Michel MichelBornens. Encyclopedia of Life Sciences: Supplementary Set. Volume 21, Wiley, 2007.
During the cell division cycle, duplication of the centrosome and segregation of the two daughter centrosomes dictate the assembly of the mitotic spindle and thus determine the plane of the cleavage furrow. Recent results have both modified and reinforced views about the role of the centrosome in the control of cell cycle progression in animal cells.
Introduction
The importance of the centrosome as a central organizer during cell division was recognized in the early days of cell biology. A century ago, Theodor Boveri would see it as the ‘division organ’, capable of coordinating cytoplasmic and nuclear division. We know now that such a role is possible because the centrosome is able to control the nucleation of microtubules. During the cell division cycle, duplication of the centrosome and segregation of the two daughter centrosomes dictate the assembly of the mitotic spindle and thus determine the plane of the cleavage furrow; hence, there is a precise control over centrosome number and coordination of its activities with other cell cycle events, which is a major focus of the current research on this organelle. In most cases, the ‘essential bipolarity’ of the cell division process rests on the reproductive capacity of the centrosome, in both animal and yeast cells.
Alternatively, the great collateral branch of multicellular organisms, the land plants, can do without a centrosome, controlling their microtubule array during interphase as well as during mitosis. The most challenging question raised by the centrosome is certainly to unravel what this organelle adds to the cell economy that explains its evolution among divergent species. The structural evolution of the centrosome, which has been a major difficulty for the definition of this organelle, will certainly be explained when the real framework is drawn, in which a comprehensive view of the centrosome microtubule network is developed.
A striking feature of centrosome reproduction is that it generally involves structural continuity, appearing as a true duplication process: one needs a centrosome to assemble a new centrosome. Centrosome continuity is an important feature for fertilization in metazoa, as the centrosome is uniparentally inherited, most often from the male. For Boveri, this was the reason why parthenogenetic development was not observed in these species, as unfertilized eggs could not divide in the absence of the ‘division organ’. In agreement with this early view, it has now been shown that a complete parthenogenetic development of the frog embryo can take place provided the sperm cell is replaced by a centrosome, even if this centrosome is heterologous (for a review see Tournier and Bornens, 1994).
Centrosome continuity has long been a matter of speculation: the possibility that centrosome reproduction could rest on the duplication of an extranuclear nucleic acid was proposed but has now been abandoned. Centrosome reproduction has also been compared in the past to the growth of a crystal. This view has recently received some support in the case of the yeast spindle pole body (SPB), a structurally simple centrosome, in which a controlled protein crystallization could be an important step in the initiation of the duplication process (Bullitt et al., 1997). More recently, the centrosome has become a focus for understanding the origin of genetic instability in tumours, particularly of chromosomal instability (for a review see Nigg, 2002). This was foreseen in the early days of experimental research on polyspermy in marine eggs by Boveri.
Structural and Biochemical Changes During the Centrosome Cycle
The considerable differences in the structure of centrosomes among divergent species has precluded a general definition of this organelle on a structural basis. In all cases, however, it is a tiny organelle which maintains a physical association with the nucleus. The two main properties, to nucleate microtubules and to reproduce by duplication once per cell cycle, have provided the functional basis of a general definition of the centrosome. There are now reasons to believe that, beyond structural differences, centrosomal functions, like most of the major cellular functions, are based on conserved molecular mechanisms among highly divergent species. It is becoming increasingly common for progress in one experimental system to be rapidly followed by progress in others. Genetically tractable unicellular model systems, such as budding or fission yeasts, or flagellates and ciliates, and multicellular systems such as the fly Drosophila melanogaster, have been very important in identifying major components. Marine eggs have also been quite important systems at the forefront of centrosome research. Cultured vertebrate cells are also a useful model in which centrosome isolation has allowed structural and biochemical analysis.
In animal cells, the centrosome is a structurally complex organelle (for a review see Bornens, 2002) composed of two centrioles tightly associated with a matrix, which consists of a complex thin filament network. Centrioles are cylindrical structures with a diameter of 0.2 mmandalengthof 0.5 µm, which display a virtually perfect nine-fold radial symmetry—a highly conserved structural motif among widely divergent eukaryotic species. The walls of the centrioles are themselves composed of microtubules. In addition, two sets of appendages surround the mother centriole. In several lower unicellular eukaryotes, it is a structurally simple plaque-like organelle, either inserted in the nuclear envelope, like the SPB in baker’s yeast (Bullitt et al., 1997), or present in the cytoplasm, as in myxamoeba (Ueda et al., 1999).
Recent results have demonstrated that the centriole pair in the centrosome of animal cells is instrumental in organizing centrosomal components into a structurally stable organelle (Bobinnec et al., 1998). Accordingly, the ultrastructural observation of isolated centrosomes has revealed that the centrosomal matrix, which is capable of important structural changes, is tightly associated with the two centrioles, which appear as the organizational core of the centrosome. Physiological loss of centrioles can happen, for example during differentiation of myoblasts into myotubes. It is accompanied by the disappearance of the centrosome and the redistribution of the microtubule-nucleating activity around the nucleus.
In the centrosome of animal cells, the two centrioles are not equivalent, either structurally or functionally (Piel et al., 2000). The structural asymmetry of the centriole pair is due to the generation process. Early electron micro-scopic investigations revealed the very peculiar manner in which the reproduction of the centrosome is achieved: procentrioles assemble close to each centriole and grow orthogonally to the respective parental centriole. A slow process of maturation of the younger centriole takes place, transforming it into a fully differentiated centriole, apparently similar in all respects to the older centriole. At the same time, the centrosome matrix changes in size or structure, displaying a varying number of satellite structures, the significance of which is not clear. Consistent with the general observation that procentriole orthogonal budding from each centriole is the earliest structural event in centrosome reproduction, it has been demonstrated in sea urchin eggs that the reproductive capacity of the centrosome depends upon the number of centrioles present (Sluder and Rieder, 1985) and that centrosomes artificially deprived of centrioles cannot reproduce. Experiments aimed at identifying the centrosome-associated activity responsible for triggering parthenogenetic development in Xenopus have also led to similar conclusions (Tournier and Bornens, 1994). Matrix accumulates during S and G2and is redistributed at the onset of mitosis between the two separating centrosomes. Finally, it is dispersed or proteolysed at the metaphase-anaphase transition.
Because centrioles are minute structures, it has not been possible to observe their duplication in living cells until recently. Using both deoxyribonucleic acid (DNA) recombinant techniques for specifically tagging a centriole-associated protein with a fluorochrome and a digital video camera capable of tracking the centrosome in vivo, it is now possible to monitor centrosome duplication in vivo (Piel et al., 2000). This approach has revealed novel features of centrosome dynamics which has led to a reappraisal of how the centrosome cycle is coupled with the cell division cycle. Until recently, structural features obtained by electron microscopy or immunofluorescence microscopy have been difficult to integrate into a coherent and reliable model of centrosome organization and dynamics, as these observations were all obtained on fixed material. By observing the centrosome in living cells, and in spite of a much lower resolution than that of electron microscopy, it is possible to obtain a more integrated view of the centrosome. Three novel features have been demonstrated in this way. First, the splitting of centrioles in each postmitotic centrosome, which corresponds to the moment when orthogonal orientation is lost, occurs much earlier than was previously reported: instead of late G1just before S phase, it takes place soon after anaphase, long before cytokinesis is completed. Second, the two centrioles of postmitotic cells demonstrate differential movement: one maintains a central and stable location, whereas the other has a wide and eccentric trajectory. Third, the motile centriole progressively slows down from the onset of centriole duplication at the G1-S border, up to late G2; although maintaining a stable location within the cell once duplicated (in G2), the former motile centriole and its associated procentriole conserve more independence with respect to the surrounding cytoplasm until the onset of mitosis, where it stops rocking completely. Therefore, the dynamics of the centrosome in quiescent cells is different from that of a proliferative cell.
This approach has also demonstrated a specific contribution of each centriole to the activity of the centrosome: both centrioles nucleate microtubules but only the mother centriole anchors them. Therefore, a critical activity of the centrosome, to explain its behaviour, is not only to nucleate microtubules but also to control their release; it is the older centriole which actually acts as the centrosome, i.e. as an organelle maintaining itself at the cell centre, because of its capacity to anchor microtubules. A general conclusion is that the behaviour of individual centrioles is maturation-dependent, correlated with the generation process of these organelles: one centriole cannot replace the other one in a centrosome.
Molecular Mechanisms Underlying Centrosome Biogenesis
Neither the duplication cycle of the centrosome in animal cells nor that of the yeast SPB are understood at the molecular level. Most importantly however, and although their precise functions are not yet known, there is an important conservation of some gene products involved in both the yeast and the vertebrate cell (Adams and Kilmartin, 1999; Spang et al., 1993; Middendorp et al., 2000). In both cases, the new organelle assembles in close proximity of the old one, suggesting that some protein complex is associated with the proximal wall of each centriole, or to the distal end of the half-bridge of the SPB, where it is able, upon a signal, to promote the assembly of a new centriole or of a new SPB (Adams and Kilmartin, 2000).
Several experiments suggest the existence of such a complex. First, centriole assembly cannot take place in somatic cells in the absence of preexisting centrioles; cells in which the centrosome has been removed by microsurgery can reestablish an aster of microtubules, probably by motor-dependent autoorganization of microtubules, but are unable to assemble centrioles. A similar situation has been described during the first cell cycle of sea urchin eggs: when the egg is cut into two before the fusion of the two pronuclei, the part that contains the female pronucleus does not possess any functional centrosome. It does not divide and is unable to form centrioles, whereas the part containing the male pronucleus and spermaster divides. Second, in Xenopus eggs, which have inactivated their centrosome during meiosis, the injection of a centrosome is necessary and sufficient to induce the formation of centrioles and to trigger parthenogenesis (reviewed in Tournier and Bornens, 1994). It is noteworthy that the injected centrosome can be from divergent vertebrate animal species and even from echinoderms, a result that would suggest great conservation of the centriole-associated complex. Centrosomes from embryonic or somatic cells from Drosophila are, however, unable to trigger parthenogenesis in Xenopus.
The requirements for structural continuity described above can be contrasted with situations where centriole assembly occurs in the absence of preexisting centrioles. In mouse embryos, early cell division cycles take place in the absence of centrioles. In echinoderms, artificial activation of eggs by various treatments can trigger assembly of centrioles and parthenogenetic development. On the other hand, during their terminal differentiation, ciliated epithelial cells from mammalian trachea or avian oviduct assemble many centriole-like structures according to a pathway distinct from classical duplication: centriole-like structures emanate from a dense and uncharacterized body, in the vicinity of the centrosome and the Golgi apparatus, and migrate towards the apical pole to form the basal bodies necessary for ciliary growth. One may assume that, despite their apparent differences, the two pathways involve some common principles, as they both lead to centriole assembly. Finally, in several unicellular eukaryotes, the centriole structure totally disappears during some stage of the vegetative cycle and reforms upon specific signals.
Coupling Between Centrosome Duplication and Cell Cycle Progression
Centrosome duplication has to be coordinated with the progression of the cell cycle to ensure a correct cell division at the exit of mitosis. The existence of two alternative pathways for centriole/basal body biogenesis, the centriolar pathway in proliferating cells and the acentriolar pathway in postmitotic epithelial cells differentiating into ciliated cells, has long been the only evidence that centriole assembly could be uncoupled from cell cycle progression. New evidence has accumulated, mainly in early embryos but also in somatic cells.
Cell Cycle Control of the Centrosome Cycle
In species in which fertilized eggs divide by segmentation without growth during early development, such as amphibian or marine eggs which contain maternal stores of cellular components, including centrosomal precursors, that have been stockpiled ahead during oogenesis, early embryonic cell cycles do not possess feedback mechanisms able to block the progression of the cell cycle when DNA synthesis or mitotic spindle assembly are impaired. This basic feature of elementary cell cycles explains why they are so short compared with somatic cell cycles in the same species (30 min instead of more than 10 h in the frog). In this case, successive runs of autonomous centrosome duplication cycles, independent of DNA synthesis (Nagano et al., 1981, Picard et al., 1988) or of cell cycle progression (Gard et al., 1990), can be easily observed. In these conditions, one major requirement for centrosome duplication is the phosphorylating activity of the cell cycle controlling kinase cyclin E/Cdk2. This particular Cdk (cycline-dependent kinase), which depends on an interaction with cyclin E to be activated, is also required for DNA synthesis and is accumulated in the egg. Therefore, it does not need to be synthesized at each cell cycle like the related cyclin B/cdk1, which is required for mitosis and is degraded at the end of each mitosis (Hinchcliffe et al., 1999). A requirement for another kinase, which depends on the presence of Ca2+ and of calmodulin for its activity, has been recently demonstrated for the initiation of each centrosome duplication cycle (Matsumoto and Maller, 2002).
In somatic cells, a similar control might exist in the context of a more complex cell division cycle in which growth requirements have to be coupled to DNA and centrosome duplication. For example, CHO (Chinese hamster ovary) cells arrested at the G1-S border by treatment with hydroxyurea show several cycles of centrosome duplication (Balczon et al., 1995). In such a system, it has been shown that cyclin E/Cdk2 activity is required, and possibly cyclin A/Cdk2, as well as phosphorylation of the retinoblastoma protein Rb and the activation of the transcription factor E2F (for reviews see Lange, 2002; Nigg, 2002).
The coupling between SPB duplication and cell cycle progression has been genetically dissected in Saccharomyces cerevisiae: a detailed analysis of the different cyclin requirements for the activity of the unique Cdk in this species (the product of the gene CDC28), to promote and control SPB duplication, has been reported (Haase et al., 2001). In conclusion, the duplication of the centrosome in somatic cells generally appears strictly coupled with cell cycle progression, or with cell growth, which is itself coupled to cell cycle progression.
Centrosome Control of Cell Cycle Progression
Controls linking SPB duplication and cell cycle progression are suggested by results in yeasts showing that an essential protein kinase (the product of the gene MPS1)is required for both SPB duplication and for the mitotic assembly checkpoint (Weiss and Winey, 1996).
Several proteins which directly participate in the cell cycle machinery are associated with the centrosome in a cell cycle-dependent manner. This is the case for Cdk1, which is associated in part with the centrosome during G2and at the beginning of M phase (Bailly et al., 1989); for cyclin B1 and B2, which associate to the centrosome from the beginning of their accumulation in the cytoplasm at the onset of S phase; and of cyclin A, which associates transiently with separating centrosomes at the onset of mitosis. Finally, proteins participating in the degradation of cyclins by the ubiquitin pathway at the S phase entry or at the exit of mitosis are present as a large complex at the centrosome throughout the cell cycle (King et al., 1996; Freed et al., 1999). The centrosomal association of several components of the cell cycle engine could reflect the existence of a feed-back mechanism of the centrosome-microtubule system on Cdk1 activation that would ensure the phasing of the separation of the two centrosomes with the onset of mitosis. For example, Polo-like kinase, which is associated with the centrosome during mitosis and affects centrosome maturation and separation (Golsteyn et al., 1995), is also implicated in the activation of the Cdc25 phosphatase in Xenopus, which controls the final activation of Cdk1 (Kumagai and Dunphy, 1996). This suggests that this kinase could ensure a link between the pathway leading to mitotic spindle assembly and the Cdk1 maturation pathway.
Recently, several results have both modified and reinforced understanding of the role of the centrosome in the control of cell cycle progression in animal cells. First, it has been shown that apparently normal bipolar mitotic spindles, able to support anaphase, can form in vertebrate cells in which centrosomes have been disassembled by antibody injection (Bobinnec et al., 1998). This was further confirmed by two other approaches in which centrosomes were ablated either by laser irradiation (Khodjakov et al., 2000) or by microsurgery (Hinchcliffe et al., 2001). These experiments indicated the existence of a default pathway for assembling the mitotic spindle, and for karyokinesis (a conclusion which would agree with the observation that female meiotic divisions can take place without centro somes in some species). But the fact that metaphase exit can take place in these artificial conditions also suggests that the centrosome might be necessary for the mitotic exit checkpoint. Whether or not chromosome segregation was more prone to errors in that situation was not investigated. However, cells did not generally complete cytokinesis and never resumed DNA synthesis (Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001). Cytokinesis failure could be interpreted as the result of misorientation of the spindle with respect to the cleavage furrow. Another possibility comes from time-lapse microscopy: the final cleavage is correlated with a movement of the mother centriole of one of the two sister cells from the cell centre toward the intercellular bridge (Piel et al., 2001). When the centriole moves back to the cell centre, the intercellular bridge cleaves. Moreover, Drosophila cells lacking centrioles exhibit a high rate of cytokinesis failure. Thus the centrosome could either activate cytokinesis or could release cells from cytokinesis arrest. Results from yeast support the latter possibility.
In agreement with the demonstration that the centrosome is an absolute requirement for egg cleavage in amphibians, the centrosome therefore appears to be a critical organelle for cytokinesis in somatic cells. In addition, it is required for cell cycle progression from G1 to S phase, suggesting the intriguing possibility that the ‘checklist’ before reaching the end of cell division also recapitulates the conditions necessary for initiating a new cell division cycle. Involvement of the centrosome in this temporal control could reveal the need for integrating spatial constraints into the decision. Being at the centre of an aster of micro-tubules, the centrosome could be well suited to such a role.