Results Probl Cell Differ (42) P. Kaldis: Cell Cycle Regulation DOI 10.1007/b136685/Published online: 14 July 2005 © Springer-Verlag Berlin Heidelberg 2005 The Centrosome Cycle Christopher P. Mattison · Mark Winey (✉) MCD Biology, University of Colorado-Boulder, CB347, Boulder, CO 80309-0347, USA Mark.Winey@Colorado.edu Abstract Centrosomes are dynamic organelles involved in many aspects of cell function and growth. Centrosomes act as microtubule organizing centers, and provide a site for concerted regulation of cell cycle progression. While there is diversity in microtubule organizing center structure among eukaryotes, many centrosome components, such as centrin, are conserved. Experimental analysis has provided an outline to describe cen- trosome duplication, and numerous centrosome components have been identified. Even so, more work is needed to provide a detailed understanding of the interactions between centrosome components and their roles in centrosome function and duplication. Precise duplication of centrosomes once during each cell cycle ensures proper mitotic spindle formation and chromosome segregation. Defects in centrosome duplication or function are linked to human diseases including cancer. Here we provide a multifaceted look at centrosomes with a detailed summary of the centrosome cycle. 1 Introduction 1.1 History The centrosome is a unique organelle, first described and fully appreciated for its importance in the cell cycle by Theodor Boveri in early 1900 (for a re- view of Boveri’s work, see Moritz and Sauer 1996). Working with roundworm and purple sea urchin embryos, he carefully documented the assembly of the mitotic spindle and thoughtfully described the centrosome in detail. He noted the presence of two centrioles engulfed by a dense sphere of material andalsoprovidedadetailedoutlineofthecentrosomecycle.Hisexperimen- tation indicated that centrosomes duplicate once per cell cycle and act as equivalent force generating anchors for the mitotic spindle. In his thorough observations, he also noted the occurrence of multipolar and monopolar spindles during the cell cycle as spontaneously occurring abnormalities. No- tably, in the analysis of his own work, he insightfully conceptualized the idea that centrosome malfunction and the resulting chromosome inequal- ity might lead to malignant tumors, as documented in his book The Origin of Malignant Tumors (1914) (for a review, see Manchester 1995). The past 15 years has seen a surge in interest and activity in centrosome study and 112 C.P. Mattison · M. Winey an increase in understanding of this organelle. In this review, we consider the progress made in understanding centrosome complexity, function, link to disease, and duplication. As you will see, much of the recent progress made in understanding centrosome duplication and function serves to re- inforce Boveri’s conclusions and highlight the importance of his pioneering contributions. The past 15 years has seen a surge in interest and activity in centrosome study and an increase in the understanding of this complex organelle. In this review, we first provide a brief look at centrosome related organelles and sur- vey the growing list of centrosome functions. We also describe centrosome structure, complexity and link to disease. Next, we delve into the main body of this text and provide a comprehensive review of the components and regu- lators involved in centrosome duplication. 1.2 Microtubule Organizing Centers The centrosome is one example of a broad class of structures called micro- tubule organizing centers (MTOCs). MTOCs from different organisms are morphologically distinct, but serve the same function to nucleate micro- tubules. These structures also contain many conserved components such as centrin and tubulins. There are centrosome related organelles such as basal bodies in ciliates, asters in plants, and spindle pole bodies (SPBs) in yeast. Each of these organelles is used to arrange microtubules into distinct func- tional arrays. Basal bodies are also found in specialized ciliated and flagel- lated cells in the kidney, lung and sperm in mammals. Chlamydomonas and sperm basal bodies are converted into centrosomes. This observation serves to reinforce the equivalent nature of these organelles. The yeast spindle pole body (SPB) serves as the centrosome, but differs in that it is a membrane bound organelle and is duplicated during G1 rather than S-phase as in mam- malian cells. There are also differences among the centrioles of metazoans. For example, D. melanogaster centrioles contain doublet microtubule pairs while in C. elegans they are singlets. Studies of different systems and or- ganelles have complemented one another in many instances and provided the building blocks for our current understanding of centrosome function and duplication. 1.3 Centrosome Functions Centrosomes are unique organelles that function to organize cellular micro- tubules, and they are important for mitotic spindle positioning, cell division, and cell cycle progression (Heald et al. 1997; Hinchcliffe et al. 2001; Khod- jakov and Rieder 2001; Piel et al. 2001). However, bipolar mitotic spindles The Centrosome Cycle 113 can form in the absence of centrosomes (Khodjakov et al. 2000). More re- cently, centrosomes have been shown to serve as a signaling platform for many cell cycle decisions (Kramer et al. 2004; Doxsey 2005). Centrosomes also play a role in organization of interphase microtubules to maintain proper cell shape and influence nuclear translocation and cell migration (Schliwa et al. 1999; Abal et al. 2002; Malone et al. 2003). 1.4 Centrosome Dysfunction and Cancer/Disease Proper duplication of centrosomes is essential for bipolar spindle formation and equal segregation of chromosomal DNA to daughter cells. Centrosome defects can lead to genetic imbalance, and centrosome abnormalities have been shown to be a marker for cancer [see reviews by Nigg (2002); Sluder and Nordberg (2004)]. For example, studies involving a panel of cells from both normal breast and breast tumor tissue have shown a direct relationship between centrosome abnormalities and chromosome instability/aneuploidy (Lingle et al. 2002). Similarly, a survey of prostate and other cancers has shown that centrosome defects can be found at the earliest detectable stages of cancer and increase with chromosome instability, and more importantly with tumor grade (Pihan et al. 2003). An interesting observation from these analyses is the lack of correlation between p53 mutation and cen- trosome defects or chromosome instability (Lingle et al. 2002; Pihan et al. 2003). However, p53 mutation is correlated with increased microtubule nu- cleation by centrosomes, and p53 status can dramatically affect centrosome number, both by affecting centrosome duplication and cytokinesis (Fuka- sawa et al. 1996, 1997; Lingle et al. 2002; Tarapore and Fukasawa 2002). In addition to p53, the retinoblastoma protein (RB) and breast cancer 1 (BRCA1) tumor suppressor proteins localize to centrosomes with cell cycle specific timing, suggesting an important link between centrosomes, proper cell cycle regulation, and genetic stability (Thomas et al. 1996; Hsu and White 1998). Thus there is a clear link between centrosome defects and can- cer. It should be noted that additional mechanisms are probably involved in mediating compensation for prolonged cell division from a progenitor cell(s) derived from multipolar spindles and cancer progression [see re- views by Nigg (2002); Sluder and Nordberg (2004)]. Finally, there are at least eight ciliated cell types in humans, and diseases including Bardet-Biedl syndrome and polycystic kidney disease, and several neuronal disorders have been linked to mutations in basal body, centriolar, and pericentrio- lar material (PCM) proteins (Afzelius 2004; Snell et al. 2004; Bodano et al. 2005) providing additional links to centriole/basal body dysfunction and disease. 114 C.P. Mattison · M. Winey 1.5 Centrosome Structure Electron microscopy studies have provided informative details of centrosome structure and offered an outline of the centrosome cycle (Chretien et al. 1997; Kuriyama and Borisy 1981; Vorobjev and Nadezhdina 1987; see Figure 1). The centrosome of vertebrates contains two orthogonally spaced, loosely connected, cylindrically shaped centrioles at its core. The centrioles are com- posed of nine-triplet microtubule bundles symmetrically organized around a circular hub. It is estimated that centrioles are ∼ 1 µM 3 in size. At the base or proximal end of each centriole is a cartwheel structure, and this end an- chors the minus ends of the centriole microtubules. On the mother centriole, this end also serves as the initiation platform for daughter centriole con- struction. The distal end contains the plus end of centriole microtubules and is the site for the assembly of distal appendages. Distal appendages are fin- like structures giving centrioles a rocket-like shape, but are not found in all centrioles. They function in microtubule nucleation, membrane attachment of primary cilia, and serve as a maturation marker. Surrounding the paired centrioles is the PCM, a dense cloud of structured matter (On et al. 2004). In- teractions between PCM components and centrioles are important for proper centrosome function and duplication. The PCM is composed of proteins that anchor the gamma-tubulin ring complex (γ-TuRC) and other components in- volved in microtubule nucleation. Lastly, some PCM components are shared with centrioles and are also important for accurate centrosome duplication. 2 The Centrosome Cycle 2.1 Introduction This chapter is focused on the centrosome cycle, the changes that occur within the centrosome throughout the cell cycle, and the molecules that reg- ulate these processes. For additional material reviewing centrosomes, see Ou and Rattner (2004). The typical mammalian G1 cell contains a single centro- some with two centrioles, an older mother and younger daughter, at its core. As the cell progresses from G1 through S-phase, the centriole pair within the centrosome lose their orthogonal position, although centriole disorientation has been observed to occur as early as telophase. During S-phase, centriole duplication ensues with each centriole serving as the template for a daugh- ter centriole. In general centriole duplication is initiated first in the mother centriole (White et al. 2000), although untemplated centriole duplication has The Centrosome Cycle 115 Fig. 1 Thecentrosomecycle.a ThecentrosomeinanearlyG1cellcontainsamother and daughter centriole pair in an orthogonal orientation. b In late G1 or at the G1/S transition, the centriole pair lose their tight association and disorient. c In S-phase, cartwheel structures form at the proximal end of both centrioles. d Procentrioles form and e continue to elongate in S-phase. f–h Centrosome maturation begins in late S-phase and continues throughout G2. Maturation includes the recruitment of additional PCM components, increased microtubule nucleation, and the addition of distal appendages to the oldest centriole. i At the G2/M transition centrosomes separate, j move apart to form the mitotic spindle poles, and mitosis ensues. Following mitosis, centrosomes lose much of the additional PCM and return to a G1 state been observed (Khodjakov et al. 2002). Centrioles continue to elongate, and it is not until after G2 that two complete centrosomes have formed. These centrosomes are not equivalent, as one contains a grandmother/daughter pair of centrioles and the other a mother/daughter pair. During G2 and the G2/M transition, proteins are added to the two parental centrioles within a centrosome in a maturation process that causes morphological and func- tional distinctions between the centriole pairs. In addition, the amount of PCM increases. Concomitantly, duplicated centrosomes separate and migrate to opposite sides of the nucleus in preparation for mitotic spindle assem- bly. After mitosis, centrosomes return to a G1 state in which they have a reduced/altered microtubule nucleation capacity. Our description will be based on the canonical mammalian centrosome cycle (Fig. 1), but where 116 C.P. Mattison · M. Winey helpful we incorporate information from other systems to provide a more complete picture. 2.2 Centrosome Duplication As mentioned, EM studies of centrosome duplication have provided an ex- cellent guide for dissecting the centrosome duplication cycle into discrete steps (Kuriyama and Borisy 1981; Vorobjev and Nadezhdina 1987; Chre- tien et al. 1997) and are summarized in Hinchcliffe and Sluder (2001), Meraldi and Nigg (2002) and Delattre and Gonczy (2004). Experiments using labeled tubulin show that only the newly forming centriole incorporates tubulin during duplication of centrioles, indicating a conservative mech- anism of duplication (Kochansky and Borisy 1990). Once centrosome sep- aration occurs, centrioles are partitioned such that each cell receives ei- ther the grandmother/daughter or mother/daughter centriole pair (semi- conservative distribution), and therefore each cell has a unique centrosome assembly (Kochanski and Borisy 1991). Importantly, using cell fusion assays, it has been shown that a G1 cell duplicates its centrosome when fused to S or G2 phase cells, but G2 centrosomes cannot duplicate in G1/Scytoplasm (Wong and Stearns 2003). This indicates there are factors inherent to the cen- trosome that prevent reduplication during a normal cell cycle. 2.2.1 Cyclin-Dependent Kinase 2, Cdk2 Mammalian centrosome duplication occurs once each cell cycle and normally begins in late G1 with centriole disorientation. Centriole duplication proceeds through S-phase, and the duplicated centrosomes mature during G2 and M. Regulated expression of different cyclins in association with specific mem- bers of the conserved serine/threonine cyclin-dependent kinase (Cdk) family is important for the timing and progression of cell cycle events, including centrosome duplication (Hinchcliffe and Sluder 2002). Several studies have implicated cyclin-dependent kinase 2 (Cdk2) as an important regulator of ini- tiation and progression of the centrosome cycle. Cells arrested in S-phase are permissive for extra rounds of centrosome duplication, and this observation has provided an assay to demonstrate the requirement of factors in centro- some duplication (Kuriyama et al. 1986; Balczon et al. 1995). Cdk2 activity has a role in DNA replication (reviewed in Woo and Poon 2003) and also partici- patesincentrosomere-duplicationinS-phasearrestedcells(Matsumotoetal. 1999; Meraldi et al. 1999). Similarly, Cdk2/cyclin E activity, which drives the G1/S transition, contributes to centrosome duplication in X. laevis extracts (Hinchcliffe et al. 1999; Lacey et al. 1999; Matsumoto et al. 1999). Cdk2 also associates with cyclin A, and centrosomes can reduplicate in CHO cells under The Centrosome Cycle 117 conditions thought to be specific for Cdk2/cyclin A activity (Meraldi et al. 1999) or in HeLa cells overexpressing cyclin A (Balczon 2001). While the role of Cdk2 is not completely understood, these and other studies clearly demon- strate a role for Cdk2 coupled to cyclin A or E in centrosome duplication (Tarapore et al. 2002). Proper regulation of centrosome duplication requires the function of sev- eral transcription factors. For example, phosphorylation of the Rb protein and release of bound E2F transcription factors is required for centrosome duplication (Meraldi et al. 1999). Release of E2Fs presumably leads to the up- regulation of cyclins, other cell cycle regulators, and centrosome structural components. In addition, the status of the p53 protein/transcription factor can affect centrosome number. Careful studies of p53-/-mouseembryonicfi- broblasts(MEFs)haveshownthatcentrosomeabnormalitiesintheabsenceof p53 can arise from cytokinesis defects, but the major route for p53-dependent centrosome number defects arises from inappropriate initiation of centro- some duplication and/or prevention of reduplication (Fukasawa et al. 1996; Fukasawa et al. 1997; Tarapore and Fukasawa 2002). The p53 protein is known to control transcription of the Cdk inhibitor p21, and this indirectly influ- ences centrosome duplication; however, p53 localizes to centrosomes and can also directly affect centrosome duplication (Tarapore et al. 2001a,b; Tarapore and Fukasawa 2002). Cdk2/cyclin E or A phosphorylates p53 at serine 315 (Wang and Prives 1995), and while mutations at this site do not affect p53 transcriptional activity (Crook et al. 1994; Fuchs et al. 1995), they can affect localization of p53 to centrosomes. The p53-S315A mutation prevents local- ization to unduplicated centrosomes, suggesting a direct link to regulation of centrosome duplication (Tarapore et al. 2001b; Tarapore and Fukasawa 2002). 2.2.2 Cdk2 Substrates While there is clearly a role for Cdk2-cyclin A/E in centrosome duplication, the mechanism by which Cdk2 controls centrosome duplication is not com- pletely understood. There are currently four known Cdk2 substrates relevant to centrosome duplication, including p53 (described above), Nucleophosmin, CP110, and Mps1. Nucleophosmin (NPM/B23/numatrin) is a nucleolar pro- tein involved in ribosome biogenesis and also regulates the stability and transcriptional activity of p53 (Colombo et al. 2002). NPM localizes to undu- plicated centrosomes and is phosphorylated on Thr199 by Cdk2/cyclin E late in G1 (Okuda et al. 2000; Tokuyama et al. 2001). This phosphorylation dissociates NPM from unduplicated centrosomes and is required to allow duplication to proceed (Okuda et al. 2000; Tokuyama et al. 2001; Tarapore et al. 2002). Conversely, there is evidence that phosphorylation of NPM on two threonine residues, amino acid 234 and 237, by Cdk1/cyclin B is im- portant for its recruitment to centrosomes after nuclear envelope breakdown 118 C.P. Mattison · M. Winey (Cha et al. 2004). This may impart some change in microtubule dynamics at the centrosome for spindle assembly, or it may provide a regulatory circuit to reset each centrosome prior to duplication for the next cell cycle. These observations suggest that phosphorylation of NPM by different kinases is expression of a non-phosphorylatable important for multiple stages of centro- some duplication. Another Cdk2 substrate is CP110, which was isolated in a screen for pro- teins that bound to a dominant negative Cdk2 allele. CP110 is a substrate for Cdk2/cyclin A or E and Cdk1/cyclin B (Chen et al. 2002). CP110 is an integral coiled-coil centrosome component whose protein level peaks during S-phase. Treatment of cells with CP110 RNA interference (RNAi) or CP110 allele leads to premature centrosome separation. In addition, CP110 RNAi treatment pre- vents centrosome reduplication in S-phase arrested cells (Chen et al. 2002). Taken together, these data suggest that CP110 is a structural component that may be important for centriole disorientation, or play a role in the timing of centrosome separation. Lastly, the Mps1 protein kinase has been demonstrated to be a Cdk2 sub- strate whose stability during S-phase is regulated by Cdk2 phosphorylation (Fisk and Winey 2001). The mouse and human Mps1 proteins (mMps1 and hMps1) localize to centrosomes throughout the cell cycle and their over- expression can drive centrosome re-duplication (Fisk and Winey 2001; Fisk et al. 2003; Liu et al. 2003; Quintyne et al. 2005). Conversely, expression of kinase inactive Mps1 or treatment with MPS1 RNAi prevents the normal du- plication of centrosomes during S-phase (Fisk and Winey 2001; Fisk et al. 2003). The threshold level of hMps1 activity required for centrosome duplica- tion seems to be very low, and a severe decrease in hMps1 level is required to reveal its role in centrosome duplication (Fisk et al. 2003). Experiments that do not sufficiently reduce hMps1 activity have no effect on centrosome dupli- cation (Stucke et al. 2002). In addition, hMps1 autophosphorylation compli- cates the analysis of immunofluorescence microscopy localization data, due to the varying efficacy with which numerous antibodies recognize Mps1 (Stucke et al. 2004). Consequently, while there are data implicating mammalian Mps1 in centrosome duplication, conclusions about its role must be tempered while waiting for continued analysis. In some other systems, it seems Mps1 is not involved in centrosome duplication. For example, no role for the S. pombe and Drosophila Mps1 proteins in SPB/centrosome duplication has been de- tected (He et al. 1998; Bettencourt-Dias et al. 2004; Fischer et al. 2004). In S. cerevisiae however, there are clearly multiple roles for Mps1 in SPB duplica- tion (Jaspersen and Winey 2004). Further, analogous to mMps1, S. cerevisiae Mps1 is also a Cdc28 (Cdk homolog) substrate, and this interaction stabi- lizes the Mps1 protein (Fisk and Winey 2001; Jaspersen et al. 2004). However, even in S. cerevisiae the role of Mps1 in centrosome duplication is only partly understood, and there are only three identified Mps1 substrates relevant to centrosome duplication/function (reviewed in Jaspersen and Winey 2004). The Centrosome Cycle 119 The only potential centrosome related substrate described to date for hMps1 is the transforming acidic coiled-coil protein-2 (TACC2). TACC2 interacts with hMps1 in mitotic lysates, and its centrosome localization is disrupted by expression of kinase inactive hMps1 (Dou et al. 2004). While this interac- tion may be important for spindle stability, it is not clear what role, if any, it plays in centrosome duplication. It is important to note that Mps1 proteins also play a highly conserved role in the mitotic spindle checkpoint (Abrieu et al. 2001; Martin-Lluesma et al. 2002; Poss et al. 2002; Stucke et al. 2002, 2004; Liu et al. 2003; Fischer et al. 2004; Fisk and Winey 2004). Stucke et al. (2004) also observed that microtubules can increase hMps1 kinase activity in vitro, suggesting a possible regulatory mechanism in vivo. While these studies strongly suggest an essential role for Cdk2 in centro- some duplication, it seems likely that there is redundancy among Cdk/cyclin complexes and that other Cdk/cyclin complexes can function to drive centro- some duplication. In support of this, Cdk2 is dispensable for normal mouse development (Berthet et al. 2003; Ortega et al. 2003). In addition, mice lacking both cyclin E1 and E2 have problems with DNA replication in some special- ized cells required for complete gestation, but appear to have normal early embryonic development (Geng et al. 2003). Nonetheless, there is sufficient data to indicate that Cdk2 and its associated cyclins are important for the regulated duplication of centrosomes. Undoubtedly there are additional Cdk substrates important for centrosome duplication, and further analysis is re- quired to identify them. Moreover, the interactions between Cdk2 and other kinases such as Mps1 must be better understood to provide a more complete picture of how centrosome duplication is regulated. Lastly, the differences in the ability of Cdk2 cyclin A or E associated activity to regulate centrosome duplication in the various model systems may provide clues to the regulation of the process or its coupling to the cell cycle. 2.2.3 Centriole Disorientation In G1, a cell has a single centrosome containing a mother/daughter pair of centrioles. Each of these centrioles can provide a template site for the initiation of a new centriole. The first step in centrosome duplication is dis- orientation (also called splitting or loss of orthogonal positioning) of the centrioles just prior to initiation of daughter centriole formation (Fig. 1). Centriole disorientation can occur as early as anaphase/telophase in the pre- vious cell cycle, but is most often thought to occur late in G1 or at the G1/S transition. Several proteins have been implicated in disorientation, but it is not clear what specific cues are required. First, Cdk2 activity is important for cen- trosome duplication, and in human cells Cdk2 paired with cyclin A or E overexpression can induce disorientation of parental centrioles (Meraldi and 120 C.P. Mattison · M. Winey Nigg 2001). This may reflect an early role for Cdk2 prior to centriole dupli- cation. Secondly, a highly conserved component of centrioles is the centrin protein (discussed below), and human centrin2 is phosphorylated by pro- tein kinase A (PKA). Further, elevated PKA expression can cause interphase centrioles to separate, suggesting that this phosphorylation event may be an important cue for initiation of centriole disorientation (Lutz et al. 2001). Thirdly, a protein linkage is thought to connect paired centrioles within a centrosome, and this linkage may need to be modified or broken to allow centriole disorientation. Thus, it is likely that proteolysis may be import- ant for this early step in centriole duplication. In support of this, embryos from Drosophila Fizzy mutants (a homolog of Cdc20 involved in proteolysis during the metaphase to anaphase transition) have delayed centriole dis- orientation (Vidwans et al. 1999). Further, mammalian components of the Skp1-Cullin-F-box (SCF) complex (important for entry into S-phase) localize to centrosomes, and inhibition of SCF components in Xenopus extracts blocks centriole disorientation (Freed et al. 1999). While these observations estab- lish a role for proteolysis in centriole disorientation, the targets are not yet known. In summary, centriole disorientation is the first identifiable step in the process of centriole duplication, and occurs at or prior to the G1 to S-phase transition. It is thought that proteolysis or other modification of proteins link- ing paired centrioles in their orthogonal orientation is required for this step. Further research will hopefully clarify this step and provide a more complete list of the proteins involved and their regulation. 2.2.4 Daughter Centriole Formation We favor the idea that centriole assembly/elongation resembles the self- assembly of viral capsids. That is, once centriole duplication has been initi- ated with a template, it is propagated by the sequential addition of proteins independently of the mother centriole. An initial step thought to occur is the generation of a cartwheel structure (Fig. 1). The coiled-coil Bld10 protein has been localized to the cartwheel structure by EM, and it is the only identified cartwheel component (Matsuura et al. 2004). Bld10 was isolated as a flagella- less mutant from Chlamydomonas rheinhardtii and characterization of Bld10 mutants shows an absence of any basal body structures, indicating that it is required for the earliest steps of basal body assembly (Matsuura et al. 2004). Following cartwheel formation, procentrioles form and centriole duplication continues in S-phase. Not all the genes described in the following sections have been shown to function solely in centriole elongation. Some of them may function prior to elongation or at multiple steps in the pathway, but further investigation is required to determine this. [...]... the list is sure to grow Several proteins critical to cell cycle regulation localize to centrosomes, and their localization is thought to be important for the coordinated relationship between cell cycle progression and the centrosome cycle The Centrosome Cycle 135 Continued study of centrosomes is essential as centrosome/ basal body abnormalities are connected to a growing number of diseases For instance,... microtubule arrays and determines centrosome position EMBO J 18:6786–6792 Kramer A, Lukas J, Bartek J (2004) Checking out the centrosome Cell Cycle 3:1390–1393 Kuriyama R, Borisy G (1981) Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle J Cell Biol 91:822–826 The Centrosome Cycle 141 Kuriyama R, Dasgupta... PCM, prior to and during duplication, but during maturation is also recruited to the daughter centrosome (Chang and Stearns 2000) EM analysis shows that human ε-tubulin localizes specifically to the sub- distal appendages of the grandmother centriole in the older centrosome, and is recruited to the mother centriole of the new centrosome only after S-phase X laevis ε-tubulin depleted extracts fail to duplicate... to the next cell cycle The presence of NPM at the centrosome would prevent centrosome duplication until Cdk2/cyclin E/A phosphorylation removes it from the centrosome in the following S-phase (Okuda 2002) 2.6 Post-Mitosis Return to G1 After mitosis centrosomes return to a G1 state, decreasing in size and microtubule nucleation capacity as cells prepare for duplication in the next cell cycle How this... complexes are important for microtubule nucleation at centrosomes 2.4 Centrosome Separation 2.4.1 NIMA-Related Kinase-2 (NEK2) Once duplicated, the tether between grandmother and mother centrioles must be severed for the duplicated centriole pairs/centrosomes to separate prior to mitosis The conserved NIMA-related kinase 2 (NEK2) is a homolog of the Aspergillus never in mitosis A (NIMA) protein involved... with the kinesin Eg5, and potentially other motor proteins, contributes to the proper separation of centrosomes and the maintenance of spindle pole separation Several other kinases and phosphatases have also been implicated in regulating centrosome cohesion For example, inhibition of the PCM localized Rho-dependent kinase p160ROCK leads to stable G1 centriole separation and movement of the mother centriole... characterizing the interactions between these proteins will hopefully answer this question 2.3 Centrosome Maturation Centrosome maturation occurs during late S/G2 and early mitosis and continues as cells enter mitosis It is characterized by a significant increase in centrosome size and microtubule nucleation through the recruitment of ad- The Centrosome Cycle 127 ditional PCM proteins (Palazzo et al 2000) The. .. Cdk1/cyclin B in regulating the deconstruction of mitotic centrosomes Lastly, an interesting aspect of centrioles is their migration after telophase Using GFP labeled centrin as a centriole marker Piel et al (2000) observed movement of the mother and daughter centrioles within the cell The mother centriole migrated to the intercellular bridge during cell narrowing but moved back to the cell center before... guides the organization of PCM The PCM components that engulf centrioles work in concert with centrioles and are essential for the intricate microtubule patterns formed during cell growth The network of interactions between centriole and PCM components must be further characterized to fully understand their role in centrosome duplication and the organization of microtubule networks guided by the centrosome. .. and Schiebel 1998) In humans, the pericentrin protein, a homolog of Spc110, functions to anchor γ -TuRC to the centrosome (Doxsey et al 1994; Li et al 2001; Flory and Davis 2003; Zimmerman et al 2004) Pericentrin is not the only protein thought to recruit the γ -TuRC to centrosomes Another Spc110 homolog, the A-kinase anchoring protein-450 (AKAP450 or CG-NAP), shares the centrosome targeting PACT domain . accurate centrosome duplication. 2 The Centrosome Cycle 2.1 Introduction This chapter is focused on the centrosome cycle, the changes that occur within the centrosome. untemplated centriole duplication has The Centrosome Cycle 115 Fig. 1 Thecentrosomecycle.a ThecentrosomeinanearlyG1cellcontainsamother and daughter centriole pair