The coordination of centrosome reproduction with nuclear events during the cell cycle

DEVELOPMENTAL BIOLOGY V49 - AP - 0513 / C12-267 / 06-15-00 17:25:27

12 The Coordination of Centrosome Reproduction with Nuclear Events during the Cell Cycle Greenfield Sluder and Edward H. Hinchcliffe Department of Cell Biology University of Massachusetts Medical School Worcester, Massachusetts 01605

I. Introduction II. The Events of Centrosome Reproduction III. Controls for Centrosome Reproduction A. Intrinsic Controls B. Extrinsic Controls IV. Coordination of Centrosome Reproduction with Nuclear Events in the Cell Cycle A. Cell Cycle Stage Dependency of Centrosome Reproduction B. Cyclin-Dependent Kinases in the Control of Centrosome Reproduction References

I. Introduction The centrosome is the ensemble of structures that nucleates the interphase array of cytoplasmic microtubles and assembles the poles of the mitotic spindle (for comprehensive reviews on centrosome structure, composition, and function, see Brinkley, 1985, and Kellogg et al., 1994). Since the equal segregation of daughter chromosomes depends upon the spindle being strictly bipolar, it is of obvious importance for the cell to coordinate the events of centrosome doubling with nuclear events during the cell cycle, thereby limiting centrosome copy number at the start of mitosis to two and only two. If the centrosome fails to fully duplicate before the onset of mitosis, the cell may eventually return to interphase without dividing and becomes polyploid. If the centrosome duplicates more than once in a cell cycle, a multipolar spindle may be assembled, and the chromosomes will be unequally distributed to the daughter cells (Fig. 1). Although many daughter cells with abnormal chromosome number will be inviable, progeny that accumulate chromosomes with growth promoting genes and/or lose chromosomes containing tumor suppressor genes will produce a population of cells with aggressive growth characteristics (reviewed in Orr-Weaver and Weinberg, 1998; Brinkley and Goepfert, 1998). Indeed, many human Current Topics in Developmental Biology, Vol. 49 Copyright 䉷 2000 by Academic Press. All rights of reproduction in any form reserved. 0070-2153/00 $35.00

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Figure 1 Random chromosome segregation by tetrapolar spindles in PtK cells. (a-b) First cell: This tetrapolar cell has all four poles located at the cell periphery. (a) Metaphase, all chromosomes bioriented to pairs of spindle poles. (b) Late anaphase, the normal chromosome compliment is randomly distributed to four spindle poles. (c-d) Second cell: This cell has one spindle pole located at the cell center and three poles at the cell periphery. (c) Anaphase onset. (d) Telophase. Phase contrast optics. Bar in (b) 20 애m.

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tumor cells, which are genetically unstable, have abnormally high numbers of centrosomes (Pihan et al., 1998; Lingle et al., 1998). This is a real problem because neither somatic or embryonic cells appear to have a checkpoint for the metaphase–anaphase transition that monitors spindle bipolarity, as long as all chromosomes establish bipolar attachments to the spindle (Sluder et al., 1997).

II. The Events of Centrosome Reproduction At the end of mitosis each daughter cell inherits a single centrosome, and by the onset of the next mitosis it contains just two centrosomes. This precise doubling of the interphase centrosome in preparation for mitosis is called centrosome reproduction. At the functional level centrosome reproduction in higher animal cells consists of at least four morphological events that are distinct and experimentally separable (Mazia et al., 1960; Sluder and Begg, 1985). Figure 2 illustrates the sequence of these events. The first event is the splitting apart of the pair of inherited mother–daughter centrioles, which coincides with the loss of their orthogonal relationship within the centrosome (also referred to as centriole disorientation; see Kuriyama and Borisy, 1981). The splitting event is followed by duplication, in which the centrosome goes from a single unit capable of organizing one centrosome to two units that have the ability to form two centrosomes. Duplication is correlated with the formation of daughter centrioles at right angles to each mother or mature centriole. The third event entails the splitting apart or disjunction of the two sister centrosomes, each containing a pair of mother–daughter centrioles. The fourth and final step is the physical separation of the now disjoined sister centrosomes through the action of microtubule-based motor proteins. For normal cells, be they embryonic or somatic, the steps of centrosome reproduction are correlated with distinct stages of the cell cycle (Fig. 2). In mammalian somatic cells the start of centrosome reproduction is commonly thought to begin in late G1, when the inherited centriole pair loses its orthogonal arrangement (Kuriyama and Borisy, 1981). Depending upon the cell type, daughter centrioles are first seen between late G1 and midS phase with the appearance of short, annular centrioles (often called procentrioles) at right angles to and separated slightly from the proximal end of each mature centriole (Robbins et al., 1968; reviewed in Hinchcliffe and Sluder, 1998). These daughter centrioles elongate during S and G2, and depending on the cell type, may reach their mature length as late as the following G1 (Kuriyama and Borisy, 1981; Lange and Gull, 1995). The completion of centrosome reproduction typically occurs when the daughter centrosomes disjoin and separate at a variable time in G2, with

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Figure 2 Schematic illustrating the centrosome reproduction cycle. At each stage of the cycle the centrioles are represented as barrels. Clockwise from the top right: (I) The single centrosome inherited at the end of mitosis; the mother–daughter pair of centrioles are arranged at right angles to and in close proximity with each other. (II) During late G1 or early S phase, the mother–daughter centriole pair splits apart, and (III) concurrently, the centriole pair loses its orthogonal orientation (termed ‘‘disorientation’’). (IV) The centrosome then duplicates once, as seen by the appearance of short, annular daughter centrioles, called procentrioles, at right angles to the preexisting centrioles. The duplicated centrosome now consists of two mother–daughter pairs of centrioles. (V) During S phase the reproductive capacity of the duplicated centrosome is restored (termed ‘‘licensing’’). (VI) The duplicated centrosome disjoins during G2 with pairs of mother–daughter centrioles in each sister centrosome. (VII) The sister centrosomes physically separate from each other through the action of microtubule-based motor proteins. (VIII) At the time of the G2/M transition the sister centrosomes assemble the poles of the mitotic spindle. As the cell completes mitosis and divides into two, each daughter cell inherits exactly one centrosome containing a mother–daughter pair of centrioles.

pairs of mother–daughter centrioles going to each daughter centrosome (Aubin et al., 1980; Kochanski and Borisy, 1990). However, the extent to which daughter asters have separated during prophase can vary greatly between cells in the same population. In some cases the two centrosomes remain close together until nuclear envelope breakdown, while in others both asters are well separated around the nucleus before the end of prophase (reviewed in Rieder, 1990).

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III. Controls for Centrosome Reproduction Control of centrosome reproduction is exercised at two levels: (i) by limits that are intrinsic to the centrosome itself; and (ii) by extrinsic controls imposed by changing cytoplasmic conditions. Limits intrinsic to the centrosome determine centrosome copy number at the onset of mitosis; cytoplasmic controls determine when the centrosome reproduces in relation to the progression of nuclear events, such as DNA synthesis and the onset of mitosis. A. Intrinsic Controls Functional analyses of centrosome reproduction in sea urchin zygotes indicate that there is a ‘‘counting’’ mechanism within each centrosome that limits the number of daughters that can arise from the parent centrosome. The key finding is that it is possible to experimentally manipulate the reproductive capacity of centrosomes. When mitosis is prolonged by any of several independent methods (Fig. 3), the two spindle poles split during mitosis to yield four functional poles that will not further subdivide even when mitosis is prolonged to 20 times its normal duration (Mazia et al., 1960; Sluder and Begg, 1985; Hinchcliffe et al., 1998). Ultrastructural analysis of such tetrapolar spindles reveals that each pole contains only one centriole, confirming that the centrosomes have split, not duplicated (Fig. 3). In effect, the two mitotic centrosomes with normal reproductive capacity have subdivided into four centrosomes with half the normal reproductive capacity (and half the complement of centrioles). After the cell divides into four and as the daughter cells go through interphase, these half centrosomes each assemble a daughter centriole, thus becoming normal centrosomes with full reproductive capacity (Sluder and Rieder, 1985). Since they do not split during interphase, each daughter cell assembles a monopolar spindle at next mitosis (Fig. 4a). If a daughter cell with a monopolar spindle remains in mitosis longer than normal, as they often do, the centrosome of the monopolar spindle will split to give two functional spindle poles with one centriole apiece (Fig. 4b). These poles duplicate but do not double in interphase and monopolar spindles are once again formed at the following mitosis (Fig. 4b). Thus, the splitting of the mother–daughter centrioles and their reproduction are distinct events that can be experimentally uncoupled by prolonging mitosis (Sluder and Begg, 1985; Sluder and Rieder, 1985). The reproduction of a spindle pole cannot simply be the subdivision of the centrosomal microtubule organizing center (MTOC), because such a fission mechanism should always give two smaller centrosomes, each of which should have the same reproductive capacity as the parent pole.

Figure 3 Microinjection of cyclin B ⌬-90 mRNA at first prophase arrests zygotes in mitosis. (a) The zygote enters mitosis and forms a normal bipolar spindle. The refractile sphere in the lower left portion of the zygote is a drop of oil used to cap the micropipet. (b) Anaphase onset occurs at the normal time. (c) This zygote shortly before fixation for serial section ultrastructural analysis. Both of the spindle poles have split and a tetrapolar spindle forms. (e) Each spindle pole of this zygote contains only a single centriole. All four centrioles in this zygote are shown in this frame. (a-c) Polarization optics, 10 microns per scale division. (From Journal of Cell Biology (1986), with copyright permission of the Rockefeller University Press, 103, 1873–1881).

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Figure 4 Diagrammatic representation of the experimental manipulation of the reproductive capacity of spindle poles in zygotes. (a) The first division spindle has a pair of orthogonally arranged centrioles at each spindle pole. During prolonged prometaphase the centriole pairs (and centrosomes) split without duplicating, and the four spindle poles separate from each other, each containing a single centriole. In telophase, as the cell divides into four, the singlet centrioles replicate but do not separate. The result is the formation of monopolar spindles at the next mitosis. Each centrosome has the normal complement of two centrioles. When a cleavage furrow fails to form, two monopolar spindle come together to assemble a functional bipolar spindle with poles that reproduce in a normal fashion. (b) Centriole behavior during prolonged prometaphase in a cell containing a monopolar spindle. The single pole splits and a bipolar spindle forms when prometaphase is prolonged, as it often is in such cells. Each sister aster contains only one centriole. After anaphase, the cell divides and the singlet centrioles duplicate but do not split during interphase. At the next mitosis monopolar spindles are again assembled.

The mechanism that limits the number of daughters that can be formed from the parent centrosome is a mystery. One possibility is that centrosome duplication depends upon the formation of a template assembly that seeds or nucleates the daughter centrosome in association with an existing centrosome. Not all centrosomal structures need be templated, only the new entities around which the familiar structures of the centrosome are later elaborated (discussed in Sluder and Reider, 1996). Possibly this ‘‘seed’’ represents the molecular assemblies that pattern the daughter centrioles, if centrioles in fact organize the centrosome in higher animal cells as a number of studies suggest (Sluder and Rieder, 1985; Sluder et al., 1989; reviewed in Marshall and Rosenbaum, 1999). Even if the nine triplet microtubules of centrioles per se do not do not participate in the organization of the centrosome in animal cells, some specific activity associated with the

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centriole is required for the assembly of a complete daughter centrosome from nonlimiting subunit pools. Evidence for templating activity in the centrosome can be found at both the functional and morphological levels. Even though before fertilization, sea urchin, clam, and Xenopus eggs all contain enough centrosomal subunits to assemble many complete centrosomes (Sluder et al., 1986, 1990; Gard et al., 1990; Palazzo et al., 1992), they normally assemble only one new centrosome in close spatial association with each preexisting centrosome at each cell cycle. Somatic cells, after microsurgical removal of the centrosome, reform an asterlike array of microtubules, yet this ‘‘aster’’ does not reassemble centrioles or double even though the cell grows to a larger than normal size and enters S phase, the expected time of centrosome reproduction (Maniotis and Schliwa, 1991). Structural studies reveal that procentrioles form only at the proximal ends on parenting centrioles, and in some cases specific precursor structures have been described (Dippel, 1968; Gould, 1975).

B. Extrinsic Controls The formation of a new centrosome through a templating mechanism is not sufficient, by itself, to fully explain the tight temporal coordination between the nuclear and centrosome cycles. Tests of the possibility that the coordination of centrosome reproduction with nuclear events in the cell cycle is determined by nuclear activities started with an examination of centrosome reproduction in enucleate sea urchin zygotes (Lorch, 1952; Sluder et al., 1986). The approach was to remove the nucleus and one centrosome from individual prophase zygotes with a micropipet and determine if the centrosome left behind could reproduce. After the nucleus was removed, the cell cycle appeared to continue, as seen by the cyclical rise and fall of the astral microtubule assembly in normal coordination with the initiation of cleavage furrows. Importantly, the single centrosome reproduced in a precise 1-2-4-8 fashion in proper coordination with cycles of astral microtubule assembly/disassembly and cleavage furrow initiation (Fig. 5). The fact that all daughter centrosomes were found to contain two centrioles indicated that centrosome reproduction was normal and complete. These observations reveal that nuclear activities, such as the synthesis of DNA and/or the timed transcription of limiting RNAs, do not control when the centrosome reproduces. The finding that all the centrosomes reproduced in synchrony within an enucleated zygote suggests that the temporal control of their reproduction is exercised by a cyclical change in the state of the cytoplasm.

Figure 5 Repeated centrosome reproduction in an enucleated sea urchin zygote. (a) Mitotic zygote after the nucleus and one centrosome were removed with a micropipet. The remaining centrosome organizes a birefringent aster in the center of the cell; the oil drop expelled from the micropipet is seen in the upper left portion of the egg. (b) During second mitosis two birefringent daughter asters are visible. (c) Third mitosis; the two centrosomes have reproduced to four. (d) Fourth mitosis; the zygote contains eight asters, seven of which are visible at this plane of focus. All centrosomes contain a pair of centrioles indicating that centrosome reproduction is complete. Polarization microscopy; 10 microns per scale division. (From Journal of Cell Biology (1990), with copyright permission of the Rockefeller University Press, 110, 2025–2032).

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These results were obtained with zygotes, which are admittedly specialized cells that store relatively large pools of proteins to be used for the rapid divisions of early development. At a minimum, these findings reveal that control of centrosome reproduction by limitations on the availability of centrosomal subunits is not a conserved control strategy utilized by all cells. Nevertheless, it is clear that centrosome reproduction in somatic cells ultimately does require the transcription and translation of messages for centrosomal components, because these cells must grow between divisions. Evidence for the possible role of subunit synthesis in the control of centrosome reproduction in somatic cells comes from the finding that when CHO cells are arrested in S phase, centrosomes continue to reproduce (Balczon et al., 1995). This is also the cell cycle phase in which the levels of PCM1 mRNA, which codes for a protein associated with the pericentriolar material (Balczon et al., 1994), are normally maximal. Importantly, during the prolonged S phase block the levels of this mRNA remained elevated, suggesting that there is continued synthesis of this centrosome-associated protein. Control experiments revealed that the increase in centrosome number was not simply the consequence of blocking the cell cycle, because cells arrested in G2 contained only two centrosomes, the normal number for this cell cycle phase. G2 is also a time that the levels of PCM-1 mRNA levels are normally minimal. At face value these findings suggest that the more centrosomal subunits that are synthesized, the greater the number of centrosomes that are assembled. Whether or not limits on the synthesis of new subunits at each cell cycle per se could act as a control that has sufficient finesse to ensure the formation of just one daughter centrosome per cell cycle is an important question (discussed in Sluder and Rieder, 1996). To limit centrosome number by this mechanism, the cell would have to precisely control transcript and protein pools within very tight tolerances.

IV. Coordination of Centrosome Reproduction with Nuclear Events in the Cell Cycle At first glance the coordination of the centrosome and nuclear cycles might seem to be straightforward; the events of centrosome reproduction are linked to particular cell cycle stages and coordination is thereby assured. However, the appealing simplicity of this notion is clouded by findings that the cycle of centrosome reproduction can occur repeatedly in the absence of cell cycle progression. For example, when somatic or embryonic cells are arrested in S phase by inhibitors of DNA synthesis, the interphase centrosome undergoes multiple rounds of duplication (Sluder and Lewis, 1987; Raff and Glover, 1988; Balczon et al., 1995). In addition, the centrosomes of sea urchin and Xenopus zygotes undergo repeated rounds of

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complete reproduction when protein synthesis is completely blocked and the cell cycle is arrested before mitosis due to the lack of cyclin A or B synthesis (Fig. 6a; and Sluder et al., 1990; Gard et al., 1990). Such findings

Figure 6 Patterns of centrosome reproduction in sea urchin zygotes treated with translation inhibitors before fertilization (a) or at first prophase (b). Diagrammatic representation of the experiment and its results with photographs of cells at corresponding stages. (a) Translation inhibitors (emetine plus anisomycin: E/A) were added to the zygotes at the time of fertilization. The zygotes enter S phase and contain two asters closely associated with the nucleus. After 6 hr, the zygotes are still in S phase, and some zygotes contain 10 or more asters. These zygotes were extracted with microtubule-stabilizing buffer, and astral birefringence was augmented by hexylene glycol. (b) E/A is added to zygotes at prophase of first mitosis, and they complete M phase and cleave at the normal time. These zygotes arrest at the two-cell stage prior to the onset of second S phase; each cell of the embryo contains exactly two asters positioned on either side of the reformed nuclear envelope. Each aster contains a pair of centrioles. The number of asters or centrioles never increased in these zygotes, even after 6 hr. The astral birefringence in this zygote was augmented by hexylene glycol. Polarization microscopy. (From Journal of Cell Biology (1998), with copyright permission of the Rockefeller University Press, 140, 1417–1426).

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raised the possibility that the nuclear and centrosomal cycles are regulated by independent pathways (Sluder et al., 1990). Nevertheless, the normal tight linkage between centrosomal events and nuclear events in the cell cycle forces a search for the mechanisms that coordinate these important preparations for mitosis.

A. Cell Cycle Stage Dependency of Centrosome Reproduction One avenue used to investigate how centrosome reproduction and nuclear events are coordinated in the cell cycle has been to characterize which cell cycle stages support centrosome reproduction and which do not. In general, the approach used has been to arrest cells in a particular phase of the cell cycle and then determine if the centrosome will reproduce one or more times without further experimental intervention. The results of several studies have clearly demonstrated that the centrosome inherited at the end of the previous mitosis does not reproduce during prolonged G0 (Tucker et al., 1979; reviewed in Wheatley, 1982). However, it is not clear to what extent the centrosome will begin to reproduce during G1, once the cell has passed the restriction point and is committed to prepare for division, because of the difficulty of credibly arresting cells in the G1 phase of the cell cycle. The conventional view is that the reproduction of centrioles, and hence the centrosome, is controlled by the cytoplasmic conditions of S phase. This notion comes from well-documented observations that the disorientation of mother–daughter centrioles is not seen until late in G1 and procentrioles are not evident until S phase in a wide variety of cultured cells (Robbins et al., 1968; Kuriyama and Borisy, 1981; Vorobjev and Chentsov, 1982; Alvey, 1985). Also, treatment of cultured cells with mimosine, a plant amino acid, blocks the cell cycle in G1, but the centrosome does not appear to double, as seen by the number of gamma tubulin immunoreactive spots (Matsumoto et al., 1999). However, the temporal correlation between procentriole formation and onset of S phase does not appear to be universal. For example, a high proportion of L929 cells in culture form procentrioles 4 hr before the onset of DNA synthesis (Rattner and Phillips, 1976). Also, in sea urchin zygotes arrested in interphase before the onset of DNA synthesis (a cell cycle phase presumably equivalent to G1), the centrosome inherited by each blastomere reproduces just once to form two centrosomes, each containing a pair of centrioles (Hinchcliffe et al., 1998; also see Fig. 6b). In addition, Fukasawa et al. (1996) reported that p53⫺/⫺ mouse embryonic fibroblasts (MEFs) may also support centrosome duplication in G1. For MEFs arrested in G0 by serum starvation, 17% of G0 p53⫺/⫺ MEFs had greater than two centrosomes, whereas 100% of p53⫹/⫹ MEFs had only one centrosome. After addition of serum to release

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the cell cycle arrest, none of the p53 MEFs had more than two centrosomes. However, multiple centrosomes were observed in 49% of the p53⫺/⫺ MEFs, at times well before the cell cycle had progressed into S phase. Thus, it may be premature to eliminate the possibility that the early steps of centriole duplication, such as the formation of precursor structures, occur well before DNA synthesis begins; the assembly of procentrioles to the point when they are visible in the electron microscope may mark the end of the initial reproductive processes that began in G1. This issue is important because any effort to experimentally identify cell cycle regulatory pathways that control centrosome duplication will be facilitated by an understanding of when the reproductive processes actually begin. In contrast, it is clear that the cytoplasmic conditions of S phase are permissive for the complete cycle of centrosome reproduction. In fact, prolongation of this phase in both zygotes and somatic cells through the inhibition of DNA synthesis allows multiple rounds of centrosome duplication to occur (Hinchcliffe et al., 1998; discussed in Winey, 1999) Although the daughter centrosomes normally disjoin and separate during G2, this phase of the cell cycle does not appear to support centrosome duplication. This notion comes from the observation that CHO cells arrested in G2 with the topoisomerase inhibitor etopiside contain only two sister centrosomes, whereas the same cells arrested in S assemble multiple centrosomes (Balczon et al., 1995). Importantly, the disjunction of the sister centrosomes is correlated with the rise in activity of Nek2 (Fry et al., 1998a,b), the mammalian homolog of the Aspergillus protein NIMA—a cell cycle regulatory kinase that contributes to driving entry into mitosis (Osmani et al., 1988). This suggests that the activity of Nek2 may function to coordinate sister centrosome disjunction with progression of the cell cycle into mitosis. Finally, several studies have demonstrated that the cytoplasmic conditions of mitosis do not support centrosome reproduction. When in sea urchin zygotes are microinjected with mRNA coding for a nondegradable form of sea urchin cyclin B (cyclin B ⌬90; Glotzer et al., 1991; Holloway et al., 1993), the cell cycle is arrested in M phase (Hinchcliffe et al., 1998). Anaphase onset occurs at the normal time, indicating that proteolysis at the metaphase–anaphase transition occurred, yet the cells do not exit mitosis or undergo cytokinesis. Shortly after anaphase onset the two spindle poles split apart, but no further doubling of the spindle poles occurs regardless of how long the zygotes remain in mitosis. Since each of the four poles contains only one centriole, this doubling represents a splitting of the mother–daughter centrioles, not a duplication of the centrosomes themselves (Fig. 3). Although ubiquitin-mediated proteolysis at anaphase onset happened, this does not appear to signal when centrosome reproduction begins.

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Similar results were obtained with somatic cells. Transfection of a nondegradable form of cyclin B into cultured cells arrests the cell cycle in mitosis and leads to the formation of a tetrapolar spindle (Gallant and Nigg, 1992). However, since ultrastructural analyses of these spindles were not performed, we do not know if this doubling of the spindle poles represents a splitting or full duplication of the centrosomes. At this point the simplest explanation for normal tight coordination of centrosome reproduction with other cell cycle events would be to propose that centrosomes can start to reproduce only when the cell cycle reaches or enters S phase. However, a recent study with sea urchin zygotes indicates that the situation is somewhat more complex (Hinchcliffe et al., 1998). The key observation came from an experiment in which zygotes were treated with translation inhibitors in first prophase, after they had synthesized all the necessary proteins to support first mitosis (Fig. 6b). As expected, they completed mitosis and arrested before second nuclear envelope breakdown because no new cyclin A or B was synthesized. Importantly, the two daughter blastomeres did not initiate DNA synthesis and were thus arrested in a cell cycle phase equivalent to G1 (sea urchin zygotes in the rapid early divisions do not normally have a detectable G1). The surprise was that the centrosome inherited by each daughter blastomere at the end of mitosis completely reproduced during G1 arrest. Each daughter cell contained just two centrosomes, one on each side of the nucleus, and each centrosome contained a pair of centrioles (Fig. 6b). Importantly, neither the number of centrosomes nor the number of centrioles increased thereafter. These results indicate that the G1 phase of the cell cycle supports the morphological aspects of centrosome reproduction, such as the assembly of daughter centrioles and the splitting/separation of the duplicated centrosomes. However, the finding that the sister centrosomes do not reproduce again under such supportive conditions indicates that they are lacking a component or activity necessary for reproduction. This finding, coupled with the observation that centrosomes repeatedly reproduce in second division blastomeres when they are arrested in S phase, suggests that there is an event in S phase that restores this component to the daughter centrosomes, thereby preparing them to reproduce in the next cell cycle. Although we have no information on the nature of this event, possibilities include a ‘‘licensing’’ of the centrosome for reproduction through the posttranslational modifications of key centrosomal components and/or the assembly of essential precursor structures needed for the formation of the next generation of daughter centrosomes (see Fig. 2). Taken together, these observations have led to the following working model for how centrosome reproduction is coordinated with nuclear events in the cell cycle. The centrosome inherited by a daughter cell in telophase is competent to reproduce (i.e., it was ‘‘licensed’’ during the previous S

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phase), and there is a G1 signal, conceivably passage through the restriction point, that triggers the expression of the morphological events of centrosome reproduction. These events include the splitting of the mother and daughter centrioles, as well as the assembly of procentrioles. If progress through the remainder of G1 is fast enough, the procentrioles would not be detectable until the onset of S or shortly thereafter. In addition, the centrosome cycle is linked to the nuclear cycle by a ‘‘licensing’’ event during S phase that prepares the daughter centrosomes for morphological duplication in the following G1. Disjunction and separation or the sister centrosomes are normally associated with the conditions during G2 (possibly the activity of the NIMA kinase homolog Nek2), which could couple bipolarization of the spindle with entry in M phase (Fry et al., 1998a,b). Finally, as the cell cycle enters mitosis, centrosome duplication is prevented, and can only resume after the cell has divided. Thus, the perceived independence of centrosome reproduction from progression of the cell cycle suggested by a number of previous studies (Sluder et al., 1990; Gard et al., 1990; Balczon et al., 1995) is simply due to greatly prolonging the cytoplasmic conditions of S phase, which allows both the expression of centrosome reproduction and the acquisition of reproductive capacity. Observations that S-phase arrest supports multiple rounds of centrosome reproduction raises the important question of why centrosomes do not reduplicate during S phase in the normal cell cycle. A possible answer comes from observations on sea urchin zygotes and CHO cells that the period of centrosome reduplication during S phase arrest is on average more than twice as long as the entire cell cycle (Hinchcliffe et al., 1998; Balczon et al., 1995). Under normal circumstances, therefore, S phase does not last long enough for centrosomes to reduplicate. Thus, when the cell cycle proceeds at the normal rate, progress into late G1 and entry into S are rate limiting for centrosome reproduction, and the essential coordination between centrosomal and nuclear events is assured.

B. Cyclin-Dependent Kinases in the Control of Centrosome Reproduction The link between cell cycle stage and centrosome reproduction raises the question of what control pathways establish this link. In principle, the sequential activation and inactivation of one or more of the cyclindependent kinases (Cdks) could function as the cytoplasmic mechanism that coordinately drives centrosome duplication and nuclear events in the cell cycle. Although logical, this was initially a problematic proposal because centrosomes repeatedly reproduce when progression of the cell cycle is blocked before mitosis by complete inhibition of protein synthesis (Sluder

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et al., 1990; Gard et al., 1990). Thus, the reproduction of the centrosome cannot be driven by the cyclic rise and fall of Cdk1–cyclin A or Cdk1-B activities, which depend upon the synthesis of new cyclin proteins during each cell cycle. Further investigation of this issue has also established that the absolute value of Cdk1-B activity does not establish cytoplasmic conditions that favor or inhibit centrosome reproduction (Hinchcliffe et al., 1998). Since mitotic Cdk1-cyclin combinations did not appear to control the events of centrosome reproduction, attention then shifted to whether or not Cdk2 activity plays a role in this process. Cdk2, bound to cyclin A and cyclin E, is required for the G1/S transition and the maintenance of S phase progression (Strausfeld et al., 1996; Stillman, 1996), points in the cell cycle that are correlated with the assembly of procentrioles (Kuriyama and Borisy, 1981). In addition, the levels of both the Cdk2 and cyclin E proteins remain constant from the time of fertilization until the mid-blastula transition in early Xenopus embryos (Rempel et al., 1995; Hartley et al., 1997), thus providing a possible explanation for why repeated centrosome reproduction in Xenopus zygotes is not dependent upon cyclin protein synthesis (Gard et al., 1990). Several recent studies have demonstrated that Cdk2 activity is required for centrosome reproduction in both early zygotes and somatic cells. First, Hinchcliffe et al. (1999) developed a Xenopus egg extract system (arrested in S phase with aphidicolin) that supports multiple rounds of centrosome duplication (Fig. 7). Asters organized by added sperm centrosomes doubled three times in 1-2 fashion, which is characteristic of centrosome duplication and inconsistent with either centrosome splitting or the fragmentation of the centrosomal MTOC (see Sluder and Begg, 1985). The fact that the extract is already in S means that the role of Cdk-E in the control of centrosome reproduction could be tested without the concern, applicable to live cells and cycling extracts, that inhibition of Cdk2–cyclin E activity could potentially arrest the cell cycle at a point before centrosomes are normally scheduled to reproduce. In these extracts, Cdk2-E activity was selectively inhibited by adding purified recombinant ⌬34Xic-1. This is the NH3-terminal truncated variant of Xic-1p27, the Xenopus cyclin-dependent kinase inhibitor (CKI) that inhibits Cdk2-E activity but not Cdk1-cyclin A or B at the concentration used (Su et al., 1995; Hartley et al., 1997). Since Cdk2 does not complex with cyclin A until after the mid-blastula transition in Xenopus (Rempel et al., 1995; Hartley et al., 1997), Cdk2-cyclin A activity was not a factor in these experiments. Importantly, the inhibition of Cdk2-E should not drive the cell cycle out of S phase, since the majority of S phase promoting activity is provided by Cdk1-A activity (Strausfeld et al., 1996), which is not inhibited by the concentrations of ⌬34Xic-1 used. Inhibiting Cdk2-E activity did not block the first round of aster doubling, but did prevent subsequent rounds over the duration of the experiment.

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Figure 7 (A) Repeated rounds of aster doubling in an aphidicolin-treated extract (a–e) Frames from a time-lapse sequence showing an individual aster undergoing three rounds of doubling in vitro. Polarization optics. Bar in (e) ⫽ 10 애m. (B) Analysis of aster doubling in aphidicolin-treated extracts. Each vertical line illustrates the maximum number of rounds of duplication seen for the progeny of individual asters followed for the duration of the experiment. Ordinate: Total rounds of duplication. Abscissa: Percentage for each category shown. Data taken from three experiments (N ⫽ 59 asters).

When an excess of purified Cdk2-E was added to the ⌬34Xic-1 treated extracts, multiple rounds of aster duplication took place. These observations reveal that Cdk2-E activity is required for repeated centrosome reproduction during prolonged S phase. Whether a single round of aster doubling in ⌬34Xic-1 treated extracts reflects splitting of centriole pairs or complete centrosome reproduction is an important issue to address by serial section ultrastructural analysis. Second, Lacey and colleagues (Lacey et al., 1999) arrested living Xenopus embryos in S phase with protein synthesis inhibitors, which allows repeated centrosome duplication (Gard et al., 1990). They then microinjected individual blastomeres in these arrested embryos with the CKIs p21 or p27, which bind to and block the activity of Cdk2-E (Elledge and Harper, 1994; Jackson et al., 1995). The injected blastomeres did not show the repeated centrosome duplication found in the uninjected cells of the same zygote (Lacey et al.,

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1999). In addition, these workers developed an in vitro assay that uses fluorescence light microscopy to monitor mother–daughter centriole splitting in Xenopus egg extracts treated with cylohexamide. They found that Cdk2-E activity is required to promote centriole splitting, thought to be the earliest event in the centrosome reproduction cycle (Kuriyama and Borisy, 1981). Their finding raises the question of why Hinchcliffe et al., (1999) observed one round of astral doubling in ⌬34Xic-1 treated extracts in which Cdk2-E activity was low. One explanation for this apparent discrepancy is that the Hinchcliffe et al. study used Xenopus sperm centrosomes, which may start with separated centrioles (see Felix et al., 1994), whereas Lacey et al. used isolated centrosomes from cultured cell in which the mother–daughter centriole pairs may be functionally linked to each other. Matsumoto et al. (1999) also provided evidence that repeated centrosome duplication in Chinese hamster ovary (CHO) cells that are arrested in S phase with HU is dependent upon Cdk2 activity, though their experiments did not differentiate between the importance of Cdk2-A versus Cdk2-E activity (reviewed in Winey, 1999). That centrosome reproduction is driven solely by Cdk2 bound to cyclin E has been brought into question by a recent report that Cdk2-A is more effective than Cdk2-E in restoring multiple rounds of centrosome duplication in cultured mammalian cells (Meraldi et al., 1999). For these studies, the authors used CHO cells arrested in S phase with hydroxyurea (HU). To test the role of cell cycle regulatory proteins, CHO cells (before or after addition of HU) were then transfected with plasmids encoding p16ink4, to inhibit the Cdk4 and Cdk6 kinases, which in turn blocks phosphorylation of the retinoblastoma protein (Rb) and arrests the cell cycle in G1 (Lukas et al., 1997). They found that p16ink4 transfection before HU addition blocked centrosome duplication, whereas transfection after S phase arrest did not, thus ruling out a direct role for either Cdk4 and Cdk6 activity in control of the centrosome cycle. Next, these workers tested the relative importance of Cdk2-cyclin A versus Cdk2-cyclin E activity in supporting centrosome reproduction. They coexpressed plasmids for p16ink4 and either cyclin E and cyclin A. Not surprisingly, expression of these cyclin constructs could overcome the p16ink4 block, probably by phosphorylation of Rb protein, allowing the cell cycle to progress into S phase. However, when they blocked the G1/S transition by transfection with RbDCdk (a mutant form of Rb that lacks putative Cdk phosphorylation sites; Lukas et al., 1997), only coexpression of cyclin A restored significant levels of repeated centrosome duplication; overexpression of cyclin E did not. The restoration of centrosome duplication by cyclin A was dependent strictly upon Cdk2 activity, as it could be inhibited by cotransfection with a dominant negative Cdk2 construct. Taken together, these findings suggest that Cdk2-A activity is

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required for centrosome duplication and this kinase acts downstream of its role in the Rb phosphorylation pathway needed to drive the G1/S transition. These findings are interesting, because they suggest that somatic cells and early cleavage stage zygotes use different Cdk2–cyclin complexes to regulate centrosome reproduction. However, this may not reflect fundamentally different control strategies; perhaps centrosomes, or the downstream pathways that regulate them, are responsive to both Cdk2-A and Cdk2-E. Since Cdk2 does not complex with cyclin A until the mid-blastula transition in Xenopus zygotes (Rempel et al., 1995), Cdk2-E may be the only kinase complex available to do the job. In somatic cells that contain both Cdk2-A and Cdk2-E kinase complexes, perhaps Cdk2-A is more effective in promoting centrosome reproduction. Nevertheless, despite these early indications that the regulation of centrosome reproduction in various cell types may differ slightly in the details, it is clear that cell cycle progression into S phase and the establishment of conditions that support multiple rounds of centrosome reproduction are linked through a common pathway—Cdk2 activity. Looking forward, there are a number of fascinating issues to explore. For example, when does centrosome reproduction first begin? Is the regulation of centrosome reproduction really a two-step process starting with the onset of conditions during G1 that trigger the steps in the morphological completion of daughter centriole assembly followed by a ‘‘licensing’’ event specific to S phase (Hinchcliffe et al., 1999)? Alternatively, does the whole reproductive cycle of the centrosome depend upon a rise in Cdk2 activity, be it based in Cdk2-A and/or Cdk2-E complexes? Also, are other control pathways involved? For example, do the activities of the polo kinases (reviewed in Glover et al., 1998) or Aurora kinases (reviewed in Giet and Prigent, 1999) play a direct role in the events of centrosome reproduction, or do these kinases effect only the physical separation of duplicated centrosomes through their regulation of the activity of microtubule-based motor molecules? Is ubiquitin-mediated proteolysis, at times other than the metaphase–anaphase transition, necessary for the splitting and disorientation of the mother–daughter centrioles, as suggested by recent reports (Gstaiger et al., 1999; Freed et al., 1999); is this splitting event absolutely required for their reproduction? If proteolysis is important, is it signaled by the same activities that drive entry into S phase, or is it regulated independently? Finally, does centrosome reproduction require a microtubule network to transport subunit proteins to the cell center (Balczon et al., 1999; Purohit et al., 1999), and if so, by what mechanism does this transport occur? Although we may be overwhelmed by the emerging complexity of the events and control mechanisms involved in centrosome reproduction, we can be pleased that there are yet more intriguing avenues for further research. As Chandler Fulton (1982) aptly said of the centriole,

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‘‘Many of us have had a transient affair with centrioles, and retain a profound affection for them, but few have committed ourselves to a long-term relationship, perhaps because, in spite of our enchantment, we could not find a way to build a meaningful relationship—that is, to break through the enigmas.’’ Thus, it is gratifying to find that, after being investigated for more than a hundred years, the centrosome still retains enough mysteries to continue attracting new generations of eager suitors.

Acknowledgments We thank Jim Maller, Conly Rieder, and Bob Palazzo for stimulating conversations on the nature of centrosomes and the cell cycle. We especially appreciate the efforts of Rick Miller in preparing the figures for this article. This work was supported by grants from the NIH (GM 30758), the Cabot Family Charitable Trust, and the Trustees of the Worcester Foundation to GS. E.H.H. is supported by an NRSA Cell Biology of Development Post-Doctoral Training Grant (NIH HD 07312).

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