InteractiveFly: GeneBrief

asterless: Biological Overview | References

BIOLOGICAL OVERVIEW

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A molecular mechanism of mitotic centrosome assembly in Drosophila

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

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

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

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

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

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

Drosophila asterless and vertebrate Cep152 are orthologs essential for centriole duplication

The centriole is the core structure of centrosome and cilium. Failure to restrict centriole duplication to once per cell cycle has serious consequences and is commonly observed in cancer. Despite its medical importance, the mechanism of centriole formation is poorly understood. Asl is a centrosomal protein essential for centrosome function. This study identify mecD, a severe loss-of-function allele of the asl gene. asl is shown to be required for centriole and cilia formation. Similarly, Cep152, the Asl ortholog in vertebrates, is essential for cilia formation and its function can be partially rescued by the Drosophila Asl. The study of Asl localization suggests that it is closely associated with the centriole wall, but is not part of the centriole structure. By analyzing the biogenesis of centrosomes in cells depleted of Asl, it was found that, while pericentriolar material (PCM) function is mildly affected, Asl is essential for daughter centriole formation. The clear absence of several centriolar markers in mecD mutants suggests that Asl is critical early in centriole duplication (Blachon, 2008. Full text of article).

In addition to their role in cellular division, centrioles transform into basal bodies that give rise to cilia. In flies, cilia perform mechanosensory or chemosensory functions in neurons and a motile function as flagella. To identify genes that function in centriole and cilium formation, a collection of ~600 potential candidate mechanosensory mutants was screened. The mechanosensory mutant D (mecD) exhibited all of the characteristics of a ciliary defect with a severe phenotype of uncoordination and nonmotile sperm tails. The absence of cilia was confirmed by serial-section electron microscopy (EM) of the sensory neurons and the sperm tails. In wild-type sensory neurons, the tip of the cilium had a tubular body and a pair of basal bodies at the base of the sensory cilium. The basal bodies and sensory cilium were missing in mecD. Spermatid tails were still formed in mecD but contained abnormal mitochondrial derivatives and the axonemes were missing completely. Analysis using light microscopy showed that in control spermatids each nucleus is associated with a basal body ;GFP-PACT (pericentrin/AKAP450 centrosomal targeting) and that in mecD spermatids no basal bodies were found and the tissue is disorganized. These findings firmly implicate mecD in cilia and basal bodies biogenesis (Blachon, 2008).

This article identified a new allele of the asl gene. Asl is involved in the initiation of centriole duplication and it is dispensable for PCM recruitment in meiosis in maternally contributed centrioles. Asl is essential for centriole formation in brain cells and spermatocytes. Without centrioles, cells do not have basal bodies and cannot grow cilia, which is the cause of the adult lethality of these animals. Similar results are observed in zebrafish, indicating that Asl function in centriole duplication is conserved in animals. The absence of any intermediate centriolar structure in EM, plus the lack of centriolar markers such as Sas6, which is known to be involved in the early steps of centriole duplication, strongly suggests that Asl acts very early in the process. The possibility was excluded that the failure in centriole duplication is due to growth abnormalities of the mother centriole since it is surrounded by functional PCM and is able to elongate. Asl is closely associated with the centriole wall and could be part of the PCM tube. EM studies show that procentriole formation occurs at 30 nm from the mother centriole wall, which correlates with the localization of Asl. Therefore it will be interesting in the future to see if the role of Asl and the PCM tube is to anchor and stabilize the daughter centriole nucleation site. This would be a simple mechanism to ensure that centriole duplication occurs in the vicinity of the mother centriole (Blachon, 2008).

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).

Drosophila Ana2 is a conserved centriole duplication factor.

In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP-orthologues of ZYG-1, SAS-6, and SAS-4, respectively-are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. This study finds that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. This study shows that Anastral spindle 2 (Ana2) is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. It is proposed that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery (Stevens, 2010).

The centriole is composed of a radial array of nine microtubule (MT) triplets, doublets, or singlets depending on species and cell type. Centrioles are required to make two important cellular structures: centrosomes and cilia. The centrosome consists of a pair of centrioles surrounded by pericentriolar material (PCM) and is the major MT organizing center in many animal cells. Cilia are formed when the centriole pair migrates to the cell cortex, and the older, mother, centriole forms a basal body that nucleates the ciliary axoneme. Many different cell types possess cilia, and they have multiple roles in development (Stevens, 2010).

To ensure their inheritance by each daughter cell, centrioles duplicate precisely once per cell cycle. This process must be tightly regulated. Failure in centriole duplication leads to catastrophic errors during embryogenesis in both worms and flies, and an increasing number of human diseases have been linked to defects in centrosome and/or cilia function. Centriole overduplication can be equally damaging, as excess centrioles are frequently observed in human tumors, and there appears to be a direct causative relationship between centriole overduplication and tumorigenesis in flies (Stevens, 2010).

In canonical centriole duplication, a new daughter centriole grows at a right angle to the mother centriole. A series of genome-wide RNAi and genetic screens in worms have found just five proteins essential for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 . SPD-2 is required to recruit the kinase ZYG-1 to the centriole, and both proteins then recruit a complex of SAS-5 and SAS-6. SAS-5 and SAS-6 are mutually dependent for their centriolar localization and are in turn needed to recruit SAS-4 (Stevens, 2010 and references therein).

Although DSpd-2 is not essential for centriole duplication in flies, and no SAS-5 homologues have been identified outside worms, proteins related to ZYG-1, SAS-6, and SAS-4 have a conserved role in centriole duplication in other systems. In Drosophila, for example, the kinase Sak, which is related to ZYG-1, and the homologues of SAS-6 (DSas-6) and SAS-4 (DSas-4) are required for centriole duplication. Recently, however, several additional proteins have been identified in cultured fly cells that are potentially involved in centriole duplication. This study set out to identify which of these potential duplication factors are likely to function as upstream regulators of centriole formation (Stevens, 2010).

Genome-wide RNAi screens in cultured fly cells identified just 18 proteins that, when depleted, gave a reduced number of centrioles (Goshima, 2007; Dobbelaere, 2008). This list includes Sak, DSas-6, and DSas-4, as well as eight other proteins that specifically localize to centrosomes (Ana1, Ana2, spermatogenesis. Ultrastructural analysis showed that asl mutants have morphologically normal centrioles. However, immunostaining with antibodies directed either to γ tubulin or centrosomin revealed that these proteins do not accumulate in the centrosomes, as occurs in wild-type. Thus, asl appears to specify a function required for the assembly of centrosomal material around the centrioles (Bonaccorsi, 1998).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

asterless mutant neuroblasts undergo normal division

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

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

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

Search PubMed for articles about Drosophila Asterless

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

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

Blachon, S., et al. (2008). Drosophila asterless and vertebrate Cep152: Are orthologs essential for centriole duplication? Genetics 180: 2081-2094. PubMed ID: 18854586

Blachon. S., et al. (2009). A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. Genetics 182: 133-144. PubMed ID: 19293139

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

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

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

Dobbelaere, J., et al. (2008). A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila. PLoS Biol. 6: e224. PubMed ID: 18798690

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

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

Izraeli, S., et al. (1999). The SIL gene is required for mouse embryonic axial development and left-right specification. Nature 399: 691-694. PubMed ID: 10385121

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

Kumar, A., Girimaji, S. C., Duvvari, M. R. and Blanton, S. H. (2009). Mutations in STIL, encoding a pericentriolar and centrosomal protein, cause primary microcephaly. Am. J. Hum. Genet. 84: 286-290. PubMed ID: 19215732

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

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

Pfaff, K. L., et al. (2007). The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization. Mol. Cell. Biol. 27: 5887-5897. PubMed ID: 17576815

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

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

Stevens, N. R., Dobbelaere, J., Brunk, K., Franz, A. and Raff, J. W. (2010). Drosophila Ana2 is a conserved centriole duplication factor. J. Cell Biol. 188(3): 313-23. PubMed ID: 20123993

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

date revised: 13 October 2014

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