Centrosome Duplication and Nematodes: Recent Insights from an Old Relationship
2005; Elsevier BV; Volume: 9; Issue: 3 Linguagem: Inglês
10.1016/j.devcel.2005.08.004
ISSN1878-1551
AutoresSebastian A. Leidel, Pierre Gönczy,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoCentrosome duplication is required for proper cell division, and centriole formation is a key step in this process. This review discusses recent studies in C. elegans that have identified five core proteins required for centriole formation, thus shedding light into the mechanisms underlying centrosome duplication in nematodes and beyond. Centrosome duplication is required for proper cell division, and centriole formation is a key step in this process. This review discusses recent studies in C. elegans that have identified five core proteins required for centriole formation, thus shedding light into the mechanisms underlying centrosome duplication in nematodes and beyond. Centrosomes and nematodes have a long-standing relationship. Although centrosomes were initially spotted by Flemming in a fresh-water mussel (Flemming, 1875Flemming W. Studien über die Entwicklungsgeschichte der Najaden.Sitzungsgeber Akad Wissensch Wien. 1875; 71: 81-147Google Scholar), the pioneering studies of Van Beneden and Boveri were carried out in embryos of the parasitic nematode Parascaris equorum (Boveri, 1887Boveri, T. (1887). Ueber die Befruchtung der Eier von Ascaris megalocephala. Sitz-Ber. Ges. Morph. Phys. München 3.Google Scholar, Van Beneden and Neyt, 1887Van Beneden E. Neyt A. Nouvelles recherches sur la fécondation et la division mitosique chez l'Ascaride mégalocéphale.Bull. Acad. Roy. Sci. Belg. 1887; 14: 215-295Google Scholar). Thereafter, the focus of centrosome research shifted to other experimental systems for well over a century. It is only in recent years that Caenorhabditis elegans has brought back the nematode phylum to the limelight of centrosome biology. In this review, we discuss how these studies in C. elegans identified five proteins, the kinase ZYG-1 as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2, that are required for the process of centriole formation, which is key for duplication of the entire centrosome. A pair of centrioles constitutes the core of the centrosome. Centrioles are barrel-shaped microtubule-based structures that, in vertebrate cells, are ∼175 nm in diameter and ∼400 nm in length (Chretien et al., 1997Chretien D. Buendia B. Fuller S.D. Karsenti E. Reconstruction of the centrosome cycle from cryoelectron micrographs.J. Struct. Biol. 1997; 120: 117-133Crossref PubMed Scopus (104) Google Scholar, Kuriyama and Borisy, 1981Kuriyama R. Borisy G.G. Centriole cycle in Chinese hamster ovary cells as determined by whole-mount electron microscopy.J. Cell Biol. 1981; 91: 814-821Crossref PubMed Scopus (194) Google Scholar, Paintrand et al., 1992Paintrand M. Moudjou M. Delacroix H. Bornens M. Centrosome organization and centriole architecture: their sensitivity to divalent cations.J. Struct. Biol. 1992; 108: 107-128Crossref PubMed Scopus (271) Google Scholar, Vorobjev and Chentsov, 1982Vorobjev I.A. Chentsov Y.S. Centrioles in the cell cycle. I. Epithelial cells.J. Cell Biol. 1982; 98: 938-949Crossref Scopus (266) Google Scholar). Centrioles are composed of stable microtubule arrays organized in a 9-fold radial symmetry, a structure also found in basal bodies, with which centrioles can interconvert, for example, in ciliated epithelial cells (reviewed in Mogensen, 2004Mogensen M. Microtubule organizing centers in polarized epithelial cells.in: Nigg E.A. Centrosomes in Developement and Disease. Wiley-VCH, Weinheim, Germany2004: 299-319Google Scholar). Centriolar microtubule arrays usually consist of triplet microtubules, although doublets or singlets are present in some species (reviewed in Delattre and Gönczy, 2004Delattre M. Gönczy P. The arithmetic of centrosome biogenesis.J. Cell Sci. 2004; 117: 1619-1630Crossref PubMed Scopus (120) Google Scholar). Moreover, mature centrioles in vertebrate cells harbor conspicuous appendages that are thought to be important for microtubule anchoring (Chretien et al., 1997Chretien D. Buendia B. Fuller S.D. Karsenti E. Reconstruction of the centrosome cycle from cryoelectron micrographs.J. Struct. Biol. 1997; 120: 117-133Crossref PubMed Scopus (104) Google Scholar, Mogensen et al., 2000Mogensen M.M. Malik A. Piel M. Bouckson-Castaing V. Bornens M. Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein.J. Cell Sci. 2000; 113: 3013-3023PubMed Google Scholar). The pair of centrioles is embedded in the pericentriolar material (PCM), thus, forming a centrosome. Whereas the function of many PCM proteins remains to be elucidated, an important PCM constituent is the γ-TuRC complex (Gunawardane et al., 2000Gunawardane R.N. Lizarraga S.B. Wiese C. Wilde A. Zheng Y. γ-tubulin complexes and their role in microtubule nucleation.Curr. Top. Dev. Biol. 2000; 49: 55-73Crossref PubMed Google Scholar). As this complex promotes microtubule nucleation, the centrosome is the principal microtubule organizing center (MTOC) of most animal cells. Consequently, it is required for microtubule-based cellular processes, such as polarized secretion or cell division. Centrosome number is carefully coordinated with other cell cycle events. In proliferating cells, there is a single centrosome early in the cell cycle, and this centrosome duplicates during S phase to yield two centrosomes before M phase. The two centrosomes lead to bipolar spindle assembly during mitosis, which ensures faithful segregation of sister chromatids to daughter cells, which also each inherit one centrosome. The centrosome duplication cycle has been described by ultrastructural analysis conducted primarily in vertebrate tissue culture cells (Chretien et al., 1997Chretien D. Buendia B. Fuller S.D. Karsenti E. Reconstruction of the centrosome cycle from cryoelectron micrographs.J. Struct. Biol. 1997; 120: 117-133Crossref PubMed Scopus (104) Google Scholar, Kuriyama and Borisy, 1981Kuriyama R. Borisy G.G. Centriole cycle in Chinese hamster ovary cells as determined by whole-mount electron microscopy.J. Cell Biol. 1981; 91: 814-821Crossref PubMed Scopus (194) Google Scholar, Paintrand et al., 1992Paintrand M. Moudjou M. Delacroix H. Bornens M. Centrosome organization and centriole architecture: their sensitivity to divalent cations.J. Struct. Biol. 1992; 108: 107-128Crossref PubMed Scopus (271) Google Scholar, Vorobjev and Chentsov, 1982Vorobjev I.A. Chentsov Y.S. Centrioles in the cell cycle. I. Epithelial cells.J. Cell Biol. 1982; 98: 938-949Crossref Scopus (266) Google Scholar). First, the two centrioles lose their characteristic orthogonal arrangement and move slightly away from each other. Second, a small procentriole forms perpendicular to each original centriole, which is often referred to as the mother centriole. Subsequently, the procentriole elongates and forms a complete centriole, which is often referred to as the daughter centriole. Next, the PCM splits, and the two resulting centrosomes, each containing one mother and one daughter centriole, separate. While the above-described sequence is typical, formation of centrioles differs in some cases, including in early mouse embryos or in tissue culture cells following laser ablation of the centrosome; in these cases, centrioles form de novo in the absence of a preexisting one (Khodjakov et al., 2002Khodjakov A. Rieder C.L. Sluder G. Cassels G. Sibon O. Wang C.L. De novo formation of centrosomes in vertebrate cells arrested during S phase.J. Cell Biol. 2002; 158: 1171-1181Crossref PubMed Scopus (155) Google Scholar, La Terra et al., 2005La Terra S. English C.N. Hergert P. McEwen B.F. Sluder G. Khodjakov A. The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation.J. Cell Biol. 2005; 168: 713-722Crossref PubMed Scopus (143) Google Scholar, Szollosi et al., 1972Szollosi D. Calarco P. Donahue R.P. Absence of centrioles in the first and second meiotic spindles of mouse oocytes.J. Cell Sci. 1972; 11: 521-541PubMed Google Scholar). However, in most proliferating cells, the canonical centrosome duplication cycle prevails, with assembly of a procentriole and a daughter centriole (hereafter referred to collectively as "centriole formation") being central to this cycle. In contrast to the wealth of ultrastructural information, the mechanisms governing the centrosome duplication cycle remain incompletely understood. Work in frog egg extracts and vertebrate cells indicates that the cyclin-dependent kinase Cdk2 plays a central role in coupling the onset of DNA replication with that of centrosome duplication (Hinchcliffe et al., 1999Hinchcliffe E.H. Li C. Thompson E.A. Maller J.L. Sluder G. Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts.Science. 1999; 283: 851-854Crossref PubMed Scopus (431) Google Scholar, Matsumoto et al., 1999Matsumoto Y. Hayashi K. Nishida E. Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells.Curr. Biol. 1999; 9: 429-432Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, Meraldi et al., 1999Meraldi P. Lukas J. Fry A.M. Bartek J. Nigg E.A. Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A.Nat. Cell Biol. 1999; 1: 88-93Crossref PubMed Scopus (415) Google Scholar). Whether a cyclin-dependent kinase similarly ensures this coupling in C. elegans embryos is not known. In vertebrate cells, several Cdk2 substrates, including nucleophosmin and CP110 (Chen et al., 2002Chen Z. Indjeian V.B. McManus M. Wang L. Dynlacht B.D. CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells.Dev. Cell. 2002; 3: 339-350Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Okuda et al., 2000Okuda M. Horn H.F. Tarapore P. Tokuyama Y. Smulian A.G. Chan P.K. Knudsen E.S. Hofmann I.A. Snyder J.D. Bove K.E. Fukasawa K. Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication.Cell. 2000; 103: 127-140Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar), have been proposed to be central to this coupling. Kinases, including Mps1, as well as the Polo-like kinases Plk1 and Plk2, also appear to be important, as interfering with their activity by using siRNAs or dominant-negative contstructs prevents centriole formation in vertebrate cells (Fisk and Winey, 2001Fisk H.A. Winey M. The mouse Mps1p-like kinase regulates centrosome duplication.Cell. 2001; 106: 95-104Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Liu and Erikson, 2002Liu X. Erikson R.L. Activation of Cdc2/cyclin B and inhibition of centrosome amplification in cells depleted of Plk1 by siRNA.Proc. Natl. Acad. Sci. USA. 2002; 99: 8672-8676Crossref PubMed Scopus (179) Google Scholar, Warnke et al., 2004Warnke S. Kemmler S. Hames R.S. Tsai H.L. Hoffmann-Rohrer U. Fry A.M. Hoffmann I. Polo-like kinase-2 is required for centriole duplication in mammalian cells.Curr. Biol. 2004; 14: 1200-1207Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). While these findings shed some light on the coupling between cell cycle progression and centrosome duplication, knowledge about the structural basis of centriole formation remains scarce. This is due in part to the paucity of proteins identified as being directly required for this step of the duplication cycle. One interesting player in this respect is the divergent tubulin isoform ϵ-tubulin, which localizes to the appendages of mature centrioles, and whose immunodepletion from frog egg extracts likely prevents centriole formation (Chang et al., 2003Chang P. Giddings Jr., T.H. Winey M. Stearns T. ϵ-tubulin is required for centriole duplication and microtubule organization.Nat. Cell Biol. 2003; 5: 71-76Crossref PubMed Scopus (102) Google Scholar). In Chlamydomonas reinhardtii, analysis by electron microscopy established that basal body structure is perturbed in the absence of ϵ-tubulin (Dupuis-Williams et al., 2002Dupuis-Williams P. Fleury-Aubusson A. de Loubresse N.G. Geoffroy H. Vayssie L. Galvani A. Espigat A. Rossier J. Functional role of ϵ-tubulin in the assembly of the centriolar microtubule scaffold.J. Cell Biol. 2002; 158: 1183-1193Crossref PubMed Scopus (56) Google Scholar). Another protein family of interest is that of the calcium binding protein centrin. RNAi-mediated inactivation of centrin-2 prevents centriole formation in HeLa cells (Salisbury et al., 2002Salisbury J.L. Suino K.M. Busby R. Springett M. Centrin-2 is required for centriole duplication in mammalian cells.Curr. Biol. 2002; 12: 1287-1292Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), whereas mutations in the Saccharomyces cerevisiae centrin homolog Cdc31p prevent duplication of the spindle pole body, the MTOC of yeast cells (Baum et al., 1986Baum P. Furlong C. Byers B. Yeast gene required for spindle pole body duplication: homology of its product with Ca2+-binding proteins.Proc. Natl. Acad. Sci. USA. 1986; 83: 5512-5516Crossref PubMed Scopus (252) Google Scholar). Cdc31p binds a protein called Sfi1p, and Sfi1p/Cdc31p may form filaments conferring elastic properties to replicating spindle pole bodies (Kilmartin, 2003Kilmartin J.V. Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication.J. Cell Biol. 2003; 162: 1211-1221Crossref PubMed Scopus (144) Google Scholar). There is a human Sfi1p homolog, but it remains to be determined whether it interacts with centrin-2 and is needed for centriole formation. The above-described findings notwithstanding, the mechanisms governing centriole formation remain poorly understood. There are a number of important open questions. What ensures that each mother centriole gives rise to only one daughter centriole at each cell cycle? Cell fusion experiments indicate that human G2 cells possess a centrosome-intrisic mechanism that prevents formation of additional centrosomes, but the underlying molecular tenets remain to be identified (Wong and Stearns, 2003Wong C. Stearns T. Dispatch. Centrosome biology: a SAS-sy centriole in the cell cycle.Curr. Biol. 2003; 13: R351-R352Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). What determines the invariable location on the mother centriole from which the procentriole grows? Perhaps there is a seed that, albeit not apparent by electron microscopy, serves as a mark for procentriole formation. And, what limits the extent of centriolar growth? Centrioles can form in as little as 4 min in Spisula solidissima egg extracts (Palazzo et al., 1992Palazzo R.E. Vaisberg E. Cole R.W. Rieder C.L. Centriole duplication in lysates of Spisula solidissima oocytes.Science. 1992; 256: 219-221Crossref PubMed Scopus (83) Google Scholar), and microtubule polymerization rates should enable centrioles to reach several microns given the duration of S phase in most cell types. Nevertheless, daughter centriole growth stops when the size of the mother centriole is reached. How is this achieved? While the recent findings in C. elegans have not yet addressed these important questions, they have led to the identification of five proteins that may eventually provide some of the answers. Two main concerns have been evoked when considering whether findings that pertain to centriole formation in C. elegans are generally applicable (discussed in Azimsadeh and Bornens, 2004Azimsadeh J. Bornens M. The centrosome in evolution.in: Nigg E.A. Centrosomes in Development and Disease. Wiley-VCH, Weinheim, Germany2004: 93-122Google Scholar). First, centrioles in C. elegans embryos are atypical in several aspects: they are ∼200 nm long and thus shorter than those in vertebrate cells, they are comprised of microtubule singlets, and they do not exhibit prominent appendages (O'Toole et al., 2003O'Toole E.T. McDonald K.L. Mantler J. McIntosh J.R. Hyman A.A. Muller-Reichert T. Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans.J. Cell Biol. 2003; 163: 451-456Crossref PubMed Scopus (117) Google Scholar). Such structural divergences may be accompanied by variations in the mechanisms underlying centriole formation. A second concern is that several proteins important for centriole formation in other species, including ϵ-tubulin and centrin, are not present in the C. elegans proteome. Despite these considerations, C. elegans has emerged in recent years as an attractive model system in which to identify components essential for centriole formation. The one-cell-stage embryo is ∼50 μm long, which allows for an analysis of centrosome duplication in live specimens with high spatial and temporal resolution. In contrast to what is seen in vertebrate cells, there is a clear size difference between centrioles and the PCM in C. elegans embryos, enabling one to readily determine by immunofluorescence whether a protein localizes to centrioles or the PCM (compare Figures 1A and 1C ). Importantly, powerful genetic and functional genomic tools are available in C. elegans. In particular, RNAi-mediated inactivation permits the systematic identification of genes required for cell division processes. Genes required specifically for centrosome duplication can be identified, because interference with S phase progression results in phenotypic manifestations that are distinguishable from centrosome duplication failure. In addition, cell cycle checkpoints are relaxed in early C. elegans embryos (Brauchle et al., 2003Brauchle M. Baumer K. Gönczy P. Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos.Curr. Biol. 2003; 13: 819-827Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, Encalada et al., 2005Encalada S.E. Willis J. Lyczak R. Bowerman B. A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo.Mol. Biol. Cell. 2005; 16: 1056-1070Crossref PubMed Scopus (60) Google Scholar), such that phenotypes that may not be detected in other systems due to a block in cell cycle progression can be uncovered in the nematode. Furthermore, as in embryos of many species, early blastomeres in C. elegans oscillate between S phase and M phase, with the entire cell cycle lasting ∼10–15 min (Edgar and McGhee, 1988Edgar L.G. McGhee J.D. DNA synthesis and the control of embryonic gene expression in C. elegans.Cell. 1988; 53: 589-599Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Consequently, it is plausible that centrosome duplication occurs without the regulatory mechanisms present in more complex cell cycles. As a result, proteins uncovered as being essential for centriole formation in C. elegans embryos may identify core components of this process. ZYG-1 was the first protein identified as being essential for centriole formation in C. elegans, and its analysis yielded key insights for understanding the role of subsequently identified components. Conditional alleles of zyg-1 (for zygote defective: embryonic lethal) indicated that the gene is required in proliferating tissues (Hirsh and Vanderslice, 1976Hirsh D. Vanderslice R. Temperature-sensitive developmental mutants of Caenorhabditis elegans.Dev. Biol. 1976; 49: 220-235Crossref PubMed Scopus (111) Google Scholar, O'Connell et al., 1998O'Connell K.F. Leys C.M. White J.G. A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.Genetics. 1998; 149: 1303-1321PubMed Google Scholar). However, it was the analysis of zyg-1 in the early embryo that revealed its requirement for centriole formation (O'Connell et al., 2001O'Connell K.F. Caron C. Kopish K.R. Hurd D.D. Kemphues K.J. Li Y. White J.G. The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.Cell. 2001; 105: 547-558Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). As in other metazoans, there is a dual parental contribution to forming the centrosome in the wild-type one-cell-stage C. elegans embryo. The sperm provides a pair of centrioles stripped of PCM components, whereas the oocyte is devoid of centrioles but provides an abundant source of PCM proteins. After fertilization, the paternally contributed centrioles recruit the PCM from maternal stores, thus reconstituting one centrosome in the zygote. This single centrosome undergoes duplication during S phase, yielding two centrosomes, each containing one mother and one daughter centriole, which assemble a bipolar spindle during mitosis (Figure 2A ). If paternal zyg-1 function is lacking (Figure 2B), centriole formation fails during spermatogenesis, and mature sperm contain a single centriole. After fertilization, a daughter centriole forms during the first cell cycle because the oocyte provides normal zyg-1 function. This pair of centrioles recruits PCM components, but a single centrosome is present at mitosis, resulting in monopolar spindle assembly and cell division failure. During the second cell cycle, centrosome duplication occurs normally, resulting in bipolar spindle assembly, but embryos are tetraploid and eventually die. If maternal zyg-1 function is lacking (Figure 2C), the sperm provides a pair of centrioles as in the wild-type. The two paternally contributed centrioles split, but centriole formation does not occur in the absence of zyg-1 function. Nevertheless, each single centriole recruits PCM components, allowing for bipolar spindle assembly in the one-cell stage. The phenotype becomes apparent only at the two-cell stage, when the absence of centriole formation results in monopolar spindle assembly in each blastomere. If both paternal and maternal zyg-1 function are lacking (Figure 2D), a single centriole is contributed, and no further centrioles are formed in the embryo, leading to monopolar spindle assembly during both the first and second cell cycles. Thus, zyg-1 has a dual paternal and maternal requirement to ensure proper centriole formation in the embryo. These results mirror classical work in sea urchin embryos establishing that the reproductive capacity of centrosomes correlates with the presence of a pair of centrioles at the onset of the duplication cycle (Mazia et al., 1960Mazia D. Harris P. Bibring T. The multiplicity of the mitotic centers and the time-course of their duplication and separation.J. Bioph. Bioch. Cyt. 1960; 7: 1-20Crossref PubMed Scopus (103) Google Scholar, Sluder and Rieder, 1985Sluder G. Rieder C.L. Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes.J. Cell Biol. 1985; 76: 35-51Google Scholar). ZYG-1 is a divergent kinase that cannot be placed clearly into one of the known kinase families (O'Connell et al., 2001O'Connell K.F. Caron C. Kopish K.R. Hurd D.D. Kemphues K.J. Li Y. White J.G. The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.Cell. 2001; 105: 547-558Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) (Figure 4A). However, it may be that a kinase required for centriole formation in other organisms, such as human Mps1, Plk1, or Plk2, is functionally related to ZYG-1. In C. elegans, ZYG-1 was reported to be present at centrioles during mitosis, which immediately precedes S phase and the onset of centriole formation in these embryonic blastomeres (Leidel and Gönczy, 2003Leidel S. Gönczy P. SAS-4 is essential for centrosome duplication in C. elegans and is recruited to daughter centrioles once per cell cycle.Dev. Cell. 2003; 4: 431-439Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, O'Connell et al., 2001O'Connell K.F. Caron C. Kopish K.R. Hurd D.D. Kemphues K.J. Li Y. White J.G. The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.Cell. 2001; 105: 547-558Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). However, another study found ZYG-1 to be present at centrioles throughout the cell cycle, a result reported to be confirmed with GFP-ZYG-1 transgenic animals (Dammermann et al., 2004Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Centriole assembly requires both centriolar and pericentriolar material proteins.Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Recombinant ZYG-1 can autophosphorylate in vitro, but in vivo substrates have yet to be identified (O'Connell et al., 2001O'Connell K.F. Caron C. Kopish K.R. Hurd D.D. Kemphues K.J. Li Y. White J.G. The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.Cell. 2001; 105: 547-558Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Whereas zyg-1 was discovered through forward genetic screens, identification of the other genes known to be required for centriole formation in C. elegans benefited from comprehensive RNAi-based reverse genetic screens. As the first metazoan with a sequenced genome, and as the one in which RNAi was discovered in animals, C. elegans lent itself particularly well to RNAi-based functional genomic screens. The vast majority (∼98%) of predicted genes have been subjected to RNAi to test their requirement for cell division processes in the early embryo by using time-lapse differential interference contrast (DIC) microscopy (Sönnichsen et al., 2005Sönnichsen B. Koski L.B. Walsh A. Marschall P. Neumann B. Brehm M. Alleaume A.M. Artelt J. Bettencourt P. Cassin E. et al.Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.Nature. 2005; 434: 462-469Crossref PubMed Scopus (679) Google Scholar). Both sas-4 and sas-6 (for spindle assembly) were identified in large-scale RNAi-based screens. In sas-4(RNAi) or sas-6(RNAi) embryos, a bipolar spindle assembles at the one-cell stage, but a monopolar spindle assembles in each blastomere at the two-cell stage (Dammermann et al., 2004Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Centriole assembly requires both centriolar and pericentriolar material proteins.Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Kirkham et al., 2003Kirkham M. Müller-Reichert T. Oegema K. Grill S. Hyman A.A. SAS-4 is a C. elegans centriolar protein that controls centrosome size.Cell. 2003; 112: 575-587Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, Leidel et al., 2005Leidel S. Delattre M. Cerutti L. Baumer K. Gönczy P. SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells.Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (271) Google Scholar, Leidel and Gönczy, 2003Leidel S. Gönczy P. SAS-4 is essential for centrosome duplication in C. elegans and is recruited to daughter centrioles once per cell cycle.Dev. Cell. 2003; 4: 431-439Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). This phenotype is identical to that of embryos lacking zyg-1 strictly maternally, which may be expected given that sperm is not affected by using standard RNAi conditions. Serial-section electron microscopy confirmed that each spindle pole contains a single centriole in sas-4(RNAi) and sas-6(RNAi) embryos (Dammermann et al., 2004Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Centriole assembly requires both centriolar and pericentriolar material proteins.Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Kirkham et al., 2003Kirkham M. Müller-Reichert T. Oegema K. Grill S. Hyman A.A. SAS-4 is a C. elegans centriolar protein that controls centrosome size.Cell. 2003; 112: 575-587Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Thus, like zyg-1, sas-4 and sas-6 are required for centriole formation. SAS-4 and SAS-6 are both coiled-coil proteins (Figure 4A) enriched in a tiny dot at the center of centrosomes throughout the cell cycle, suggestive of centriolar localization (Dammermann et al., 2004Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Centriole assembly requires both centriolar and pericentriolar material proteins.Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Kirkham et al., 2003Kirkham M. Müller-Reichert T. Oegema K. Grill S. Hyman A.A. SAS-4 is a C. elegans centriolar protein that controls centrosome size.Cell. 2003; 112: 575-587Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, Leidel et al., 2005Leidel S. Delattre M. Cerutti L. Baumer K. Gönczy P. SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells.Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (271) Google Scholar, Leidel and Gönczy, 2003Leidel S. Gönczy P. SAS-4 is essential for centrosome duplication in C. elegans and is recruited to daughter centrioles once per cell cycle.Dev. Cell. 2003; 4: 431-439Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) (Figures 1C and 1D). That this is the case was demonstrated by immunoelectron microscopy (Dammermann et al., 2004Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Centriole assembly requires both centriolar and pericentriolar material proteins.Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Kirkham et al., 2003Kirkham M. Müller-Reichert T. Oegema K. Grill S. Hyman A.A. SAS-4 is a C. elegans centriolar protein that controls centrosome size.Cell. 2003; 112: 575-587Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Insights into the dynamics of centriolar recruitment were gained through two lines of experiments conducted with transgenic animals expressing GFP-SAS-4 or GFP-SAS-6. First, fluorescence recovery after photobleaching (FRAP)
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