. K., Jallepalli, P. V., Hendrickson, R. C. and Tsou, M. B. (2016). Promotion and suppression of centriole duplication are catalytically coupled through PLK4 to ensure centriole homeostasis. Cell Rep. PubMed ID: 27425613

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

Kitagawa, D., Busso, C., Flückiger, I., Gönczy, P. (2009). Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos. Dev. Cell 17(6): 900-7. PubMed ID: 20059959

Kitagawa, D., et al. (2011). PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos. Dev. Cell. 20: 550-562. PubMed ID: 21497765

Klebba, J. E., Buster, D. W., Nguyen, A. L., Swatkoski, S., Gucek, M., Rusan, N. M. and Rogers, G. C. (2013). Polo-like Kinase 4 autodestructs by generating its Slimb-binding phosphodegron. Curr Biol. 23(22): 2255-61. PubMed ID: 24184097

Kleylein-Sohn, J., et al. (2007). Plk4-induced centriole biogenesis in human cells. Dev. Cell 13(2): 190-202. PubMed ID: 17681131

Ko, M. A., et al. (2005). Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis. Nat. Genet. 37(8): 883-8. PubMed ID: 16025114

Kotadia, S., et al. (2008). PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen. Oncogene 27: 6334-6346. PubMed ID: 18663356

Leidel, S., Delattre, M., Cerutti, L., Baumer, K. and Gonczy, P. (2005). SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells. Nat. Cell Biol. 7(2): 115-25. PubMed ID: 15665853

Leung, G. C., et al. (2002). The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization. Nat. Struct. Biol. 9(10): 719-24. PubMed ID: 12352953

Lowery, D. M., Lim, D. and Yaffe, M. B. (2005). Structure and function of Polo-like kinases. Oncogene 24(2): 248-59. PubMed ID: 15640840

Martinez-Campos, M., Basto, R., Baker, J., Kernan, M. and Raff, J. W. (2004). The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis. J. Cell Biol. 165(5): 673-83. PubMed ID: 15184400

Novak, Z. A., Conduit, P. T., Wainman, A. and Raff, J. W. (2014). Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos. Curr Biol 24(11): 1276-82. PubMed ID: 24835456

O'Connell, K. F., et al. (2001). The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo. Cell 105(4): 547-58. PubMed ID: 11371350

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(10): 834-43. PubMed ID: 17475495

Peel, N., Iyer, J., Naik, A., Dougherty, M.P., Decker, M. and O'Connell, K.F. (2017). Protein phosphatase 1 down regulates ZYG-1 levels to limit centriole duplication. PLoS Genet [Epub ahead of print]. PubMed ID: 28103229

Rodrigues-Martins, A., Riparbelli, M., Callaini, G., Glover, D. M., and Bettencourt-Dias, M. (2007). Revisiting the role of the mother centriole in centriole biogenesis. Science 316: 1046-1050. PubMed ID: 17463247

Shimanovskaya, E., Viscardi, V., Lesigang, J., Lettman, M. M., Qiao, R., Svergun, D. I., Round, A., Oegema, K. and Dong, G. (2014). Structure of the C. elegans ZYG-1 cryptic polo box suggests a conserved mechanism for centriolar docking of Plk4 kinases. Structure. PubMed ID: 24980795

Song, M. H., Aravind, L. Müller-Reichert, T. and O'Connell, K. F. (2008). The conserved protein SZY-20 opposes the Plk4-related kinase ZYG-1 to limit centrosome size. Dev. Cell 15: 901-912. PubMed ID: 19081077

Song, M. H., et al. (2011). Protein phosphatase 2A-SUR-6/B55 regulates centriole duplication in C. elegans by controlling the levels of centriole assembly factors. Dev. Cell. 20: 563-571. PubMed ID: 21497766

Swallow, C. J., et al. (2005). Sak/Plk4 and mitotic fidelity. Oncogene 24(2): 306-12. PubMed ID: 15640847

Vidwans, S. J., Wong, M. L. and O'Farrell, P. H. (2003). Anomalous centriole configurations are detected in Drosophila wing disc cells upon Cdk1 inactivation. J. Cell Sci. 116: 137-143. PubMed ID: 12456723

date revised: 5 August 2023

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

The Interactive Fly resides on the
Society for Developmental Biology's Web server.