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Centriole continuity: out with the new, in with the old
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ScienceDirect Centriole continuity: out with the new, in with the old Alice Meunier1,2,3 and Nathalie Spassky1,2,3 Centrioles are essential microtubule-based organelles, typically present in pairs, which organize cilia and centrosomes. Their mode of biogenesis is unique for a subcellular organelle since, during cell division, each pre-existing centriole guides the formation of a new one, a process that is coordinated with DNA replication. After centriole duplication, the new centrosomes migrate in opposite direction and localize at each pole of the mitotic spindle. This singular dynamics led to think that centrioles were permanent self-replicating structures coordinating cytoplasm and nuclear division. This vision then fell gradually into disuse when centrioles were shown to be capable to form de novo, in the absence of a pre-existing structure, and to be actually dispensable for cell division. However, new data, which are reviewed here, have breathed new life into the old ideas. Addresses 1 Ecole Normale Supe´rieure, Institut de Biologie de l’ENS, IBENS, F-75005 Paris, France 2 CNRS, UMR8197, F-75005 Paris, France 3 Inserm, U1024, F-75005 Paris, France Corresponding author: Meunier, Alice ([email protected])
Current Opinion in Cell Biology 2016, 38:60–67 This review comes from a themed issue on Cell architecture
observed by two cytologists, Van Beneden and Boveri in the late 18’s who hypothesized its role as an organizer of cell division by its capacity to replicate once per cell cycle and to associate with the mitotic spindle [5–8]. This hypothesis was rapidly challenged when proper mitosis was found to occur in flowering plants without centrioles [9,10]. In animal cells, the hypothesis was tackled experimentally long after that, when the disturbance of centrosome–spindle pole interaction [11] or the removal of centrosomes [12–15] was shown not to affect proper spindle formation. It was hypothesized [11], and a long time later confirmed [16–17,18], that the microtubules have the ability to self-organize into bipolar spindles around chromosomes. Since then, it has been largely confirmed that centrioles are not required for mitosis. Yet, the removal of centrioles in animal cells disturbs the timeliness, fidelity and polarity of cell division suggesting that, in organisms possessing centrioles, these structures have been co-opted to optimize chromosome segregation and cytokinesis and to drive asymmetric division [13,15,17,18,19–25]. The same experiments showing that centrioles were dispensable for mitosis suggested that they were instead required for G1/S progression [13–15]. This hypothesis has been disproved later on and will be discussed here [17,20–23,26].
Edited by Margaret L Gardel and Matthieu Piel
http://dx.doi.org/10.1016/j.ceb.2016.02.007 0955-0674/# 2016 Elsevier Ltd. All rights reserved.
Introduction Centrioles are half micron-sized structures usually composed of nine triplets of microtubules organized in a barrelshaped conformation. These organelles are conserved across the eukaryotic tree although they are absent in higher plants and higher fungi [1]. In animal cells, centrioles are typically present by pairs. The ancestral function of centrioles is to nucleate cilia, which are sensory, and/or motile cell projections essential for the processing of numerous signaling pathways [2] and for the propelling of physiological fluids [3]. Ciliopathies, which lead to developmental alterations of several organs, are often caused by defects in centriole-associated proteins [4]. Associated to the pericentriolar material (PCM), the centriole pair forms the centrosome, the main microtubuleorganizing center of the cell. The centrosome was first Current Opinion in Cell Biology 2016, 38:60–67
Like chromosomes, the pair of centrioles is inherited during cell division and after duplication of the preexisting structures. Centriole nucleation occurs through the formation of a procentriole orthogonally to the proximal part of each parental centriole, giving rise to two centrosomes. This stereotypical duplication pattern is observed in all the cells with a pre-existing centriolar structure. Intriguingly, unlike DNA, the pre-existing structure seems to be dispensable since centrioles are proposed to appear de novo after elimination of the preexisting ones [24,27,28,29], in acentrosomal species such as lower plants, amebo-flagellates or planarian flatworms when cilia are needed [30–32], in parthenogenetic species [33,34] or in vertebrate multiciliated cells amplifying massively their centrioles [35,36]. Here we review recent data supporting evidence that, in vertebrates, cell cycle progression is actually hindered in the absence of centrioles, thus restricting the arising of acentriolar cells. We further discuss new evidences showing that even in multiciliated cells in which tens of centrioles are formed, the two pre-existing centrioles scaffold the formation of the new ones. Together, these new studies suggest the existence of mechanisms favoring the perpetuation of continuity over centriole generation. We further outline important questions that remain to be addressed to elucidate the rationale of this apparent continuity. www.sciencedirect.com
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Centrioles and cell cycle progression A long-standing and controverted hypothesis
The first experiments suggesting the existence of a checkpoint monitoring the presence of a centrosome during cell division were achieved by removing centrosomes by microsurgery or laser ablation. If acentrosomal cells were able to go through one round of mitosis without centrioles, they were nevertheless unable to progress through S phase and arrested in G1 [13,15]. These experiments in monkey cells changed the paradigm of the role of the centrosome in cell division: it was not necessary to build the mitotic spindle but appeared to be required for cell cycle progression. In the mean time, studies in which centrosomal proteins were knocked-down highlighted a P38–P53 dependent G1 arrest suggesting that not only centrosome presence but centrosome integrity are needed for G1/S progression [37–39]. Interestingly, centrosome removal in transformed HeLa cells, which do not express P53, did not impede G1/S transition [40]. In these
cells, centrioles appeared de novo during the following divisions. These studies led to propose the existence of a new P53-dependent cell cycle checkpoint monitoring the absence or dysfunction of the centrosome. This view was then challenged by a study using optimized microsurgery and laser ablation experiments [26]. The authors showed that, when diminishing exposure to 488-nm light, non-transformed human and monkey cells were able to progress through the G1/S transition without a centrosome, suggesting that the previously proposed checkpoint was a stress-artifact. In the mean time, genetic approaches in chicken and mouse, knocking out core regulators of centriole biogenesis allowed the elimination of centrioles and the inhibition of their de novo formation, thus assessing the long-term consequences of centriole absence in cells and tissues during development. Acentriolar chicken cells in culture display a normal G1/S progression but a delay in spindle assembly and an
Figure 1
drosophila
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organism die after birth due to cilia sensory defects
human
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duplication inhibition
mouse - chicken
human - monkey P53-dependant G1-arrest
G1 arrest
P53-dependant apoptosis due to delay in mitosis
Current Opinion in Cell Biology
(a) Penetrance of centriole requirement for cell division in different animal species. Genetically modified Drosophila without centriole were shown to develop into morphologically normal adults. They die because their sensory neurons lack cilia. This indicates that in flies, centrioles are dispensable for somatic cell division. Interestingly though, the spindle assembly is slowed but chromosome segregation is accurate, cytokinesis failures are increased and neuroblast asymmetric divisions are altered [20,21]. It has been recently demonstrated that robust compensatory mechanisms buffer the effect of centrosome loss and account for the near normal development of Drosophila without centriole [85]. In mouse and chicken, cells also display a normal proliferation profile without centriole, except for mitosis which is prolonged due to a delay in spindle assembly [17,22]. This eventually leads to an increase in apoptosis which was shown to be P53-induced in mouse [22,23]. Interestingly, although chicken acentriolar cells exhibit a high rate of chromosomal instability, such defects are not reported in mouse, suggesting that mice has a better machinery to perform acentriolar mitosis [17,22,23]. This is interesting since mouse first zygotic divisions are acentriolar [42,43]. In human and monkey, mechanical removal of centrioles and inhibition of centriole duplication have shown that absence of centriole leads to a quasi-immediate and irreversible P53-dependent G1 arrest [13,15,24,29,40]. Human P53-depleted cells proliferate indefinitely but with a delay in spindle assembly and an increase in cytokinesis failures. Unlike mouse cells, an increase in chromosome segregation errors is also observed [24,29]. (b) Scheme depicting the effects of centriole duplication failure on cell cycle progression in human culture cells based on [24,29]. Inhibition of Plk4 or its inducible degradation induces duplication failure and a loss of centrioles over cell generation. Single-cell lineage tracing reports that the progressive loss of centrioles is accompanied by an increasing proportion of cells arresting in G1. If one study suggests that a large proportion of the cells with one centriole already stops in the next G1 [29], the other suggests that the arrest occurs in cells deprived of centrioles, depending on the content of the mother cell [24]. www.sciencedirect.com
Current Opinion in Cell Biology 2016, 38:60–67
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increase in cell apoptosis [41]. Consistent with the finding that the first divisions of the mouse zygote are acentriolar [42,43], genetically modified mice for the core components of centrioles, SAS-4 and STIL, showed that acentriolar cells undergo normal cell cycle progression. However, here again, mitotic delay and P53-dependent apoptosis were observed, leading to embryo death at midgestation [22,44]. Consistently acentriolar neural progenitor of conditional SAS-4 mutant mice undergo a widespread P53-dependent apoptosis leading to microcephaly [23]. In chicken and mice, the G1/S ‘acentriolar’ checkpoint does not seem to exist. However, significant proportion of acentriolar cells, either more sensitive or having accumulated stress over several cell cycles, undergo apoptosis (Figure 1a). Altogether, these mechanical or genetic ablations of centrioles in human, chicken and mice put an end to the hypothesis, supported by the first studies, of the existence of a checkpoint monitoring the absence/integrity of the centrosome. New tools: back to the old hypothesis
Recently, two studies significantly furthered our understanding by developing new tools driving an inducible, persistent and reversible centriole depletion in large populations of human cultured cells. In the first study, the authors inhibited the kinase activity of Plk4, a core regulator of centriole biogenesis, using a newly developed pharmacological compound [24]. In the second study, the researchers induced a rapid degradation of Plk4 through an auxin-inducible degron system [18]. Both approaches induced a failure in centriole duplication leading to the progressive and traceable loss of fluorescently tagged centrioles. They led to the same conclusion: centriole loss leads to an irreversible P53-dependent G1 arrest in human cultures. Consistent with the P53 dependency, cancerous cell lines do not arrest in G1, as suggested by earlier experiments [40], and proliferate indefinitely without centriole, albeit at a lower rate due to prolonged mitosis and apoptosis induced by increased mitotic errors. Cancerous cells thus divide more efficiently with centrioles but, in sharp contrast with healthy cells, do not require centriole to divide. In these studies, cells were allowed to proceed through several rounds of division after inhibiting centriole duplication (Figure 1b). The first generation of daughter cells harbors one centriole. While the first study suggests that they all progress through S-phase and division [24], the other suggests that 50% already stops in G1 [18]. At the second generation, cells harbor either one or zero centriole. Tracing individually each cell indicates that, regardless of their centriolar content, around 30% of them arrest in G1 [24]. Then, after the third generation, 70% of the progeny of the acentriolar cells observe a G1-arrest [24]. In other terms, depending on whether the mother cell has a centriole or not, the acentriolar daughter will stop in 30% or 70% of the time, respectively. This suggests that Current Opinion in Cell Biology 2016, 38:60–67
the G1 arrest is determined in the mother cell and depends on its centriolar content. Laser ablation experiments had previously shown that centrosome removal in metaphase is sufficient to trigger G1 arrest in the daughter cells [14] suggesting that the decision could be made during late mitosis in the mother cell. Interestingly, both studies showed that the G1-arrest is irreversible. Indeed, recuperation of Plk4 activity does not lead to S-phase reentry. This is consistent with the notion that centrioles are needed to downregulate P53 and transit to S-phase; and with previous observation that Plk4 dependent de novo formation would need a S-phase to occur [26]. Interestingly, the reported G1-arrest is independent of the P38 stress pathway and is not due to previously described P53-inducing stress (DNA damage, mitotic delay, aneuploidy due to decreased mitotic fidelity, hippo pathway, excessive oxidative stress) [18,24]; which suggests that a new type of P53-dependent cell cycle checkpoint drives the G1-arrest. The PCM has been recently shown to be a highly organized network of proteins [45–47], endowed with self-assembly properties [48], but structured by the centrioles [17,49,50]. One hypothesis could be that the absence of one or both mature centrioles could differentially alter microtubule anchorage/organization, which could be sensed as deleterious by the P53 pathway during mitosis. An alternative explanation of the ‘dose dependency’ of the G1-arrest may rely on centriole bound regulators that would negatively regulate P53. Re-expression of centriolar/centrosomal components in acentriolar cells to test if and which minimal components can trigger the S phase could be a way to unravel this new ‘checkpoint’. The penetrance of centriole requirement for cell cycle progression seems to depend on organisms (Figure 1a), although inducible centriole depletion still need to be assessed in non-human vertebrates. In human at least, it seems now clear that P53 senses the centriole content and stops quasi-immediately cell cycle progression in the absence of centrioles (Figure 1b). This mechanism favors the motherly inherited pathway for centriole acquisition over a de novo pathway, known to drive erratic production of a varying number of centrioles [18,24,26]. Centriole loss or supernumerary centrioles decrease mitotic fidelity and affect primary cilium nucleation, defects associated with cancer and developmental disorders [51]. This checkpoint may thus be a key protector of tissue homeostasis.
Centriole continuity Templated or spatially restricted de novo generation of centrioles?
As we have mentioned above, in human at least, a checkpoint ensures that centrioles are inherited from the mother cell. Yet, they must be transmitted with their correct 9-fold symmetrical structure, template of the cilium axoneme. The ‘preservation’ of centriole structure www.sciencedirect.com
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was initially proposed to be granted by the growth and division of the pre-existing organelle [8]. A paradox arrived when centrioles were found to appear de novo in plants suggesting that other mechanisms were ensuring centriolar structure integrity [8]. The advent of electron microscopy revealed that duplicating centrioles grow orthogonally and at a distance from the wall of the preexisting centriolar structure, which definitively excluded the growth and division hypothesis. Then, de novo centriole formation after laser ablation of the pre-existing ones [27,28] together with the finding that the cartwheel structure that scaffolds the 9-fold centriole symmetry was able to self assemble through the oligomerization property of its major component SAS6 [52,53,86], put the final nail into the coffin of the hypothesis that structural information is provided by the pre-existing organelle. Today, the classical view is that the ‘parental’ centriole constitutes a cellular platform, at the center of the microtubule network, where the emergence of an ‘initiation site’, driven by a local equilibrium of regulatory and structural proteins, is facilitated. This would allow the spatial, temporal and numerical control of daughter centriole formation [54]. However two recent studies 55,56 provide data reviving the old theory by supporting a model proposed 20 years ago [57] in which the parental organelle would function as a reservoir transmitting structural information to its progeny. Using super-resolution microscopy in human cells, the authors show that SAS-6, or its self-oligomerization mutant, is recruited to the lumen of the parental centriole during S-phase, before being released at the onset of centriole duplication. Blocking the recruitment or release of luminal SAS-6 prevents subsequent centriole duplication thus suggesting that the cartwheel is shaped by the parental centriole, released and pasted onto the centriolar wall to grow the procentriole (Figure 2a). Consistently, following experiments show that, in SAS-6 depleted acentriolar cells, de novo centriole formation upon SAS6 re-expression is 30% error prone, contrasting with the error-free canonical duplication. This error rate is not increased when a SAS-6 self-oligomerization mutant is expressed instead of the normal protein, challenging the cartwheel self-assembly model [56]. The ordering of new by old cell structures, a process called cytotaxis [58], constitutes an attractive mechanism for centriole formation since it would ensure the transmission of their circumferential polarity [59]. This polarity is critical for cell and tissue development as it is further transmitted to primary or motile cilia axonemes which in turn determine cell polarity and flow directionality [60]. One way of proving this ‘cytotaxis hypothesis’ would be to test whether altered 9-fold structures are inherited by growing procentrioles. Such hypothesis however bumps into the many examples in life diversity where centriole formation occurs in the www.sciencedirect.com
absence of a pre-existing organelle (i.e. parthenogenetic [33,34] or acentrosomal [30–32] species). Would these species be more permissive to centriole structure aberrations? Would additional proteins be expressed to secure an otherwise delicate cartwheel self-assembly? Alternatively, a long-standing hypothesis is that physical continuity would be maintained by undetectable centriole remnants transmitted over cell division in case of need [8]. Arguments standing for this hypothesis are that physiological or induced de novo centriole formation can occur very rapidly [61,62], and often occur in predetermined sites, associated with nuclear envelope [34,42,63,64] or below the plasma membrane [65–67]. Yet hard to prove, one could hypothesize that membrane-stored nanoscopic seeds are inherited through cell division and stabilized to serve as template when centrioles are needed. Aside from taking a possible structural information from the pre-existing centriole, duplicating procentrioles are engaged, that is, tightly linked to their mother until the end of mitosis [68]. The mother centrioles organize the PCM and interact with microtubules, characteristics that drive them to the spindle poles at mitosis. Dragged by their mothers, the daughters follow. This ensures an equal and semi-conservative distribution of the four centrioles between the two daughter cells. Since mother centrioles have an age difference of at least one generation and daughter centrioles are born during the same cell cycle, daughter cells therefore inherit organelles that are half twin/half age asymmetric. At the cellular level, the mother and daughter centrioles grant the cell with different characteristics that are exploited to drive differential functions [69–71]. At the tissue level, age difference between mother centrioles of the daughter cells (defining in this case the mother and daughter centrosomes) causes asynchronous primary cilium growth [72,73] and can be exploited to drive asymmetric division and determine cell fate during development [74]. Centriole semi-conservative inheritance pattern is thus critical to drive different aspects of cell dynamics and tissue development. The physical continuity brought by the duplication process may therefore be a way to ensure centriole lineage conservation. Centriole continuity: how far?
This mode of biogenesis and inheritance begs the question of how vertebrate differentiating multiciliated cells produce up to 200 centrioles to nucleate motile cilia and propel physiological fluids in the oviducts, airways and brain epithelia. The first cytologists hypothesized that the centrioles at the base of motile cilia derived from the multiplication of the original centrosomal pair (reviewed in [8]). Then, electron microscopists in the late 1960s analyzed centriole amplification in chick, Xenopus and mammalian multiciliated cells [35,36,75–77] concomitantly with the canonical centriole duplication process in cycling cells [78,79]. In multiciliated cells, multiple Current Opinion in Cell Biology 2016, 38:60–67
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Figure 2
(a)
SAS6 S
G1 (b)
Multiciliated cell maturation Current Opinion in Cell Biology
(a) Model for the templated mode of centriole duplication in cycling cells proposed in [55,56]. SAS-6 dimers (red) are recruited to the lumen of the parental centriole. They organize in a cartwheel configuration driven by their interaction with the 9-fold inner structure of the centriole. The assembled cartwheel is then released from the lumen and relocates on the proximal wall of the parental centriole where it templates procentriole formation. (b) Model for centriole amplification in vertebrate multiciliated cells based on [70,83,84]. An unknown mechanism leads to the accumulation of the core deuterosome proteins Deup1 and Ccdc78 (green) at the centrosomal daughter centriole. Deup1 and Ccdc78 recruit centriole biogenesis regulators Cep152 and Plk4 (not illustrated). This promotes the nucleation of the first procentriole. Accumulation continues from the same active site. The deuterosome grows and the second procentriole arises and so on, until the spherical deuterosome detaches from the centriolar wall. The process starts again to form another deuterosome and nucleate new procentrioles. Multiple deuterosomes can form concurrently around the parental centriole. Once the final number of centriole is reached, the procentrioles grow synchronously from all the deuterosomes, and mature to nucleate the motile ciliary tuft. Deuterosomes circumvent steric constraints imposed by the parental centriole wall and allow a massive albeit centriole-guided formation of new centrioles.
centrioles were seen growing on electron dense spherical structures called deuterosomes evoking a de novo generation of centrioles like in plant male gametes [30,80]. Yet, they observed that granule-like material accumulated around centrosomal centrioles at the beginning of the process, as during canonical duplication. Most of the authors thus retained the hypothesis that the multiple centrioles had a ‘centriolar origin’ [75–77]. Indeed, one study on chick tracheal epithelium provided very convincing morphological evidence supporting the hypothesis that the clusters of centrioles growing on the deuterosomes were primarily nucleated from centrosomal centrioles [75]. Then, the lack of dynamic observation Current Opinion in Cell Biology 2016, 38:60–67
made this process difficult to understand further. Consistent with the notion that only two pre-existing centrioles could not sustain the nucleation of more than a hundred of centrioles, the following generations of researchers kept the de novo hypothesis for massive centriole production in vertebrates. Recently, new studies in Xenopus and mice, taking advantage of the development of new imaging techniques, have led to reconsider the old ideas. First, the transcriptional profile of ciliated cells together with functional experiments showed that common regulators are involved in both centriole duplication and massive centriole www.sciencedirect.com
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amplification [81,82,83]. Most importantly, a paralogue of the canonical centriole duplication protein Cep63 was identified as being the core component of deuterosomes and named Deup1 [84]. Deup1 has the singularity, when overexpressed, even in bacteria, to drive the formation of deuterosome-like structures suggesting a self-oligomerizing activity [84]. Like Cep63 in cycling cells, Deup1 in multiciliated cells recruits Cep152 and Plk4, core regulators of centriole biogenesis, and promotes centriole nucleation [84]. The similarity with centriole duplication does not stop here since superresolution microscopy showed that, during multiciliated cell maturation, Deup1 accumulates at the proximal part of a centrosomal centriole, where its paralogue Cep63 normally localizes during centriole duplication in cycling cells [70]. By correlative electron microscopy, deuterosomes are indeed seen growing from a proximal pre-existing centriolar wall. Consistent with the recruitment of Cep152 and Plk4, this accumulation is rapidly followed by the apparition of procentrioles on the growing deuterosomes [70]. Live imaging showed that deuterosomes loaded with multiple procentrioles then detach from the parental centriolar wall allowing a new one to form and so on until the final number of centrioles is reached [70]. It thus seems that the same toolbox is used, at the same location, for centriole duplication and amplification. Interestingly though, amplification occurs on the wall of the daughter centriole suggesting that only the microenvironment provided by the younger parental centriole is permissive for this amplification [70]. This dynamics of formation and release of deuterosome shuttles enables to circumvent the steric constraint imposed by the parental centriolar wall and to drive a massive, albeit centrioleguided, production of centrioles (Figure 2b).
new centriole formation, even when centrioles are massively produced. These new evidences suggest the existence of mechanisms favoring the perpetuation of an apparent uninterrupted lineage from the first centriole of the zygote, inherited from the spermatozoon, to all the centrioles of the organism. The development of drugs and inducible constructs to reversibly eliminate centrioles opens exiting perspectives to understand the functional relevance of this continuity in different cellular and tissular contexts.
Thus, physical continuity between old and new centrioles is preserved, even in the case of massive centriole production. The fact that hundreds centrioles are formed on the wall of a single centriole can be explained by the requirement of a structural information provided by the pre-existing centriole or, maybe more likely, by the hijacking of the duplication process through the exploitation of a permissive characteristic of the younger pre-existing structure.
Concluding remark The attempts to clarify the role of centrioles in cell cycle progression, or in centriole biogenesis, feature ‘‘the complementary forces in the production of scientific knowledge: how techniques [. . .] shape the production of data and theory, and how theoretical expectations shape interpretation of reported observations’’ [8]. The availability of new molecular tools and imaging techniques showed that the absence of centriole is sensed by the P53-pathway as a deleterious stress triggering cell cycle arrest, at least in human cells. In addition, it appears that preexisting centrioles are always used as a building tool for www.sciencedirect.com
Conflict of interest statement The authors declare no competing financial interests.
Acknowledgements The authors thank N Delgehyr for her comments on the manuscript. The work of the authors is supported by the Centre National de la Recherche Scientifique, the Ecole Normale Supe´rieure and the Institut National pour la Sante´ Et la Rercherche Me´dicale, the Agence Nationale de la Recherche (ANRJCJC-15-CE13-0005-01 and ANR-12-BSV4-0006), the European Research Council (Consolidator grant 647466), the Association de Recherche pour le Cancer (ARC, PJA-20131200184), the Fondation pour la Recherche Me´dicale (20140329547) and the Fondation Pierre-Gilles de Gennes (FPGG03).
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24. Wong YL et al.: Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science 2015, 348:1155-1160. The authors developed a highly selective inhibitor against Plk4, a protein kinase required for centriole biogenesis. This allowed a rapid and reversible Plk4 inhibition leading to centriole duplication failure and progressive and traceable centriole depletion in human culture cells. They show that centriole depletion leads to an irreversible P53-dependent cell cycle arrest in G1. Interestingly, the authors have also tested the consequences of centriole depletion in cancer cell lines that normally form supernumerary centrioles. They found that, like other cancerous cells, they divide indefinitely without centrioles and that no correlation exists between centriole amplification state before drug treatment and proliferation potential after centriole depletion.
43. Courtois A, Schuh M, Ellenberg J, Hiiragi T: The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development. J Cell Biol 2012, 198:357-370.
25. Piel M, Nordberg J, Euteneuer U, Bornens M: Centrosomedependent exit of cytokinesis in animal cells. Science 2001, 291:1550-1553. 26. Uetake Y et al.: Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells. J Cell Biol 2007, 176:173-182. 27. Khodjakov A et al.: De novo formation of centrosomes in vertebrate cells arrested during S phase. J Cell Biol 2002, 158:1171-1181. 28. Marshall WF, Vucica Y, Rosenbaum JL: Kinetics and regulation of de novo centriole assembly: implications for the mechanism of centriole duplication. Curr Biol 2001, 11:308-317. 29. Lambrus BG et al.: P53 protects against genome instability following centriole duplication failure. J Cell Biol 2015, 210:63-77. See Ref. [18] 30. Hepler PK: The blepharoplast of Marsilea: its de novo formation and spindle association. J Cell Sci 1976, 21:361-390. Current Opinion in Cell Biology 2016, 38:60–67
36. Steinman RM: An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis. Am J Anat 1968, 122:19-55. 37. Srsen V, Gnadt N, Dammermann A, Merdes A: Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle. J Cell Biol 2006, 174:625-630.
44. David A et al.: Lack of centrioles and primary cilia in STILS/S mouse embryos. Cell Cycle 2014, 13:2859-2868. 45. Lawo S, Hasegan M, Gupta GD, Pelletier L: Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat Cell Biol 2012, 14:11481158. 46. Sonnen KF, Schermelleh L, Leonhardt H, Nigg EA: 3D-structured illumination microscopy provides novel insight into architecture of human centrosomes. Biol Open 2012, 1:965-976. 47. Fu J, Glover DM: Structured illumination of the interface between centriole and peri-centriolar material. Open Biol 2012, 2:120104. 48. Woodruff JB et al.: Regulated assembly of a supramolecular centrosome scaffold in vitro. Science 2015, 348:808-812. 49. Madarampalli B et al.: ATF5 connects the pericentriolar materials to the proximal end of the mother centriole. Cell 2015, 1–13 http://dx.doi.org/10.1016/j.cell.2015.06.055. The authors show that ATF-5, a previously described transcription factor, forms a 9-foci symmetrical ring around the mother centriole and is responsible for the mother centriole-directed pericentriolar material accumulation. Interestingly, they found that ATF-5 depletion causes structural defects of mother and daughter centrioles suggesting that PCM is important to maintain structural integrity of both mother and daughter centrioles. 50. Bobinnec Y et al.: Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells. J Cell Biol 1998, 143:1575-1589. www.sciencedirect.com
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51. Nano M, Basto R: The Janus soul of centrosomes: a paradoxical role in disease? Chromosome Res 2015 http:// dx.doi.org/10.1007/s10577-015-9507-3. 52. Kitagawa D et al.: Structural basis of the 9-fold symmetry of centrioles. Cell 2011, 144:364-375. 53. van Breugel M et al.: Structures of SAS-6 suggest its organization in centrioles. Science 2011, 331:1196-1199. 54. Song MH, Miliaras NB, Peel N, Connell KFO: Centrioles: some self-assembly required. Curr Opin Cell Biol 2009, 20:688-693. 55. Fong CS, Kim M, Yang TT, Liao JC, Tsou MFB: SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication. Dev Cell 2014, 30:238-245. Using various SAS-6 constructs and super-resolution microscopy, the authors show that SAS-6 is recruited to the lumen of the parental centriole during S-phase. Blocking the recruitment or release of the luminal SAS-6 hinders the subsequent centriole growth from the parental centriole wall. They also show that during procentriole growth, the lumen of the parental centriole is inaccessible and suggest that it may prevent the formation of additional procentrioles. 56. Wang W et al.: De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly. Elife 2015, 6:1-13. The authors generate P53-/- human acentriolar cell lines through CRISP/ Cas9 SAS-6 depletion to study de novo centriole formation upon SAS-6 re-expression. They found that structurally normal centrioles can be produced de novo after SAS-6 self-oligomerization mutant re-expression. In addition they report that both self-oligomerization mutant and wild-type protein re-expression drive centriole biogenesis with a 30% error rate. The authors suggest that centriole biogenesis does not strictly depend on SAS-6 self-assembly and that pre-existing centriolar structures are needed to avoid centriole mis-shaping.
70. Al Jord A et al.: Centriole amplification by mother and daughter centrioles differs in multiciliated cells. Nature 2014, 516:104107. Combining live imaging and high-resolutive microscopic approaches, the authors unveil the dynamics of centriole amplification in mouse brain multiciliated cells. They report that centrioles are massively amplified from the daughter centriole of the centrosome through successive rounds of deuterosome formation. 71. Pelletier L, Yamashita YM: Centrosome asymmetry and inheritance during animal development. Curr Opin Cell Biol 2012, 24:541-546. 72. Anderson CT, Stearns T: Centriole age underlies asynchronous primary cilium growth in mammalian cells. Curr Biol 2009, 19:1498-1502. 73. Paridaen JTML, Wilsch-Bra¨uninger M, Huttner WB: Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division. Cell 2013, 155:333-344. 74. Reina J, Gonzalez C: When fate follows age: unequal centrosomes in asymmetric cell division. Philos Trans R Soc Lond B Biol Sci 2014, 369:20130466. 75. Kalnins VI, Porter KR, Kalnins PK VI: Centriole replication during ciliogenesis in the chick tracheal epithelium. Z Zellforsch Mikrosk Anat 1969, 100:1-30. 76. Dirksen ER: Centriole morphogenesis in developing ciliated epithelium of the mouse oviduct. J Cell Biol 1971, 51:286-302. 77. Anderson R, Brenner R: The formation of basal bodies (centrioles) in the rhesus monkey oviduct. J Cell Biol 1971, 50:10-34.
57. Mignot JP: New hypothesis on the replication of centrioles and basal bodies. C R Acad Sci III 1996, 319:1093-1099.
78. Murray RG, Murray AS, Pizzo A: The fine structure of mitosis in rat thymic lymphocytes. J Cell Biol 1965, 26:601-619.
58. Beisson J, Sonneborn TM: Cytoplasmic inheritance of the organization of the cell cortex in Paramecium Aurelia. Proc Natl Acad Sci U S A 1965, 53:275-282.
79. Robbins E, Jentzsch G, Micali A: The centriole cycle in synchronized Hela cells. J Cell Biol 1968, 36:329-339.
59. Beisson J, Jerka-Dziadosz M: Polarities of the centriolar structure: morphogenetic consequences. Biol Cell 1999, 91:367-378. 60. Pearson CG: Choosing sides — asymmetric centriole and basal body assembly. J Cell Sci 2014, 127:2803-2810. 61. Palazzo RE, Vaisberg E, Cole RW, Rieder CL: Centriole duplication in lysates of Spisula solidissima oocytes. Science 1992, 256:219-221. 62. Peel N, Stevens NR, Basto R, Raff JW: Overexpressing centriolereplication proteins in vivo induces centriole overduplication and de novo formation. Curr Biol 2007, 17:834-843. 63. Renaud FL, Swift H: The development of basal bodies and flagella in Allomyces arbusculus. J Cell Biol 1964, 23:339-354. 64. Perkins FO: Formation of centriole and centriole-like structures during meiosis and mitosis in Labyrinthula sp. (Rhizopodea Labyrinthulida). J Cell Sci 1970, 6:629-653. 65. Fleury A, Le Guyader H, Iftode F, Laurent M, Bornens M: A scaffold for basal body patterning revealed by a monoclonal antibody in the hypotrich ciliate Paraurostyla weissei. Dev Biol 1993, 157:285-302. 66. Cavalier-Smith T: Basal body and flagellar development during the vegetative cell cycle and the sexual cycle of Chlamydomonas reinhardii. J Cell Sci 1974, 16:529-556. 67. Dingle AD, Fulton C: Development of the flagellar apparatus in Naegleria. J Cell Biol 1966, 31:43-54. 68. Agircan FG, Schiebel E, Mardin BR: Separate to operate: control of centrosome positioning and separation. Philos Trans R Soc B Biol Sci 2014, 369:20130461. 69. Piel M, Meyer P, Khodjakov A, Rieder CL, Bornens M: The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells 7. J Cell Biol 2000, 149:317-329.
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80. Mizukami I, Gall J: Centriole replication II. Sperm formation in the Fern, Marsilea, and the Cycad, Zamia. J Cell Biol 1966, 97–111. 81. Vladar EK, Stearns T: Molecular characterization of centriole assembly in ciliated epithelial cells. J Cell Biol 2007, 178:31-42. 82. Hoh RA, Stowe TR, Turk E, Stearns T: Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease. PLoS One 2012, 7:e52166. 83. Klos Dehring DA et al.: Deuterosome-mediated centriole biogenesis. Dev Cell 2013, 27:103-112. The authors identify the deuterosomes as a site for the localization of the core regulator of centriole biogenesis Cep152 and Plk4. They identify a new protein Ccdc78 that is associated with deuterosomes and procentrioles. They show that Ccdc78-mediated recruitment of Cep152 to the deuterosomes is required for centriole amplification in multiciliated cells. 84. Zhao H et al.: The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis. Nat Cell Biol 2013, 14:1-13. The authors identify Ccdc67 as the first deuterosome-specific protein and called it Deup1. They show that Deup1 diverged from the core regulator of centriole duplication Cep63 during vertebrate evolution. Interestingly, they show that, upon high expression in bacteria, this protein assemble ring-like structures resembling deuterosomes, thus suggesting that Deup1 alone is able to govern deuterosome formation. Like Cep63, Deup1 recruits Cep152 and Plk4, thereby allowing massive centriole biogenesis from deuterosomes in vertebrate multiciliated cells. 85. Poulton JS, Cuningham JC, Peifer M: Acentrosomal drosophila epithelial cells exhibit abnormal cell division, leading to cell death and compensatory proliferation. Dev Cell 2014, 30:731-745. 86. Hilbert M, Erat MC, Hachet V, Guichard P, Blank ID, Flu¨ckiger I, Slater L, Lowe ED, Hatzopoulos GN, Steinmetz MO, Go¨nczy P, Vakonakis I: Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry. Proc Natl Acad Sci U S A 2013, 110:11373-11378.
Current Opinion in Cell Biology 2016, 38:60–67
ScienceDirect Centriole continuity: out with the new, in with the old Alice Meunier1,2,3 and Nathalie Spassky1,2,3 Centrioles are essential microtubule-based organelles, typically present in pairs, which organize cilia and centrosomes. Their mode of biogenesis is unique for a subcellular organelle since, during cell division, each pre-existing centriole guides the formation of a new one, a process that is coordinated with DNA replication. After centriole duplication, the new centrosomes migrate in opposite direction and localize at each pole of the mitotic spindle. This singular dynamics led to think that centrioles were permanent self-replicating structures coordinating cytoplasm and nuclear division. This vision then fell gradually into disuse when centrioles were shown to be capable to form de novo, in the absence of a pre-existing structure, and to be actually dispensable for cell division. However, new data, which are reviewed here, have breathed new life into the old ideas. Addresses 1 Ecole Normale Supe´rieure, Institut de Biologie de l’ENS, IBENS, F-75005 Paris, France 2 CNRS, UMR8197, F-75005 Paris, France 3 Inserm, U1024, F-75005 Paris, France Corresponding author: Meunier, Alice ([email protected])
Current Opinion in Cell Biology 2016, 38:60–67 This review comes from a themed issue on Cell architecture
observed by two cytologists, Van Beneden and Boveri in the late 18’s who hypothesized its role as an organizer of cell division by its capacity to replicate once per cell cycle and to associate with the mitotic spindle [5–8]. This hypothesis was rapidly challenged when proper mitosis was found to occur in flowering plants without centrioles [9,10]. In animal cells, the hypothesis was tackled experimentally long after that, when the disturbance of centrosome–spindle pole interaction [11] or the removal of centrosomes [12–15] was shown not to affect proper spindle formation. It was hypothesized [11], and a long time later confirmed [16–17,18], that the microtubules have the ability to self-organize into bipolar spindles around chromosomes. Since then, it has been largely confirmed that centrioles are not required for mitosis. Yet, the removal of centrioles in animal cells disturbs the timeliness, fidelity and polarity of cell division suggesting that, in organisms possessing centrioles, these structures have been co-opted to optimize chromosome segregation and cytokinesis and to drive asymmetric division [13,15,17,18,19–25]. The same experiments showing that centrioles were dispensable for mitosis suggested that they were instead required for G1/S progression [13–15]. This hypothesis has been disproved later on and will be discussed here [17,20–23,26].
Edited by Margaret L Gardel and Matthieu Piel
http://dx.doi.org/10.1016/j.ceb.2016.02.007 0955-0674/# 2016 Elsevier Ltd. All rights reserved.
Introduction Centrioles are half micron-sized structures usually composed of nine triplets of microtubules organized in a barrelshaped conformation. These organelles are conserved across the eukaryotic tree although they are absent in higher plants and higher fungi [1]. In animal cells, centrioles are typically present by pairs. The ancestral function of centrioles is to nucleate cilia, which are sensory, and/or motile cell projections essential for the processing of numerous signaling pathways [2] and for the propelling of physiological fluids [3]. Ciliopathies, which lead to developmental alterations of several organs, are often caused by defects in centriole-associated proteins [4]. Associated to the pericentriolar material (PCM), the centriole pair forms the centrosome, the main microtubuleorganizing center of the cell. The centrosome was first Current Opinion in Cell Biology 2016, 38:60–67
Like chromosomes, the pair of centrioles is inherited during cell division and after duplication of the preexisting structures. Centriole nucleation occurs through the formation of a procentriole orthogonally to the proximal part of each parental centriole, giving rise to two centrosomes. This stereotypical duplication pattern is observed in all the cells with a pre-existing centriolar structure. Intriguingly, unlike DNA, the pre-existing structure seems to be dispensable since centrioles are proposed to appear de novo after elimination of the preexisting ones [24,27,28,29], in acentrosomal species such as lower plants, amebo-flagellates or planarian flatworms when cilia are needed [30–32], in parthenogenetic species [33,34] or in vertebrate multiciliated cells amplifying massively their centrioles [35,36]. Here we review recent data supporting evidence that, in vertebrates, cell cycle progression is actually hindered in the absence of centrioles, thus restricting the arising of acentriolar cells. We further discuss new evidences showing that even in multiciliated cells in which tens of centrioles are formed, the two pre-existing centrioles scaffold the formation of the new ones. Together, these new studies suggest the existence of mechanisms favoring the perpetuation of continuity over centriole generation. We further outline important questions that remain to be addressed to elucidate the rationale of this apparent continuity. www.sciencedirect.com
Centriole continuity: out with the new, in with the old Meunier and Spassky 61
Centrioles and cell cycle progression A long-standing and controverted hypothesis
The first experiments suggesting the existence of a checkpoint monitoring the presence of a centrosome during cell division were achieved by removing centrosomes by microsurgery or laser ablation. If acentrosomal cells were able to go through one round of mitosis without centrioles, they were nevertheless unable to progress through S phase and arrested in G1 [13,15]. These experiments in monkey cells changed the paradigm of the role of the centrosome in cell division: it was not necessary to build the mitotic spindle but appeared to be required for cell cycle progression. In the mean time, studies in which centrosomal proteins were knocked-down highlighted a P38–P53 dependent G1 arrest suggesting that not only centrosome presence but centrosome integrity are needed for G1/S progression [37–39]. Interestingly, centrosome removal in transformed HeLa cells, which do not express P53, did not impede G1/S transition [40]. In these
cells, centrioles appeared de novo during the following divisions. These studies led to propose the existence of a new P53-dependent cell cycle checkpoint monitoring the absence or dysfunction of the centrosome. This view was then challenged by a study using optimized microsurgery and laser ablation experiments [26]. The authors showed that, when diminishing exposure to 488-nm light, non-transformed human and monkey cells were able to progress through the G1/S transition without a centrosome, suggesting that the previously proposed checkpoint was a stress-artifact. In the mean time, genetic approaches in chicken and mouse, knocking out core regulators of centriole biogenesis allowed the elimination of centrioles and the inhibition of their de novo formation, thus assessing the long-term consequences of centriole absence in cells and tissues during development. Acentriolar chicken cells in culture display a normal G1/S progression but a delay in spindle assembly and an
Figure 1
drosophila
(a)
organism die after birth due to cilia sensory defects
human
(b)
duplication inhibition
mouse - chicken
human - monkey P53-dependant G1-arrest
G1 arrest
P53-dependant apoptosis due to delay in mitosis
Current Opinion in Cell Biology
(a) Penetrance of centriole requirement for cell division in different animal species. Genetically modified Drosophila without centriole were shown to develop into morphologically normal adults. They die because their sensory neurons lack cilia. This indicates that in flies, centrioles are dispensable for somatic cell division. Interestingly though, the spindle assembly is slowed but chromosome segregation is accurate, cytokinesis failures are increased and neuroblast asymmetric divisions are altered [20,21]. It has been recently demonstrated that robust compensatory mechanisms buffer the effect of centrosome loss and account for the near normal development of Drosophila without centriole [85]. In mouse and chicken, cells also display a normal proliferation profile without centriole, except for mitosis which is prolonged due to a delay in spindle assembly [17,22]. This eventually leads to an increase in apoptosis which was shown to be P53-induced in mouse [22,23]. Interestingly, although chicken acentriolar cells exhibit a high rate of chromosomal instability, such defects are not reported in mouse, suggesting that mice has a better machinery to perform acentriolar mitosis [17,22,23]. This is interesting since mouse first zygotic divisions are acentriolar [42,43]. In human and monkey, mechanical removal of centrioles and inhibition of centriole duplication have shown that absence of centriole leads to a quasi-immediate and irreversible P53-dependent G1 arrest [13,15,24,29,40]. Human P53-depleted cells proliferate indefinitely but with a delay in spindle assembly and an increase in cytokinesis failures. Unlike mouse cells, an increase in chromosome segregation errors is also observed [24,29]. (b) Scheme depicting the effects of centriole duplication failure on cell cycle progression in human culture cells based on [24,29]. Inhibition of Plk4 or its inducible degradation induces duplication failure and a loss of centrioles over cell generation. Single-cell lineage tracing reports that the progressive loss of centrioles is accompanied by an increasing proportion of cells arresting in G1. If one study suggests that a large proportion of the cells with one centriole already stops in the next G1 [29], the other suggests that the arrest occurs in cells deprived of centrioles, depending on the content of the mother cell [24]. www.sciencedirect.com
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62 Cell architecture
increase in cell apoptosis [41]. Consistent with the finding that the first divisions of the mouse zygote are acentriolar [42,43], genetically modified mice for the core components of centrioles, SAS-4 and STIL, showed that acentriolar cells undergo normal cell cycle progression. However, here again, mitotic delay and P53-dependent apoptosis were observed, leading to embryo death at midgestation [22,44]. Consistently acentriolar neural progenitor of conditional SAS-4 mutant mice undergo a widespread P53-dependent apoptosis leading to microcephaly [23]. In chicken and mice, the G1/S ‘acentriolar’ checkpoint does not seem to exist. However, significant proportion of acentriolar cells, either more sensitive or having accumulated stress over several cell cycles, undergo apoptosis (Figure 1a). Altogether, these mechanical or genetic ablations of centrioles in human, chicken and mice put an end to the hypothesis, supported by the first studies, of the existence of a checkpoint monitoring the absence/integrity of the centrosome. New tools: back to the old hypothesis
Recently, two studies significantly furthered our understanding by developing new tools driving an inducible, persistent and reversible centriole depletion in large populations of human cultured cells. In the first study, the authors inhibited the kinase activity of Plk4, a core regulator of centriole biogenesis, using a newly developed pharmacological compound [24]. In the second study, the researchers induced a rapid degradation of Plk4 through an auxin-inducible degron system [18]. Both approaches induced a failure in centriole duplication leading to the progressive and traceable loss of fluorescently tagged centrioles. They led to the same conclusion: centriole loss leads to an irreversible P53-dependent G1 arrest in human cultures. Consistent with the P53 dependency, cancerous cell lines do not arrest in G1, as suggested by earlier experiments [40], and proliferate indefinitely without centriole, albeit at a lower rate due to prolonged mitosis and apoptosis induced by increased mitotic errors. Cancerous cells thus divide more efficiently with centrioles but, in sharp contrast with healthy cells, do not require centriole to divide. In these studies, cells were allowed to proceed through several rounds of division after inhibiting centriole duplication (Figure 1b). The first generation of daughter cells harbors one centriole. While the first study suggests that they all progress through S-phase and division [24], the other suggests that 50% already stops in G1 [18]. At the second generation, cells harbor either one or zero centriole. Tracing individually each cell indicates that, regardless of their centriolar content, around 30% of them arrest in G1 [24]. Then, after the third generation, 70% of the progeny of the acentriolar cells observe a G1-arrest [24]. In other terms, depending on whether the mother cell has a centriole or not, the acentriolar daughter will stop in 30% or 70% of the time, respectively. This suggests that Current Opinion in Cell Biology 2016, 38:60–67
the G1 arrest is determined in the mother cell and depends on its centriolar content. Laser ablation experiments had previously shown that centrosome removal in metaphase is sufficient to trigger G1 arrest in the daughter cells [14] suggesting that the decision could be made during late mitosis in the mother cell. Interestingly, both studies showed that the G1-arrest is irreversible. Indeed, recuperation of Plk4 activity does not lead to S-phase reentry. This is consistent with the notion that centrioles are needed to downregulate P53 and transit to S-phase; and with previous observation that Plk4 dependent de novo formation would need a S-phase to occur [26]. Interestingly, the reported G1-arrest is independent of the P38 stress pathway and is not due to previously described P53-inducing stress (DNA damage, mitotic delay, aneuploidy due to decreased mitotic fidelity, hippo pathway, excessive oxidative stress) [18,24]; which suggests that a new type of P53-dependent cell cycle checkpoint drives the G1-arrest. The PCM has been recently shown to be a highly organized network of proteins [45–47], endowed with self-assembly properties [48], but structured by the centrioles [17,49,50]. One hypothesis could be that the absence of one or both mature centrioles could differentially alter microtubule anchorage/organization, which could be sensed as deleterious by the P53 pathway during mitosis. An alternative explanation of the ‘dose dependency’ of the G1-arrest may rely on centriole bound regulators that would negatively regulate P53. Re-expression of centriolar/centrosomal components in acentriolar cells to test if and which minimal components can trigger the S phase could be a way to unravel this new ‘checkpoint’. The penetrance of centriole requirement for cell cycle progression seems to depend on organisms (Figure 1a), although inducible centriole depletion still need to be assessed in non-human vertebrates. In human at least, it seems now clear that P53 senses the centriole content and stops quasi-immediately cell cycle progression in the absence of centrioles (Figure 1b). This mechanism favors the motherly inherited pathway for centriole acquisition over a de novo pathway, known to drive erratic production of a varying number of centrioles [18,24,26]. Centriole loss or supernumerary centrioles decrease mitotic fidelity and affect primary cilium nucleation, defects associated with cancer and developmental disorders [51]. This checkpoint may thus be a key protector of tissue homeostasis.
Centriole continuity Templated or spatially restricted de novo generation of centrioles?
As we have mentioned above, in human at least, a checkpoint ensures that centrioles are inherited from the mother cell. Yet, they must be transmitted with their correct 9-fold symmetrical structure, template of the cilium axoneme. The ‘preservation’ of centriole structure www.sciencedirect.com
Centriole continuity: out with the new, in with the old Meunier and Spassky 63
was initially proposed to be granted by the growth and division of the pre-existing organelle [8]. A paradox arrived when centrioles were found to appear de novo in plants suggesting that other mechanisms were ensuring centriolar structure integrity [8]. The advent of electron microscopy revealed that duplicating centrioles grow orthogonally and at a distance from the wall of the preexisting centriolar structure, which definitively excluded the growth and division hypothesis. Then, de novo centriole formation after laser ablation of the pre-existing ones [27,28] together with the finding that the cartwheel structure that scaffolds the 9-fold centriole symmetry was able to self assemble through the oligomerization property of its major component SAS6 [52,53,86], put the final nail into the coffin of the hypothesis that structural information is provided by the pre-existing organelle. Today, the classical view is that the ‘parental’ centriole constitutes a cellular platform, at the center of the microtubule network, where the emergence of an ‘initiation site’, driven by a local equilibrium of regulatory and structural proteins, is facilitated. This would allow the spatial, temporal and numerical control of daughter centriole formation [54]. However two recent studies 55,56 provide data reviving the old theory by supporting a model proposed 20 years ago [57] in which the parental organelle would function as a reservoir transmitting structural information to its progeny. Using super-resolution microscopy in human cells, the authors show that SAS-6, or its self-oligomerization mutant, is recruited to the lumen of the parental centriole during S-phase, before being released at the onset of centriole duplication. Blocking the recruitment or release of luminal SAS-6 prevents subsequent centriole duplication thus suggesting that the cartwheel is shaped by the parental centriole, released and pasted onto the centriolar wall to grow the procentriole (Figure 2a). Consistently, following experiments show that, in SAS-6 depleted acentriolar cells, de novo centriole formation upon SAS6 re-expression is 30% error prone, contrasting with the error-free canonical duplication. This error rate is not increased when a SAS-6 self-oligomerization mutant is expressed instead of the normal protein, challenging the cartwheel self-assembly model [56]. The ordering of new by old cell structures, a process called cytotaxis [58], constitutes an attractive mechanism for centriole formation since it would ensure the transmission of their circumferential polarity [59]. This polarity is critical for cell and tissue development as it is further transmitted to primary or motile cilia axonemes which in turn determine cell polarity and flow directionality [60]. One way of proving this ‘cytotaxis hypothesis’ would be to test whether altered 9-fold structures are inherited by growing procentrioles. Such hypothesis however bumps into the many examples in life diversity where centriole formation occurs in the www.sciencedirect.com
absence of a pre-existing organelle (i.e. parthenogenetic [33,34] or acentrosomal [30–32] species). Would these species be more permissive to centriole structure aberrations? Would additional proteins be expressed to secure an otherwise delicate cartwheel self-assembly? Alternatively, a long-standing hypothesis is that physical continuity would be maintained by undetectable centriole remnants transmitted over cell division in case of need [8]. Arguments standing for this hypothesis are that physiological or induced de novo centriole formation can occur very rapidly [61,62], and often occur in predetermined sites, associated with nuclear envelope [34,42,63,64] or below the plasma membrane [65–67]. Yet hard to prove, one could hypothesize that membrane-stored nanoscopic seeds are inherited through cell division and stabilized to serve as template when centrioles are needed. Aside from taking a possible structural information from the pre-existing centriole, duplicating procentrioles are engaged, that is, tightly linked to their mother until the end of mitosis [68]. The mother centrioles organize the PCM and interact with microtubules, characteristics that drive them to the spindle poles at mitosis. Dragged by their mothers, the daughters follow. This ensures an equal and semi-conservative distribution of the four centrioles between the two daughter cells. Since mother centrioles have an age difference of at least one generation and daughter centrioles are born during the same cell cycle, daughter cells therefore inherit organelles that are half twin/half age asymmetric. At the cellular level, the mother and daughter centrioles grant the cell with different characteristics that are exploited to drive differential functions [69–71]. At the tissue level, age difference between mother centrioles of the daughter cells (defining in this case the mother and daughter centrosomes) causes asynchronous primary cilium growth [72,73] and can be exploited to drive asymmetric division and determine cell fate during development [74]. Centriole semi-conservative inheritance pattern is thus critical to drive different aspects of cell dynamics and tissue development. The physical continuity brought by the duplication process may therefore be a way to ensure centriole lineage conservation. Centriole continuity: how far?
This mode of biogenesis and inheritance begs the question of how vertebrate differentiating multiciliated cells produce up to 200 centrioles to nucleate motile cilia and propel physiological fluids in the oviducts, airways and brain epithelia. The first cytologists hypothesized that the centrioles at the base of motile cilia derived from the multiplication of the original centrosomal pair (reviewed in [8]). Then, electron microscopists in the late 1960s analyzed centriole amplification in chick, Xenopus and mammalian multiciliated cells [35,36,75–77] concomitantly with the canonical centriole duplication process in cycling cells [78,79]. In multiciliated cells, multiple Current Opinion in Cell Biology 2016, 38:60–67
64 Cell architecture
Figure 2
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SAS6 S
G1 (b)
Multiciliated cell maturation Current Opinion in Cell Biology
(a) Model for the templated mode of centriole duplication in cycling cells proposed in [55,56]. SAS-6 dimers (red) are recruited to the lumen of the parental centriole. They organize in a cartwheel configuration driven by their interaction with the 9-fold inner structure of the centriole. The assembled cartwheel is then released from the lumen and relocates on the proximal wall of the parental centriole where it templates procentriole formation. (b) Model for centriole amplification in vertebrate multiciliated cells based on [70,83,84]. An unknown mechanism leads to the accumulation of the core deuterosome proteins Deup1 and Ccdc78 (green) at the centrosomal daughter centriole. Deup1 and Ccdc78 recruit centriole biogenesis regulators Cep152 and Plk4 (not illustrated). This promotes the nucleation of the first procentriole. Accumulation continues from the same active site. The deuterosome grows and the second procentriole arises and so on, until the spherical deuterosome detaches from the centriolar wall. The process starts again to form another deuterosome and nucleate new procentrioles. Multiple deuterosomes can form concurrently around the parental centriole. Once the final number of centriole is reached, the procentrioles grow synchronously from all the deuterosomes, and mature to nucleate the motile ciliary tuft. Deuterosomes circumvent steric constraints imposed by the parental centriole wall and allow a massive albeit centriole-guided formation of new centrioles.
centrioles were seen growing on electron dense spherical structures called deuterosomes evoking a de novo generation of centrioles like in plant male gametes [30,80]. Yet, they observed that granule-like material accumulated around centrosomal centrioles at the beginning of the process, as during canonical duplication. Most of the authors thus retained the hypothesis that the multiple centrioles had a ‘centriolar origin’ [75–77]. Indeed, one study on chick tracheal epithelium provided very convincing morphological evidence supporting the hypothesis that the clusters of centrioles growing on the deuterosomes were primarily nucleated from centrosomal centrioles [75]. Then, the lack of dynamic observation Current Opinion in Cell Biology 2016, 38:60–67
made this process difficult to understand further. Consistent with the notion that only two pre-existing centrioles could not sustain the nucleation of more than a hundred of centrioles, the following generations of researchers kept the de novo hypothesis for massive centriole production in vertebrates. Recently, new studies in Xenopus and mice, taking advantage of the development of new imaging techniques, have led to reconsider the old ideas. First, the transcriptional profile of ciliated cells together with functional experiments showed that common regulators are involved in both centriole duplication and massive centriole www.sciencedirect.com
Centriole continuity: out with the new, in with the old Meunier and Spassky 65
amplification [81,82,83]. Most importantly, a paralogue of the canonical centriole duplication protein Cep63 was identified as being the core component of deuterosomes and named Deup1 [84]. Deup1 has the singularity, when overexpressed, even in bacteria, to drive the formation of deuterosome-like structures suggesting a self-oligomerizing activity [84]. Like Cep63 in cycling cells, Deup1 in multiciliated cells recruits Cep152 and Plk4, core regulators of centriole biogenesis, and promotes centriole nucleation [84]. The similarity with centriole duplication does not stop here since superresolution microscopy showed that, during multiciliated cell maturation, Deup1 accumulates at the proximal part of a centrosomal centriole, where its paralogue Cep63 normally localizes during centriole duplication in cycling cells [70]. By correlative electron microscopy, deuterosomes are indeed seen growing from a proximal pre-existing centriolar wall. Consistent with the recruitment of Cep152 and Plk4, this accumulation is rapidly followed by the apparition of procentrioles on the growing deuterosomes [70]. Live imaging showed that deuterosomes loaded with multiple procentrioles then detach from the parental centriolar wall allowing a new one to form and so on until the final number of centrioles is reached [70]. It thus seems that the same toolbox is used, at the same location, for centriole duplication and amplification. Interestingly though, amplification occurs on the wall of the daughter centriole suggesting that only the microenvironment provided by the younger parental centriole is permissive for this amplification [70]. This dynamics of formation and release of deuterosome shuttles enables to circumvent the steric constraint imposed by the parental centriolar wall and to drive a massive, albeit centrioleguided, production of centrioles (Figure 2b).
new centriole formation, even when centrioles are massively produced. These new evidences suggest the existence of mechanisms favoring the perpetuation of an apparent uninterrupted lineage from the first centriole of the zygote, inherited from the spermatozoon, to all the centrioles of the organism. The development of drugs and inducible constructs to reversibly eliminate centrioles opens exiting perspectives to understand the functional relevance of this continuity in different cellular and tissular contexts.
Thus, physical continuity between old and new centrioles is preserved, even in the case of massive centriole production. The fact that hundreds centrioles are formed on the wall of a single centriole can be explained by the requirement of a structural information provided by the pre-existing centriole or, maybe more likely, by the hijacking of the duplication process through the exploitation of a permissive characteristic of the younger pre-existing structure.
Concluding remark The attempts to clarify the role of centrioles in cell cycle progression, or in centriole biogenesis, feature ‘‘the complementary forces in the production of scientific knowledge: how techniques [. . .] shape the production of data and theory, and how theoretical expectations shape interpretation of reported observations’’ [8]. The availability of new molecular tools and imaging techniques showed that the absence of centriole is sensed by the P53-pathway as a deleterious stress triggering cell cycle arrest, at least in human cells. In addition, it appears that preexisting centrioles are always used as a building tool for www.sciencedirect.com
Conflict of interest statement The authors declare no competing financial interests.
Acknowledgements The authors thank N Delgehyr for her comments on the manuscript. The work of the authors is supported by the Centre National de la Recherche Scientifique, the Ecole Normale Supe´rieure and the Institut National pour la Sante´ Et la Rercherche Me´dicale, the Agence Nationale de la Recherche (ANRJCJC-15-CE13-0005-01 and ANR-12-BSV4-0006), the European Research Council (Consolidator grant 647466), the Association de Recherche pour le Cancer (ARC, PJA-20131200184), the Fondation pour la Recherche Me´dicale (20140329547) and the Fondation Pierre-Gilles de Gennes (FPGG03).
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