InteractiveFly: GeneBrief

Spindle assembly abnormal 4 ortholog (C. elegans): Biological Overview | References

Gene name - Spindle assembly abnormal 4

Synonyms -

Cytological map position- 84C6-84C7

Function - centriole formation

Keywords - centriolar assembly, centriole replication, asymmetric cell division, flagellum biogenesis, cilium biogenesis, locomotion

Symbol - Sas-4

FlyBase ID: FBgn0011020

Genetic map position - 3R: 2,977,076..2,979,886 [+]

Classification - T-complex protein 10 C-terminus

Cellular location - nuclear

NCBI link: EntrezGene
Sas-4 orthologs: Biolitmine
Recent literature
Conduit, P. T., Wainman, A., Novak, Z. A., Weil, T. T. and Raff, J. W. (2015). Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP. Elife 4 [Epub ahead of print]. PubMed ID: 26530814
Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) it was shown that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. This study shows, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes. Importantly, Sas-4 may have a more indirect role in mitotic PCM assembly in fly embryos as centriolar Sas-4 (as opposed to cytoplasmic Sas-4) is required to efficiently recruit Asl molecules around maturing mother centrioles, and Asl has an important role in recruiting Spd-2 and Cnn around mother centrioles
Novak, Z. A., Wainman, A., Gartenmann, L. and Raff, J. W. (2016). Cdk1 phosphorylates Drosophila Sas-4 to recruit Polo to daughter centrioles and convert them to centrosomes. Dev Cell 37: 545-557. PubMed ID: 27326932
Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic 'centriole conversion' is regulated, but it requires Plk1/Polo kinase. This study shows that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome.
Ramani, A., Mariappan, A., Gottardo, M., Mandad, S., Urlaub, H., Avidor-Reiss, T., Riparbelli, M., Callaini, G., Debec, A., Feederle, R. and Gopalakrishnan, J. (2018). Plk1/Polo phosphorylates Sas-4 at the onset of mitosis for an efficient recruitment of pericentriolar material to centrosomes. Cell Rep 25(13): 3618-3630.e3616. PubMed ID: 30590037
Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. This study identified that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4 localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and gamma-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and gamma-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
Persico, V., Migliorini, M., Callaini, G. and Riparbelli, M. G. (2020). The Singularity of the Drosophila Male Germ Cell Centriole: The Asymmetric Distribution of Sas4 and Sas6. Cells 9(1). PubMed ID: 31947732
Drosophila spermatocytes have giant centrioles that display unique properties. Both the parent centrioles maintain a distinct cartwheel and nucleate a cilium-like region that persists during the meiotic divisions and organizes a structured sperm axoneme. Moreover, the parent centrioles are morphologically undistinguishable, unlike vertebrate cells in which mother and daughter centrioles have distinct structural features. However, immunofluorescence analysis of the parent centrioles in mature primary spermatocytes revealed an asymmetric accumulation of the typical Sas4 and Sas6 proteins. Notably, the fluorescence intensity of Sas4 and Sas6 at the daughter centrioles is greater than the intensity found at the mother ones. In contrast, the centrioles of wing imaginal disc cells display an opposite condition in which the loading of Sas4 and Sas6 at the mother centrioles is greater. These data underlie a subtle asymmetry among the parent centrioles and point to a cell type diversity of the localization of the Sas4 and Sas6 proteins (Persico, 2020).
Persico, V., Callaini, G. and Riparbelli, M. G. (2021). Sas-4 Colocalizes with the Ciliary Rootlets of the Drosophila Sensory Organs. J Dev Biol 9(1). PubMed ID: 33466292
The Drosophila eye displays peculiar sensory organs of unknown function, the mechanosensory bristles, that are intercalated among the adjacent ommatidia. Like the other Drosophila sensory organs, the mechanosensory bristles consist of a bipolar neuron and two tandemly aligned centrioles, the distal of which nucleates the ciliary axoneme and represents the starting point of the ciliary rootlets. This study reports that the centriole associated protein Sas-4 colocalizes with the short ciliary rootlets of the mechanosensory bristles and with the elongated rootlets of chordotonal and olfactory neurons. This finding suggests an unexpected cytoplasmic localization of Sas-4 protein and points to a new underscored role for this protein. Moreover, it was observed that the sheath cells associated with the sensory neurons also display two tandemly aligned centrioles but lacks ciliary axonemes, suggesting that the dendrites of the sensory neurons are dispensable for the assembly of aligned centrioles and rootlets.


Centrioles and centrosomes have an important role in animal cell organization, but it is uncertain to what extent they are essential for animal development. The Drosophila protein Sas-4 is related to the human microcephaly protein CenpJ and the C. elegans centriolar protein Sas-4 (Leidel, 2003; Kirkham, 2003). Drosophila Sas-4 is essential for centriole replication in flies. Sas-4 mutants start to lose centrioles during embryonic development, and, by third-instar larval stages, no centrioles or centrosomes are detectable. Mitotic spindle assembly is slow in mutant cells, and 30% of the asymmetric divisions of larval neuroblasts are abnormal. Nevertheless, mutant flies develop with near normal timing into morphologically normal adults. These flies, however, have no cilia or flagella and die shortly after birth because their sensory neurons lack cilia. Thus, centrioles are essential for the formation of centrosomes, cilia, and flagella, but, remarkably, they are not essential for most aspects of Drosophila development (Basto, 2006)

Since their first description more than 100 years ago, centrosomes have been recognized as important organizers of animal cells (see The centrosome cycle in mammalian cells from Azimzadeh, 2007). They consist of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM), which nucleates and organizes microtubules (MTs). Through the MTs they organize, centrosomes are thought to have important roles in establishing cell polarity, positioning organelles within cells, directing intracellular traffic, and organizing cell division (Basto, 2006).

Although centrosomes are major organizers of animal cell division, they are not essential for mitotic spindle assembly. Some animal cells normally organize their spindles without canonical centrosomes, and cultured cells that have had their centrosomes removed by laser ablation or microsurgery can still form bipolar spindles. In these cases, the mitotic chromosomes appear to initiate the assembly of a bipolar spindle and thereby compensate for the lack of centrosomes. Acentrosomal cultured cells, however, often fail in cytokinesis, and, even if these cells complete cytokinesis successfully, they usually arrest in the following G1 phase. This has led to the suggestion that cells have a checkpoint that monitors centrosome integrity in G1 (Basto, 2006)

Although centrosomes are dispensable for spindle assembly in many cell types, it is widely believed that they are essential for asymmetrical division because astral MTs directly contact cues in the cell cortex to position the mitotic spindle appropriately within the cell. Studies of centrosomin (cnn) and asterless (asl) mutants in Drosophila, however, suggest that centrosomes and astral MTs may not be essential for the asymmetric divisions of larval neuroblasts (Bonaccorsi, 2000; Giansanti, 2001; Megraw, 2001). These mutants appear to lack functional mitotic centrosomes, yet their neuroblasts have only subtle defects in aligning their spindles with cortical cues during early mitosis, and, by telophase, almost all neuroblasts appear to divide asymmetrically, just as in wild-type (wt) larvae. It remains controversial, however, whether cnn and asl mutants completely lack functional mitotic centrosomes (Basto, 2006)

Consistent with their many functions, centrosome dysfunction has been implicated in a wide variety of human diseases. Centrosome defects are believed to contribute to the genetic instability associated with many cancers, and genetic studies have implicated centrosomes in microcephaly, a condition associated with a small brain size at birth. Of the four genes so far linked to microcephaly, three (ASPM, Cdk5Rap2, and CenpJ) encode centrosomal proteins (Bond, 2005; Kouprina, 2005). It has been postulated that the small brain size in these individuals may be caused by defects in asymmetric division in the neural precursor cells that generate neurons during early fetal development (Woods, 2005). In addition, the centrioles in many animal cells are thought to have important functions that are distinct from their function as organizers of the centrosome. They form the basal bodies that nucleate the formation of cilia and flagella, and cilia defects contribute to a variety of human diseases (Basto, 2006)

Despite the likely importance of centrioles and centrosomes in so many cell processes, few experiments have addressed whether they have essential roles during animal development. The polo-like kinase Plk-4/Sak is essential for centriole replication (Bettencourt-Dias, 2005; Habedanck, 2005), and Drosophila sak hypomorphic mutant third-instar larval brains appear to lack centrioles in ~20% of their cells, yet they develop into adults at rates that are only slightly slower than normal (Bettencourt-Dias, 2005). The centrioles that are present in sak mutants, however, appear to be fully functional, so it is difficult to infer whether centrioles or centrosomes have essential roles in Drosophila development. Centrioles and centrosomes are essential for the development of C. elegans embryos. Five C. elegans proteins, Sas-4, Sas-5, Sas-6 (Leidel, 2003; Leidel, 2005a), Spd-2 (see Drosophila Spd-2), and Zyg-1 (the likely homolog of Plk-4/Sak) are required for centriole replication in worm embryos, and perturbing the function of any of them arrests embryonic development at the one or two cell stage. This early arrest, however, precludes an analysis of the importance of centrioles and centrosomes at later stages of worm development. This study show that a mutation in the Drosophila Sas-4 gene shows that centrioles and centrosomes are not essential for most aspects of Drosophila development (Basto, 2006)

The Drosophila protein encoded by the gene CG10061 shares a C-terminal domain with the human centrosomal protein CPAP/CenpJ and an N-terminal domain with the C. elegans centriolar protein Sas-4. CG10061 appears to function in a similar manner to C. elegans Sas-4, and is there for referred to as Drosophila Sas-4 (Basto, 2006)

Antibodies were raised against the N-terminal region of Sas-4. Affinity-purified anti-Sas-4 antibodies recognized a small dot at the center of the centrosome at all stages of the cell cycle in both embryos and larval neuroblasts. This staining was absent in Sas-4 mutant larval neuroblasts. Such dot-like centrosomal staining is usually indicative of centriole staining, and Sas-4 colocalizes with the Drosophila centriole markers GFP-PACT and GTU88* (Martinez-Campos, 2004) in fixed larval neuroblasts. Moreover, anti-Sas-4 antibodies stain the very large centrioles found in spermatocytes; this staining is absent in Sas-4 mutant spermatocytes. Thus, Sas-4 is closely associated with centrioles (Basto, 2006)

Sas-4 mutant cells progressively lose centrioles during embryonic development as the maternally supplied Sas-4 protein is exhausted; by first-instar larval stages, ~90% of mutant brain cells lack detectable centrioles, and by third-instar larval stages, centrioles are essentially undetectable in these cells. No centrosomes, cilia, or flagella are detected in mutant cells that lack centrioles, strongly suggesting that centrioles are essential for the formation of these structures in flies. Remarkably, these mutant flies develop at near-normal rates and are born at near normal Mendelian ratios, demonstrating that flies can proceed through the majority of development without centrioles, centrosomes, cilia, or flagella. Mutant adults, however, die shortly after birth because they lack cilia in type I sensory neurons. Thus, centrioles are essential for fly survival only because they are required for cilia formation (Basto, 2006)

In C. elegans, centrioles and centrosomes are essential for early development: mutant embryos that cannot replicate their centrioles arrest after only one or two rounds of cell division. The same would probably be true for the earliest stages of Drosophila embryogenesis, as one would expect centrosomes to be especially important in early syncytial Drosophila embryos, in which hundreds of large spindles have to assemble and disassemble very quickly within a common cytoplasm. In Sas-4 mutants, however, the heterozygous mothers contribute Sas-4 to the early embryos, which therefore contain centrioles and centrosomes (Basto, 2006)

It is clear from previous studies that centrosomes are not required for spindle assembly, since mitotic chromosomes and MT-dependent motor proteins can organize the assembly of bipolar spindles. It is nonetheless surprising that centrioles and centrosomes are dispensable for cell division during most stages of Drosophila development. Although a Drosophila cell line that lacks centrioles has been identified, these cells often fail to divide normally. Cultured mammalian cells that have had their centrosomes removed also often fail to complete cytokinesis, and those cells that do divide often then arrest in G1 of the next cycle, suggesting that centrosomes are required for both efficient cytokinesis and cell-cycle progression. One might expect, therefore, that an animal lacking centrosomes would, at the very least, be at a severe growth disadvantage compared to a normal animal. This seems not to be the case in Drosophila. Although spindle assembly is slowed in acentrosomal Sas-4 mutant cells, once assembled, these spindles make few chromosome-segregation errors. Moreover, the ~30%-40% increase in the duration of mitosis in mutant cells does not significantly delay development, probably because mitosis occupies such a small fraction of the total cell cycle. Thus, in flies at least, centrioles and centrosomes are not essential for any aspect of somatic cell-cycle progression or cell division; unlike cultured mammalian cells, fly cells do not arrest in G1 if they have no centrioles or centrosomes (Basto, 2006)

Centrioles or centrosomes were found to have an important role in asymmetric cell division in Drosophila. This contrasts with previous studies on cnn and asl mutants, which also appear to lack functional mitotic centrosomes. The cortical cues that guide asymmetric division are localized normally in these mutants, although spindles fail to align efficiently with these cues at early stages of mitosis. By telophase, however, ~90% of the mutant cells have properly aligned spindles, and the cells appear to divide asymmetrically. In Sas-4 mutants, by contrast, at least two aspects of asymmetric division are perturbed. (1) The localization of Miranda to a basal cortical crescent occurs unreliably, suggesting that centrosomes play an important part in establishing and/or maintaining cortical Miranda during asymmetric division. (2) ~30% of mutant neuroblasts either divide symmetrically or fail to complete cytokinesis. It is suspected that the explanation for the differences between the Sas-4 mutants and the cnn and asl mutants is that the centrosomes in cnn and asl mutants are partially functional, whereas they are completely absent in Sas-4 mutants. Cnn mutants, for example, have centrioles, and astral MTs can be detected in at least some cnn mutant neuroblasts using live-cell imaging techniques, which are more sensitive than those used in previous studies (Basto, 2006)

These observations fit well with recent evidence for two partially redundant mechanisms that ensure the fidelity of asymmetric division in embryonic fly neuroblasts. The first mechanism is MT independent and is initiated prior to the entry into mitosis by Insc/Par protein complexes concentrated at the apical cortex. These complexes recruit Pins/Gαi complexes, which then help drive the redistribution of proteins like Miranda to the basal cortex. In the absence of Insc/Par complexes, a MT-dependent mechanism can recruit Pins/Gαi complexes to a cortical region adjacent to one of the spindle poles. Presumably, these two mechanisms normally cooperate to ensure that the forming spindle efficiently aligns with preexisting cortical cues. If one mechanism is perturbed, however, the other is apparently sufficient to allow neuroblasts to divide asymmetrically. This redundancy presumably explains why neuroblasts carrying mutations that affect asymmetric division usually have misaligned spindles at metaphase but are “rescued” by telophase and so ultimately divide asymmetrically. The current analysis emphasizes that these two mechanisms are only partially redundant: Miranda does not consistently localize to the basal cortex in Sas-4 mutant cells, even though the Insc/Par complex almost always localizes correctly. Moreover, telophase rescue is inefficient in these cells, and ~30% of cells either fail in cytokinesis or divide symmetrically. Thus, Drosophila neuroblasts apparently have great difficulty in compensating for a lack of centrosomes (Basto, 2006)

Despite these difficulties, ~70% of acentrosomal Sas-4 mutant larval neuroblasts divide asymmetrically. How can cells that lack centrosomes and astral MTs align their spindle with cortical cues so as to divide asymmetrically? Live-cell analysis provides a potential explanation. Many acentrosomal spindles extend across the full length of the cell, so that the spindle poles are in close contact with the cortex, and this was also a noticeable feature of the mutant spindles analyzed by EM. This may allow the acentrosomal spindles to interact directly with cortical cues even in the absence of astral MTs, perhaps explaining how the majority of these spindles ultimately align correctly (Basto, 2006)

It is even more surprising that flies in which ~30% of the brain neuroblasts fail to divide properly seem to have so few developmental defects. The brain seems grossly normal in size, morphology, and histology. Moreover, the neuronal axons in the developing eye disc seem to be oriented correctly, which is unexpected, since previous studies have suggested that the initial direction of axon outgrowth in these cells is defined by the position of the centrosome (de Anda, 2005). DSas-4 mutants may well have subtle defects in neuronal development such as mild proliferation defects which would require lineage trancing experiments to be detected. However, it is clear that the developing fly brain has a remarkable ability to compensate for large-scale abnormalities in neuroblast divisions. It is unclear how the brain manages this. In humans, mutations in CenpJ, the homolog of DSas-4, results in microcephaly, which has been proposed to be caused by abnormalities in neural precursor cell divisions during fetal development (Woods, 2005). The finding that neuroblast divisions are frequently abnormal in Sas-4 mutant flies provides the first direct support for this proposal, although the brains of Sas-4 mutants appear no smaller than wt brains. Perhaps the developing human brain cannot compensate for abnormalities in neural precursor cell divisions in the way that the developing fly brain can (Basto, 2006)

It will be of great interest to determine whether centrioles and centrosomes are largely dispensable for much of development in other organisms. This may be difficult to address in other systems. In flies, only type I mechanosensory neurons and sperm have cilia and flagella, respectively, so the lack of centrioles produces only an uncoordinated phenotype. By contrast, many types of vertebrate cells have a primary cilium, which, in some cells at least, is required for the cell to respond to certain extracellular signals. Moreover, cilia in vertebrates have crucial roles in the development of organs such as the kidney. Thus, a lack of centrioles is likely to have a more devastating effect on vertebrate development than on fly development, which might make it difficult to assess whether developing vertebrates can compensate for the lack of centrioles and centrosomes in cell division in the way that Drosophila apparently can (Basto, 2006)

Centrosomes are thought to influence many aspects of cell behavior, including cell migration and cell polarity. These findings suggest that centrosomes are not essential for any of these processes during most of fly development. It remains unclear, however, whether these processes do not depend on centrosomes in Drosophila or whether they are normally dependent but can compensate for the absence of centrosomes. Sas-4 mutants should provide a useful model to explore the importance of centrosomes in many cell processes (Basto, 2006)

Phenotypic analysis of misato function reveals roles of noncentrosomal microtubules in Drosophila spindle formation

Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. In Drosophila, mutants without centrosomes can form functional anastral spindles and survive to adulthood. This study shows that mutations in the Drosophila misato (mst) gene inhibit kinetochore-driven MT growth, lead to the formation of monopolar spindles and cause larval lethality. In most prophase cells of mst mutant brains, asters are well separated, but collapse with progression of mitosis, suggesting that k-fibers are essential for maintenance of aster separation and spindle bipolarity. Analysis of mst; Sas-4 double mutants showed that mitotic cells lacking both the centrosomes and the mst function form polarized MT arrays that resemble monopolar spindles. MT regrowth experiments after cold exposure revealed that in mst; Sas-4 metaphase cells MTs regrow from several sites, which eventually coalesce to form a single polarized MT array. By contrast, in Sas-4 single mutants, chromosome-driven MT regrowth mostly produced robust bipolar spindles. Collectively, these results indicate that kinetochore-driven MT formation is an essential process for proper spindle assembly in Drosophila somatic cells (Mottier-Pavie, 2011).

This study has shown that mst function is required for chromosome-associated MT regrowth after cold exposure. Abundant evidence indicates that chromatin and kinetochores have the ability to promote MT nucleation and stabilization. Studies in Xenopus have shown that DNA injected into eggs promotes MT nucleation leading to the formation of spindle-like structures. Spindle-like structures were also observed around DNA-coated beads incubated in Xenopus egg extracts. These findings suggested that the process of spindle formation does not require the centromere-kinetochore function. However, several studies have demonstrated that in somatic cells recovering from MT poisons or cold exposure, MTs regrow from the kinetochores and not from chromosome arms. Consistent with these results, RCC1 and other factors regulating the Ran GTP-GDP cycle are enriched at mammalian kinetochores, and RanGTP accumulates at the kinetochore of cells recovering from MT depolymerization. Most importantly, there is evidence that kinetochores can drive MT growth even under physiological conditions. Studies in mammalian cells have shown that in monopolar spindles the kinetochores that face away from the centrosome can drive formation of k-fibers. Similarly, in Drosophila S2 cells, chromosomes that are distant from the astral MTs develop k-fibers from the kinetochore that does not face the centrosome. Thus, in both Drosophila and mammalian cells, chromosome-induced MT growth occurs primarily at the centromere-kinetochore region (Mottier-Pavie, 2011).

The pattern of MT regrowth observed in these experiments on wild-type neuroblasts is very similar to the pattern of kinetochore-driven formation of k-fibers observed in Drosophila S2 cells either untreated or exposed to MT-depolymerizing agents (see Bucciarelli, 2009). To explain the mechanism of kinetochore-driven k-fiber formation it has been proposed that kinetochores capture the plus ends of MTs nucleated near the centromere; these MTs continue to polymerize at the kinetochore, forming MT bundles with the minus ends pointing away from the chromosomes. Interactions between these MT bundles and the astral MTs lead to the formation of mature k-fibers that connect the chromosomes with the spindle poles. The current results are consistent with this model, and strongly suggest that mst mutants are specifically defective in kinetochore-driven but not centrosome-driven MT growth. MTs emanating from mst centrosomes eventually reach the kinetochores and form sparse k-fibers, which are much thinner than those of wild-type cells because they do not incorporate the MTs generated by the kinetochores (Mottier-Pavie, 2011).

The role of mst in kinetochore-induced MT formation is currently unknown. Studies in human cells have suggested that an Mst homologue is localized at the outer mitochondrial membrane and regulates mitochondrial distribution and morphology. However, it is unlikely that mst regulates MT growth by affecting mitochondrial functions, because this study found that Drosophila Mst is not associated with mitochondria. It was also found that Mst does not associate with MTs in pull-down assays, which is consistent with the finding that anti-Mst antibodies do not decorate the spindle MTs. In addition, preliminary co-immunoprecipitation and mass spectrometry experiments did not reveal reliable Mst interactors. Thus, the data do not provide indications on whether mst mutants are defective in chromatin-induced MT nucleation near the centromere or in the subsequent kinetochore-driven formation of k-fibers. It is also possible that the mst function is not restricted to the formation of k-fibers and that mst has functions that are general to MT behavior, such as MT dynamics or bundling. However, even if mst had a more general role in MT behavior, kinetochore-driven MTs are clearly more susceptible to the loss of mst function than are other MTs. It was found that Mst is primarily expressed during mitosis, and accumulates in dividing cells from prophase through telophase. This finding leads to a speculation that Mst might be involved in the upregulation of MT dynamics that characterizes the interphase to M-phase transition. However, the molecular mechanisms underlying the Mst-dependent MT behavior and the specific role of Mst in kinetochore-driven k-fiber formation are currently unclear and remain a matter for future studies (Mottier-Pavie, 2011).

An incomplete occupancy of the kinetochore plate by k-MTs is probably responsible for spindle assembly checkpoint SAC protein recruitment at kinetochores and SAC-mediated metaphase arrest in mst mutants. When the SAC is abrogated by a mutation in the rod gene, a fraction of the metaphases of mst mutant brains manages to undergo anaphase. However, most mutant anaphases seen in mst; rod double mutants exhibit defects in chromosome segregation that are more severe than those observed in rod single mutants, suggesting that the thin k-fibers of mst mutant cells have a reduced ability to mediate chromosome segregation. Interestingly, mst; rod double mutants displayed a lower frequency of polyploid cells than mst single mutants. A possible interpretation for this finding is that loss of rod function in an mst mutant background releases bipolar spindles from SAC-induced metaphase arrest, allowing chromosome segregation and preventing polyploid cell formation. It can be also envisaged that in mst; rod double mutants, a fraction of the mitotic cells undergoes anaphase before aster collapse, further reducing polyploid cell formation (Mottier-Pavie, 2011).

In mst mutant brains, prophase cells display normal aster separation, whereas most prometaphase-metaphase figures are monopolar. This suggests that k-fibers are required to maintain aster separation after nuclear envelope breakdown (NEB). Several studies indicate that k-fibers have a role in aster separation. However, the effects of k-fiber disruption appear to be cell-type specific. The Aurora A activator TPX2 is required for both chromosome-dependent MT formation and the assembly of a bipolar spindle. However, there is a controversy on the spindle abnormalities caused by depletion of TPX2 in human cells. A study showed that loss of TPX2 causes centrosome fragmentation and multipolar spindles in HeLa cells. In other studies on HeLa cells, TPX2 depletion resulted in two prominent asters that did not interact to form a bipolar spindle. More recent work has shown that RNAi against TPX2 in U2OS cells leads to aster collapse and monopolar spindles. In Caenorhabditis elegans embryos depleted of the TPX2 ortholog TPXL-1, asters also collapsed, but gave rise to short bipolar spindles. Another factor required for both k-fiber formation and centrosome separation is the human kinetochore protein Mcm21R/CENP-O; recent work on this protein has led to the conclusion that k-fibers use the poleward MT flux to generate forces that push centrosomes apart. Analysis of Drosophila mitosis has provided additional evidence for a role of k-fibers in centrosome separation. The conserved Drosophila Orbit/Mast protein (CLASP in vertebrates) is required for tubulin dimer addition to the MT plus ends of fluxing k-fibers and for the maintenance of kinetochore and k-fiber connection (Maiato, 2002; Maiato, 2005). Mutations in orbit/mast and RNAi-mediated knockdown of these genes cause aster collapse and frequent monopolar spindles in embryonic and S2 tissue culture cells, respectively. Finally, it has been observed that RNAi-mediated depletion of the augmin complex in S2 cells leads to a complete suppression of k-fiber regrowth after cold exposure (Bucciarelli, 2009) and to the formation of monopolar spindles. Collectively, these results indicate that in many systems, the fluxing MTs of k-fibers exert forces required to maintain proper spindle architecture. It is proposed that the thin k-fibers of mst mutant spindles are unable to generate sufficient force to keep the centrosomes apart and avoid aster collapse (Mottier-Pavie, 2011).

mst is the first Drosophila gene so far identified that appears to be specifically required for kinetochore-driven MT formation during mitosis of a living organism. The orbit/mast gene and the augmin-coding genes (wac and msd1) are not specifically involved in this process. Orbit/Mast is a microtubule-associated protein that is particularly enriched at the spindle poles and the central spindle midzone, and it is required for both spindle assembly and cytokinesis. Studies on two augmin subunits, Wac and Msd1, have shown that they are not essential for mitotic spindle formation and fly viability, but are only required for meiotic spindle organization in females. Thus, the analysis of mst mutants allow defining of the role of kinetochore-driven MT formation in living flies. Previous studies have shown that lack of centrosomal MTs results in anastral, but otherwise functional spindles, and there is evidence that flies without centrosomes can develop to adulthood. By contrast, the current results indicate that kinetochore-driven MT formation is an essential process for Drosophila mitotic division and development (Mottier-Pavie, 2011).

The specific involvement of Mst in kinetochore-driven MT formation gave led to an opportunity to ask whether Drosophila brain cells can form a spindle in the absence of both centrosomal and kinetochore-driven MTs. It was found that most mst; Sas-4 cells displayed acentrosomal MT arrays (AMTAs) that resembled monopolar spindles. MT regrowth assays showed that in mst; Sas-4 metaphases, MTs grow from multiple foci, which eventually aggregate to form a monopolar MT array that is indistinguishable from an AMTA observed in non-chilled cells. In cells returned to 25°C for 2 minutes, robust MT regrowth foci were observed that were well separated from the chromosomes. These foci are likely to contain MTs nucleated independently of the chromosomes, perhaps by MT nucleation sites associated with membrane and/or Golgi. The same cells also contained foci that appeared to lie on or near the chromosomes. However, the finding that mst single mutants are severely defective in chromosome-driven MT regrowth suggests that most, if not all, of the foci that appear to lie over the chromosomes (because of the squashing procedure used to obtain brain preparations) do in fact contain MTs that were nucleated independently of the chromatin (Mottier-Pavie, 2011).

It is thus proposed that in mst; Sas-4 mutants all MT growth foci contain MTs that were nucleated independently of the chromosomes; these foci would associate with the chromosomes through a RanGTP-dependent mechanism that attracts growing MTs towards the chromatin. Studies using an in vitro system containing Xenopus egg extracts, chromatin-coated beads and purified human centrosomes have shown that the MTs nucleated by the centrosomes grow towards the chromatin. This leads to a concomitant movement of the asters, which stops when the astral MTs are stabilized by the high RanGTP concentration around the chromatin. A Ran-GTP-mediated attraction of astral MTs has also been described in mammalian cells; mammalian chromosomes with defective kinetochores, but capable of generating a normal RanGTP gradient are unable to form k-fibers but retain the ability to attract astral MTs. Thus it is suggested that in an mst mutant background, kinetochore-driven k-fiber formation is inhibited, whereas chromosomes retain the ability to form a RanGTP gradient that attracts the MTs nucleated at either the centrosomes or other cellular sites. In the absence of centrosomes, the MTs emanating from several chromosome-independent growth foci would be attracted by the chromatin, ultimately leading to chromosome-associated AMTAs (Mottier-Pavie, 2011).

The size of acentrosomal MT arrays (AMTAs) would suggest that MTs nucleated at non-canonical sites provide a substantial contribution to mitotic spindle assembly. However, it should be considered that the relatively large size of AMTAs could be the consequence of an increase in tubulin dimer availability as a result of the inhibition of MT polymerization at both the centrosomes and chromosomes. Consistent with this view, in both mst and Sas-4 single mutants, chromosome-independent MT regrowth foci were smaller and less frequent than in the double mutants. In addition, previous studies have shown that in both mammalian and Drosophila cells, inhibition of chromosome-dependent MT formation results in overgrowth of aster. Collectively, these results indicate that centrosomes and kinetochores have dominant roles in mitotic MT nucleation over non-canonical nucleation sites (Mottier-Pavie, 2011).

Previous studies have shown that Drosophila cells depleted of factors required for proper spindle assembly can form short bipolar spindles. Even if many of the AMTAs observed in mst; Sas-4 cells comprise enough MTs to form a short bipolar spindle, in both untreated cells and cells subjected to MT regrowth, bipolar MT arrays were rare. By contrast, MT regrowth in Sas-4 single mutants mostly generated bipolar spindles. Thus, in Drosophila somatic cells the assembly of both centrosomal and acentrosomal bipolar spindles requires kinetochore-driven MT formation and cannot rely on MTs nucleated in other cellular compartments (Mottier-Pavie, 2011).

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).

The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis

Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. The COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. By establishing colcemid-induced microtubule depolymerization as a sensitive assay, this study examined the characteristics of COC activity and found that it functions uniquely in GSCs but not in their differentiating progeny. The COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types (Venkei, 2015).

Stereotypical orientation of the mitotic spindle is a widely utilized mechanism to achieve asymmetric cell division. Although considerable knowledge has accumulated regarding how spindle orientation is established, little is known about whether cells possess a mechanism that monitors successful spindle orientation. In the present study, using colcemid treatment as a sensitive assay, the nature of the COC, which is the only known orientation/polarity checkpoint in multicellular organisms, was was investigated. It was established that: (1) the COC specifically operates in GSCs, but not differentiating germ cells (GBs and SGs); (2) the COC operates in G2 phase of the cell cycle to prevent precocious entry into mitosis upon centrosome misorientation; and (3) as a checkpoint mechanism, the COC is distinct from the SAC (Venkei, 2015).

These results show that the COC is a GSC-specific checkpoint that monitors centrosome orientation and arrests cells in G2 phase when centrosomes are not correctly oriented. It remains unclear whether the COC-mediated G2 arrest might eventually undergo adaptation to allow mitotic entry, as is the case with mitotic slippage in the SAC. It is worth noting in this context that the GSC mitotic index never increased during 6 h of colcemid treatment, whereas SGs seem to undergo mitotic slippage by 6 h of colcemid treatment. This suggests that the COC-mediated G2 arrest is relatively strong. The findings that the mad2 mutation has no effect on G2 arrest of GSCs and that par-1 and cnn mutants have no effect on mitotic arrest in GBs/SGs upon colcemid treatment strongly support the notion that the COC and SAC constitute distinct checkpoint mechanisms. Although CySCs and SGs also orient their mitotic spindles, the present study shows that spindle orientation in these cells is not under the control of the COC. However, the lack of a COC in these cell types does not exclude the possibility that distinct polarity checkpoint mechanisms are in place to ensure correct spindle orientation. If the arrest points of such checkpoints are not prior to the arrest point of the SAC (i.e. metaphase), the assay using colcemid would not reveal their presence (Venkei, 2015).

Mutant analysis of multiple centrosomal components in this study revealed a selective requirement for centrosomal components in the function of the COC. Sas-4 and Cnn are crucial for COC function, whereas Spd-2 and Apc1 are not. This indicates that not all of the centrosomal proteins are involved in COC function. Conversely, not all COC components are localized to the centrosome. As shown in a previous study, an essential component of the COC, Par-1, is localized to the spectrosome, where it regulates the localization of Cyclin A to regulate mitotic entry. How the information on centrosomal orientation is communicated to the spectrosome, where Par-1 and Cyclin A localize, remains to be determined. A major outstanding question in understanding the COC is how it senses the location of the centrosome with respect to the hub cells. Previous studies have shown that the mother centrosome is anchored to the adherens junctions formed at the hub-GSC interface via MTs. Therefore, it is plausible that the COC senses aspect(s) of these interactions. It awaits future investigation to understand how the association of the centrosome with the hub-GSC interface is mechanistically sensed, and how such information is integrated with the activity of COC component(s) on the spectrosome (Venkei, 2015).

In summary, this present work establishes that the COC is a checkpoint mechanism that is distinct from the SAC and monitors correct centrosome orientation specifically in GSCs. It is speculated that a similar mechanism might be in place in other systems that rely on asymmetric cell division (Venkei, 2015).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of beta-sheets fold was determined. This unique feature of the TCP domain suggests that it could provide an 'extended surface-like' platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th beta-strands (beta9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 beta9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the beta9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, this study provides possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication (Zheng, 2014).

Sequential protein recruitment in C. elegans centriole formation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Centriole assembly in Caenorhabditis elegans

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Using electron tomography of staged C. elegans one-cell embryos this study shows that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. Centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms (Pelletier, 2006).

The structure of centrioles is conserved from ancient eukaryotes to mammals. They are barrel-shaped, 100-250 nm in diameter and 100-400 nm in length, with a ninefold symmetric array of singlet, doublet or triplet microtubules. Daughter centrioles grow orthogonally to the older (mother) centrioles, but the assembly process remains mysterious. Centrioles in C. elegans are 150 nm in length, 100 nm in diameter and consist of a central tube surrounded by nine singlet microtubules. Genomic and genetic analyses have identified proteins required for centriole duplication in C. elegans. These include the ZYG-1 kinase (O'Connell, 2001), the SAS proteins (SAS-4, SAS-5 and SAS-6) and SPD-2, all of which localize to centrioles (Pelletier, 2006 and references therein).

C. elegans oocytes arrest in meiotic prophase I, at which point they lack centrioles. Fertilization of oocytes by sperm triggers the completion of meiosis and contributes a centriole pair. After meiosis, the embryo enters the first mitotic division, which is characterized by a series of easily distinguishable events. These include pronuclear appearance (PNA), pronuclear migration (PNM), pronuclear rotation (PNR) and metaphase. Using chemical fixation and serial section electron microscopy it was possible to detect daughter centriole assembly intermediates at, but not before, PNM (Pelletier, 2006).

To determine whether the onset of daughter centriole assembly correlates with the recruitment of known centriole proteins, mating-based assays were performed. In these assays, the sperm donates an unlabelled centriole pair, whereas the green fluorescent protein (GFP)-tagged centriole proteins are contributed by the maternal cytoplasm. Their incorporation into nascent daughter centrioles can therefore be monitored. Embryos were fixed at different stages of the first cell division and stained using SAS-4 antibodies -- to mark the position of the sperm centrioles -- and GFP or ZYG-1 antibodies to monitor the recruitment of individual centriole proteins to the site of assembly. It was observed that SPD-2 and ZYG-1 are recruited to centrioles soon after fertilization, during the completion of female meiosis, whereas SAS-4, SAS-5 and SAS-6 are recruited shortly afterwards, at PNA (Pelletier, 2006).

To gain insights into the assembly relationship between these proteins, individual centriole proteins were depleted by RNA-mediated interference (RNAi) and the effect this had on the recruitment of the other proteins was determined. In one-cell spd-2(RNAi) embryos, no ZYG-1 recruitment was detected at any stage; however, in zyg-1(RNAi) embryos, SPD-2 recruitment was unaffected. These results suggest that SPD-2 acts upstream of ZYG-1. In all spd-2(RNAi) embryos analysed, no significant recruitment of maternal SAS-4, SAS-5 or SAS-6 was detected to the site of centriole assembly. Essentially the same result was observed in zyg-1(RNAi) embryos, which is in agreement with the observation that SPD-2 is required to recruit ZYG-1. In sas-4(RNAi) embryos, both SAS-5 and SAS-6 are recruited to the site of daughter centriole assembly, confirming published results in two-cell embryos. It has been shown that SAS-4 recruitment is blocked in sas-5(RNAi) and sas-6(RNAi) embryos. Together, these results suggest that in one-cell embryos SPD-2 and ZYG-1 are required for daughter centriole assembly by promoting the recruitment of the centriole components SAS-5 and SAS-6. SAS-5 and SAS-6 recruitment then promotes the recruitment of SAS-4 (Pelletier, 2006).

Thin section electron microscopy results showed that daughter centriole assembly begins during PNM. In contrast, mating assays showed that centriole proteins are recruited beforehand, during meiosis or at PNA. Two possibilities could explain these observations. First, centriole proteins may be recruited to the proximity of the mother centrioles before daughter centriole assembly. Second, the recruitment observed may coincide with the emergence of a structural intermediate that is undetectable using chemical fixation and thin section electron microscopy. To examine potential structural intermediates of centriole assembly, an approach was developed to perform high-pressure freezing and electron tomography on isolated embryos at different stages of the first cell division. Surprisingly, daughter centriole assembly intermediates were observed at PNA. The smallest structure detected was a cylindrical tube approximately 60 nm in length, similar to the central tube of mother centrioles. Electron-dense appendages emanated from the singlet microtubules of mother centrioles. During PNM, the central tubes of daughter centrioles were longer, reaching about 110 nm at the end of PNM. The central tube of daughter centrioles increased in diameter during PNM, whereas that of the mother centriole did not; this increase coincided with the appearance of an inner/outer wall structure on daughter centriole central tubes. Singlet microtubules began assembling around the central tube during PNR and by the end of PNR nine singlet microtubules were observed. Hook-like appendages were detected along the length of daughter central tubes at positions where singlet microtubules had yet to assemble. Singlet microtubule assembly did not seem to occur preferentially at distal or proximal extremities, although a slight positional bias for the distal region of the central tube was observed. In summary, tomography results show that in one-cell embryos, daughter centriole assembly begins with the formation of the central tube, which elongates and increases in diameter before the assembly of the nine singlet microtubules (Pelletier, 2006).

The effect was examined of individually depleting centriole proteins on daughter centriole assembly. In zyg-1(RNAi) embryos, no daughter centriole structures were detected, consistent with the idea that in zyg-1(RNAi) embryos, the SAS-proteins are not recruited to the site of centriole assembly. Similarly, in sas-5(RNAi) and sas-6(RNAi) embryos, no daughter centriole structures were observed. Most interestingly, in sas-4(RNAi) embryos at PNA central tubes of daughter centrioles still formed. After PNR, daughter centriole central tubes were longer, suggesting that tube elongation can still occur in sas-4(RNAi) embryos. Daughter centriole central tubes failed to increase in width, and seemed defective in the assembly of both singlet microtubules and hook-like appendages. In older embryos, daughter centriole central tubes were often difficult to discern. These results are consistent with observation for wild-type embryos that the formation of a central tube represents a structural intermediate in centriole assembly, which is unstable in sas-4(RNAi) embryos. Taken together, electron tomography and light microscopy data suggest that SAS-4 is required to assemble or maintain singlet microtubules onto the central tube, the assembly of which is, in turn, dependent on SAS-5 and SAS-6 (Pelletier, 2006).

Using electron tomography this study has shown that daughter centriole assembly in one-cell C. elegans embryos occurs in at least three distinct steps: tube formation; tube elongation; and singlet microtubule assembly (See a model for centriole formation in C. elegans; Delattre, 2006). Assembly begins at the time of PNA and is completed by the end of PNR, which is estimated to require 8-10 min. The recruitment of the SAS-proteins at PNA to the site of daughter centriole assembly is coincident with the emergence of the daughter central tube. In spd-2(RNAi) and zyg-1(RNAi) embryos, the recruitment of the SAS-proteins to the site of assembly is blocked. Accordingly, daughter centriole structures were not detected in zyg-1(RNAi) embryos. These observations, in combination with results showing that SPD-2 is required for the recruitment of the ZYG-1 kinase to the site of daughter centriole assembly, suggest that the role of SPD-2 in centriole duplication is to recruit ZYG-1 to centrioles. ZYG-1 - possibly in concert with SPD-2 - can then either function as a signal required for the initiation of daughter centriole assembly or directly drive the assembly process (Pelletier, 2006).

The fact that daughter centriole central tubes assemble in sas-4(RNAi) embryos (conditions under which SAS-5 and SAS-6 recruitment is not inhibited) indicates that SAS-5 and SAS-6 could be structural components of the central tube. SAS-5 and SAS-6 are known to physically interact, indicating that they may fulfil this structural role as a heterodimer (Leidel, 2005a). Alternatively, it is possible that SAS-5 and SAS-6 are required to maintain the structural integrity of the central tube, which is composed of other proteins. In sas-4(RNAi) embryos, central tube assembly is not perturbed although its stability and capacity to assemble singlet microtubules are compromised. Thus, a role is proposed for SAS-4 in tethering singlet microtubules to the central tube, which would be consistent with the ring-like distribution of SAS-4 around centrioles (Kirkham, 2003). It was previously shown that the amount of SAS-4 on centrioles is related to the quantity of pericentriolar material they recruit (Kirkham, 2003). It is therefore possible that the singlet microtubules on the C. elegans centriole are required to recruit and organize pericentriolar material (Leidel, 2005b). Recent evidence has shown that pericentriolar material is also required for daughter centriole assembly (Dammermann, 2004). It will be of interest to delineate the contribution of such proteins to daughter centriole assembly (Pelletier, 2006).

Published structures using chemical fixation and thin section electron microscopy on mammalian tissue culture cells are ambiguous as to whether centrioles have a central tube. It will be of importance to revisit daughter centriole assembly in mammalian tissue culture cells, and other organisms, using high-pressure freezing and time-resolved tomography to determine if daughter centriole assembly also proceeds in the same order: tube formation, elongation and microtubule assembly. Interestingly, three of the five components required for centriole duplication in C. elegans (SPD-2, SAS-4 and SAS-6) have mammalian homologues, although only human SAS-6 is known to be required for centriole duplication. It is therefore likely that some of the assembly intermediates uncovered here in C. elegans are conserved in mammals and other eukaryotes (Pelletier, 2006).

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila Centrosomin

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. This study shows that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles 'rocket' around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and only subtle defects are observed in the asymmetric divisions of mutant neuroblasts. It is concluded that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts (Lucas, 2007).

This study shows that mutations in DSas-4, which encodes the Drosophila homologue of the human microcephaly protein CenpJ/CPAP, also lead to defects in the asymmetric divisions of larval NBs. The defects were much more severe in DSas-4 mutants, which completely lack centrioles/ centrosomes (~15% of NBs divided symmetrically, whereas ~15% failed in cytokinesis). The much milder defects in asymmetric division that were observe in cnn NBs suggest that centrosomes are partially functional as MTOCs in cnn mutant somatic cells. Indeed, relatively well-focused astral MT arrays were frequently observed forming and disassembling in the cytoplasm, and these were often transiently associated with the spindle poles in cnn NBs (Lucas, 2007).

Taken together, these observations on cnn and DSas-4 mutant NBs reveal that, unlike the situation in male GSCs, the asymmetric behavior of the centrosomes is not essential for the accurate asymmetric division of larval NBs. Nevertheless, mutations in the Drosophila homologues of two of the three human centrosomal proteins implicated in microcephaly do lead to relatively subtle defects in NB divisions in flies. Drosophila cnn and DSas-4 mutants do not have small brains, suggesting that flies are able to compensate for defects in these divisions in a way that perhaps humans cannot (Lucas, 2007).

Stepwise evolution of the centriole-assembly pathway

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

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

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

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


Search PubMed for articles about Drosophila Sas-4

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

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

Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C. G., Khodjakov, A. and Raff, J. W. (2006). Flies without centrioles. Cell 125: 1375-1386. PubMed ID: 16814722

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

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

Bond, J., et al. (2005). A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat. Genet. 37: 353-355. PubMed ID: 15793586

Bucciarelli, E., et al. (2009). Drosophila Dgt6 interacts with Ndc80, Msps/XMAP215, and gamma-tubulin to promote kinetochore-driven MT formation. Curr. Biol. 19(21): 1839-45. PubMed ID: 19836241

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

Dammermann, A., et al. (2004). Centriole assembly requires both centriolar and pericentriolar material proteins. Dev. Cell 7: 815-829. PubMed ID: 15572125

de Anda, F. C., et al. (2005). Centrosome localization determines neuronal polarity. Nature 436: 704-708. PubMed ID: 16079847

Delattre, M., Canard, C. and Gonczy, P. (2006). Sequential protein recruitment in C. elegans centriole formation. Curr. Biol. 16(18): 1844-9. PubMed ID: 16979563

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

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

Kirkham, M., Muller-Reichert, T., Oegema, K., Grill, S. and Hyman, A. A. (2003). SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112: 575-587. PubMed ID: 12600319

Kouprina, N., et al. (2005). The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein, Hum. Mol. Genet. 14: 2155-2165. PubMed ID: 15972725

Leidel, S. and Göczy, P. (2003). SAS-4 is essential for centrosome duplication in C. elegans and is recruited to daughter centrioles once per cell cycle. Dev. Cell 4(3): 431-9. PubMed ID: 12636923

Leidel, S., Delattre, M., Cerutti, L., Baumer, K. and Gonczy, P. (2005a). SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells. Nature Cell Biol. 7: 115-125. PubMed ID: 15665853

Leidel, S. and Gonczy, P. (2005b). Centrosome duplication and nematodes: recent insights from an old relationship. Dev. Cell 9: 317-325. PubMed ID: 16139223

Lucas, E. P. and Raff, J. W. (2007). Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila Centrosomin. J. Cell Biol. 178: 725-732. PubMed ID: 17709428

Maiato, H., et al. (2002). MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity. J. Cell Biol. 157: 749-760. PubMed ID: 12034769

Maiato, H., Khodjakov, A. and Rieder, C. L. (2005). Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres. Nat. Cell Biol. 7: 42-47. PubMed ID: 15592460

Martinez-Campos, M., et al. (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

Megraw, T. L., et al. (2001). Zygotic development without functional mitotic centrosomes. Curr. Biol. 11: 116-120. PubMed ID: 11231128

Mottier-Pavie, V., Cenci, G., Vernì, F., Gatti, M. and Bonaccorsi, S. (2011). Phenotypic analysis of misato function reveals roles of noncentrosomal microtubules in Drosophila spindle formation. J. Cell Sci. 124(Pt 5): 706-17. PubMed ID: 21285248

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

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

Peel, N., Stevens, N. R., Basto, R. and Raff, J. W. (2007). Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation. Curr. Biol. 17(10): 834-43. PubMed ID: 17475495

Pelletier, L., et al. (2006). Centriole assembly in Caenorhabditis elegans. Nature 444(7119): 619-23. PubMed ID: 17136092

Venkei, Z. G. and Yamashita, Y. M. (2015). The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis. Development 142: 62-69. PubMed ID: 25480919

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

Woods, C. G. et al. Bond, J. and Enard, W. (2005). Autosomal recessive primary microcephaly (MCPH): a review of clinical, molecular, and evolutionary findings. Am. J. Hum. Genet. 76: 717-728. PubMed ID: 15806441

Zheng, X., Gooi, L. M., Wason, A., Gabriel, E., Mehrjardi, N. Z., Yang, Q., Zhang, X., Debec, A., Basiri, M. L., Avidor-Reiss, T., Pozniakovsky, A., Poser, I., Saric, T., Hyman, A. A., Li, H. and Gopalakrishnan, J. (2014). Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis. Proc Natl Acad Sci U S A. PubMed ID: 24385583

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date revised: 12 June 2021

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