Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology - Developmental Biology,
GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11,
Georg-August-University of Göttingen, Germany
The centrosome, located at the cell center, is the main microtubule organizing center (MTOC) of the cell. In interphase, it assembles microtubules that are then anchored with their minus ends at the centrosome. Since microtubules (MTs) build the cellular framework for the positioning of cell organelles, the centrosome is important for the whole cellular architecture. In mitotic cells, the centrosome plays a central role in organizing the mitotic spindle to correctly separate the chromosomes. Aberrant centrosomes are often associated with aneuploidy and tumorigenesis. In animal cells the centrosome consists of a pair of centrioles surrounded by the pericentriolar material (PCM). Intrinsic proteins of the centrosome are mostly characterized by their overall coiled-coil structure, but otherwise lack specific domains. Additionally, a lot of proteins show a cell cycle dependent association with the centrosome leading to the assumption, that the centrosome might orchestrate cell cycle progression. In non-cycling cells, the centrosome is transformed into the basal body to generate a primary cilium, a cellular organelle that has long been neglected. Nowadays, the importance of primary cilia for signal transduction and cell homeostasis is widely acknowledged as indicated by the constantly growing list of cilia dependent diseases. The centrosome thus is an essential organelle in most animal cells affecting cell shape, cell cycle progression, and the cellular response to environmental cues.
The centrosome is a tiny organelle approximately ~1-2 µm in diameter and positioned centrally in animal cells. It consists of two orthogonally arranged centrioles embedded in an electron dense mesh called the pericentriolar matrix (PCM) (reviewed in: Kellogg et al., 1994; Stearns and Winey, 1997; Urbani and Stearns, 1999; Zimmerman et al., 1999; Bornens, 2002) (Fig. 1). Centrioles are microtubule based organelles mostly with a characteristic radial array of nine microtubule (MT) triplets. They are usually about 0,5 µm long and 0,2 µm in diameter. Both centrioles of a typical centrosome differ in age (see below) and structure and are associated by interconnecting fibers. The older centriole, also called the mature or mother centriole, is characterized by distal and subdistal appendages. Subdistal appendages function by anchoring microtubules, whereas distal appendages seem to be involved in anchoring the centriole/basal body to the cell membrane (reviewed in: Hoyer-Fender, 2010).
The PCM consists of a large number of proteins that share a characteristic coiled-coil structure. Although the pericentriolar material has long been described as an "amorphous cloud of electron dense material" centrosomal coiled-coil proteins may form a lattice which serves as a platform to which regulatory proteins may bind (reviewed in: Fry and Hames, 2004). An integral coiled-coil protein of the PCM is, e.g., pericentrin/kendrin that plays a role in centrosome and spindle organization and forms a complex with γ-tubulin (Dictenberg et al., 1998; Doxsey et al., 1994; Li et al., 2000; Flory et al., 2000; Flory and Davis, 2003). Some other coiled-coil proteins are e.g. ninein, Cep135, centriolin, CG-NAP/AKAP350/AKAP450, and ODF2/cenexin (Bouckson-Castaing et al., 1996; Lange and Gull, 1995; Takahashi et al., 1999; Takahashi et al., 2002; Schmidt et al., 1999; Ohta et al., 2002; Gromley et al., 2003; Donkor et al., 2004). Ninein may function in microtubule minus-end capping and anchoring and may promote microtubule nucleation by docking the γ-tubulin ring complex (γ-TuRC) at the centrosome (Mogensen et al., 2000; Delgehyr et al., 2005). Cep135 is a universal component of the centrosome in a wide range of organisms and important for MT organization (Ohta et al., 2002). Organization of centrosomal microtubules in addition seems to be dependent on transforming acidic coiled-coil (TACC) proteins, a family of proteins that concentrate to centrosomes through a conserved coiled coil carboxy-terminal domain called the TACC domain (Gergely et al., 2000a; Gergely et al., 2000b; Lee et al., 2001). Centriolin localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression (Gromley et al., 2003). ODF2/cenexin is a marker of the mature centriole and essential for primary cilia formation (Nakagawa et al., 2001; Ishikawa et al., 2005).
In the interphase cell, the centrosome functions as the main microtubule organizing center (MTOC) to nucleate and anchor microtubules (MTs). Microtubules are assembled from soluble tubulin subunits and are important for the maintenance of cell morphology and the position of organelles. Whereas the centrioles may rather act as a scaffold on which to assemble the pericentriolar material, the MT nucleation and anchorage capacity of centrosomes relies on components of the PCM. MT are anchored at their minus ends by the γ-tubulin ring complex (γ-TuRC) located in the PCM (Moritz et al., 1995a; Moritz et al., 1995b; Wiese and Zheng, 2000). The γ-tubulin ring complex (γ-TuRC) consists of γ-tubulin and a couple of associated proteins (Moritz et al., 1998; Murphy et al., 1998; Tassin et al., 1998; Oegema et al., 1999; Fava et al., 1999; Murphy et al., 2001). γ-tubulin is essential for MT nucleation as has been shown by depletion experiments (Raynaud-Messina et al., 2004). γ-tubulin binds to the minus ends of MTs and by capping them prevents minus end growth (Li and Joshi, 1995; Leguy et al., 2000). Although in animal cells most MTs nucleate from centrosomes, the bulk of soluble γ-tubulin is found in the cytoplasm (about 80% in contrast to only 20% found at the centrosome) but is devoid of significant MT nucleation activity (Moudjou et al., 1996). At the onset of mitosis, concomitant with an increase in MT nucleation activity, additional γ-tubulin is recruited to the centrosome as well as numerous regulatory proteins (reviewed in: Schiebel, 2000). This structural reorganization of centrosomes in the preparation for mitosis is termed maturation and protein kinases as well as phosphatases have been implicated in its regulation (reviewed in: Meraldi and Nigg, 2002).
In addition to intrinsic centrosomal components the centrosome is a platform for the binding of a lot of different proteins and enzymes, i.e. protein kinases and phosphatases, which are brought into contact with their substrates and upstream regulators. Therefore, the centrosome plays important roles in orchestrating cell cycle progression (reviewed in: Fry and Hames, 2004).
Fig. 1 Schematic representation of a typical mammalian centrosome consisting of mother and daughter centrioles enclosed by the pericentriolar matrix (PCM). The mother centriole contains distal and subdistal appendages (not all are shown) and both centrioles are associated by interconnecting fibers. In the PCM, γ-TuRCs are located that anchor MT minus ends.
In mitosis, the two poles of the bipolar mitotic spindle are established by two centrosomes that are generated during the previous cell cycle. Since centrosomes establish the spindle poles, the presence of more than two centrosomes in a single cell may influence the formation of the bipolar spindle eventually resulting in multipolar spindles and unequal distribution of chromosomes. Centrosome duplication therefore has to be restricted to once and only once per cell cycle. The centrosome division cycle starts at G1-S phase and by the end of G2, each cell has two centrosomes, each one comprising two centrioles surrounded by its own PCM (reviewed in: Kochanski and Borisy, 1990; Delattre and Gönczy, 2004; Tsou and Stearns, 2006a; Nigg, 2007; Azimzadeh and Bornens, 2007; Bettencourt-Dias and Glover, 2009). Mitosis then separates the two centrosomes into the two daughter cells. Each daughter cell therefore receives one centrosome. Centrosome duplication starts with the separation of the two otherwise tightly opposed ("engaged") centrioles. Although both centrioles are still attached via fibers, disengagement might license subsequent duplication in the next cell cycle (Tsou and Stearns, 2006b). Both centrioles then nucleate a new daughter centriole in a perpendicular orientation at their proximal ends. Centriole duplication starts at the same time as initiation of DNA synthesis. Throughout S to G2 phases, the procentrioles elongate to full-length daughter centrioles. Eventually, each newly formed centriole pair assembles its own PCM. By the end of G2, two centrosomes are present in the cell that eventually separate to build up the spindle poles during mitosis. Mitosis then distributes each of the two centrosomes to one daughter cell. Therefore, one daughter cell receives the original mother centriole associated with a daughter centriole generated during the last cell cycle, whereas the other daughter cell received the original daughter centriole associated with a new centriole generated during the last cell cycle. The original daughter centriole then matures into a mother centriole by formation of appendages. Eventually, each new daughter cell contains one centrosome comprising one mother and one daughter centriole (Fig. 2).
Fig. 2 Canonical centrosome duplication cycle of somatic cells. Centriole disengagement in G1 phase licenses centrioles for duplication. Centriole duplication starts in S phase, at the same time as DNA replication, by the formation of procentrioles. During G2 phase procentrioles elongate to full-length daughter centrioles and each centriole pair assembles its own PCM. Centrosomes are then separated to build up the poles of the bipolar mitotic spindle in mitosis.
Centriole duplication begins at the G1/S transition with the appearance of one procentriole at the proximal end of each parental centriole. Procentrioles organise around a cartwheel, and microtubules are nucleated by γ-TuRC like structures. Centriole duplication therefore depends on the presence of γ-tubulin, and of NEDD1, which recruits γ-TuRCs to the centrosome (Haren et al., 2006; Guichard et al., 2010). During S to G2 phases of the cell cycle, γ-tubulin is targeted for degradation by BRCA1 mediated ubiquitinylation thus blocking centrosome reduplication (Kasi and Parvin, 2008; reviewed in: Parvin, 2009). The initial steps of procentriole assembly are additionally dependent on several other proteins. An intrinsic centriolar protein is centrin-2. Centrins are small Ca2+-binding proteins of the calmodulin superfamily. They are highly conserved in evolution and are required for centrosome duplication across the eukaryotic kingdom. In HeLa cells, RNAi-mediated inactivation of centrin-2 prevents daughter centriole formation (Salisbury et al., 2002). Sas-6, Cep135, Sas-4 (CPAP in humans), Cep152, CP110, and geminin are likewise essential for centriole duplication as well as an array of kinases, such as polo-like kinases Plk1, and Plk2, cyclin-dependent kinase 2 (Cdk2)/cyclin A/E, and Mps1, and the phosphatase Cdc14B (Lacey et al., 1999; Meraldi et al., 1999; Chen et al., 2002; Fisk et al., 2003; Warnke et al., 2004; Leidel et al., 2005; Tachibana et al., 2005; Dammermann et al., 2008; Wu et al., 2008; Tsou et al., 2009; Lu et al., 2009; reviewed in: Sillibourne and Bornens, 2010; Pike and Fisk, 2011). Plk2 is activated near the G1/S phase transition and regulates centriole duplication by phosphorylation of NPM/B23 (Nucleophosmin) and CPAP (Krause and Hoffmann, 2010; Chang et al., 2010). The master regulator of centriole duplication, however, is the protein kinase Plk4/SAK (Habedanck et al., 2005). Plk4 belongs to the polo-like kinase family and localizes to centrosomes. Depletion of Plk4 impairs centriole duplication, whereas overexpression of Plk4 leads to the formation of multiple daughter centrioles in a single cell. Additionally, the correct level of Plk4 is essential for cytokinesis. By regulating the localization of Sas-6, a conserved coiled-coil protein essential for centriole duplication, Plk4 may affect centriole biogenesis (reviewed in: Pearson and Winey, 2010). On the other hand, Plk4 itself is recruited to the centrosome by Cep152 along with CPAP (Cizmecioglu et al., 2010). In vitro, Cep152 can be phosphorylated by Plk4 (Hatch et al., 2010). Plk4 negatively regulates the F-box protein FBXW5 by phosphorylation to suppress ubiquitylation of Sas-6. Depletion of FBXW5 leads to centrosome overduplication (Puklowski et al., 2011). These results show, that phosphorylation and controlled protein degradation are important regulators of centriole duplication.
Even though centriole duplication is typically coupled with DNA replication, there are some important exceptions: Uncoupling of centrosome duplication from DNA replication seems to occur during meiotic prophase in male gametogenesis and after fertilization (see below). Primary spermatocytes as well as secondary spermatocytes have two centrioles in each centrosome. The same holds true for spermatids, the products of meiotic divisions. Each secondary spermatocyte (with one centriole pair) generates two spermatids (also with one centriole pair per cell) during meiosis II. Centrosome duplication therefore has to occur prior to meiosis I, most likely concurrently with the last DNA replication step, and additionally prior to meiosis II, despite the absence of DNA replication, to ensure correct centrosome segregation. At fertilization, the sperm of most mammals contributes one centriole to the acentriolar oocyte. The single paternally contributed centriole therefore must duplicate once, in spite of absence of DNA replication, prior to first mitosis in zygotes to give rise to all in all four centrioles in two centrosomes.
The centrosome is a dynamic structure that changes in size and protein composition synchronous with the cell cycle. As centrosomes are not encircled by a membrane, the definition of true centrosomal components is hampered. Centrosomal proteins could therefore roughly be classified into three groups: 1. centriolar proteins, that are intrinsic to centrioles, as e.g. α- and β-tubulin, or permanently associated with centrioles; 2. intrinsic proteins of the PCM, as e.g. γ-tubulin and; anpericentrind 3. proteins temporarily associated with the centrosome, as e.g. kinases. About 500 different proteins have been associated with the centrosome of somatic cells by proteomic studies (Andersen et al., 2003). However, not all of them may be true centrosomal proteins but co-isolated molecules that use the centrosome as a docking platform. Since most studies have been performed in somatic cells in culture, it is largely unknown whether centrosomes of somatic cells differ from those of stem cells, neither concerning their overall organisation nor their protein composition. These constrains are mainly attributed to the difficulties to identify and handle stem cells. Since the most important stem cell in development is constituted by the fusion of male and female gametes, the centrosome of gametes, zygotes, and early developmental stages have attracted major attention. This review focuses on the centrosome of germ cells, zygotes, and stem cells in mammals, and addresses centrosome aberrations in cancer.
1. Centrosomes in Germ cells
Duplication and segregation of centrosomes are based on cell-cycle dependent conservative replication of centrioles, and semi-conservative segregation during cell division to ensure equal centrosome distribution to daughter cells. Each mitotically cycling cell thus comprises one and only one centrosome. To ensure proper centrosome number in each cell of the body, even during successive generations, centrosomes have to be reduced during gametogenesis, in analogy to the reduction of DNA content. Thus, fusion of male and female gametes not only restores the diploid DNA content but also the centrosome.
It was Theodor Boveri in 1901 who introduced the theory of uniparental distribution of the centrosome by recognizing that the egg typically loses the centrosome during oogenesis whereas the sperm typically introduces this structure at fertilization (Boveri 1901). Centrosome reduction thus occurs differentially in males and females.
However, thenceforward, the picture has become much more complicated owing to the emergence of even more sophisticated methods and detection skills (comprehensively reviewed by Schatten, 1994; Manandhar et al., 2005; Schatten and Sun, 2010). Although most animals show the typical pattern of paternal contribution of the centrosome, there are remarkable exceptions. Investigation of microtubule patterns throughout fertilization in the mouse, and, later on, studies using centrosomal antibodies showed that the sperm do not contain a centrosome (Schatten et al., 1985; Schatten et al., 1986). In mice and other rodents, the centrosome, which is largely defined by its microtubule polymerizing activity, thus seems to be maternally inherited (reviewed in: Schatten et al., 1991).
The centrosome of mature spermatozoa
In early stages of post-meiotic male germ cells, the centrosome is transformed into a basal body to generate the sperm flagellum. Usually, the distal centriole which is oriented perpendicular to the membrane, functions as the basal body to give rise to the axoneme. The proximal centriole is orthogonally oriented but closely associated with the distal centriole. The proximal centriole organizes the anlage of the capitulum which will later articulate with the implantation fossa of the sperm nucleus. The distal end of the proximal centriole additionally polymerizes the centriolar adjunct, a temporary structure no longer found in mature spermatozoa. Both distal and proximal centrioles are engaged in assembly of the striated columns of the connecting piece (reviewed in: Fawcett, 1981). Later during spermiogenesis, the distal centriole, which formerly has initiated outgrowth of the sperm flagellum, disintegrates and is no longer present in the mature spermatozoon whereas the proximal centriole usually persists (Fig. 3).
Fig. 3 Centrioles in elongating spermatids and mature spermatozoa of most mammals including human. In elongating spermatids both proximal and distal centrioles are present. The distal centriole functions in assembling the sperm tail. In mature spermatozoa the distal centriole is degenerated and no longer discernible.
The disintegration of only the distal centriole in mature spermatozoa is typical for most mammals including humans and rhesus monkeys (Manandhar et al., 2000). Therefore, in these species the centriole of the prospective zygotic centrosome is paternally inherited at fertilization. Paternal inheritance of the centriolar component has been confirmed for human (Sathananthan et al., 1991; Simerly et al., 1995), sheep (Le Guen and Crozet, 1989; Crozet, 1990), pig (Szöllösi and Hunter, 1973) and cow (Sathananthan et al., 1997; Long et al., 1993; Navara et al., 1994). Albeit centrioles are present in mature spermatozoa, the composition and accessibility of centrosomal proteins seem to be altered. Centrin and γ-tubulin are both found in human as well as in bovine mature spermatozoa. However, γ-tubulin is largely inaccessible in the mature spermatozoa in vitro until after disulfide bond reduction. It is therefore assumed, that the sperm centrosome must be primed by the reducing environment within the mammalian oocyte's cytoplasm to be transformed into an active centrosome. Since γ-tubulin is not only present in the oocyte, but also introduced by the sperm at fertilization, it is biparentally inherited (Simerly et al., 1999). Remarkably, anti-centrin staining of human and bull mature spermatozoa revealed two adjacent immunoreactive spots at the base of the sperm head which is reminiscent of two adjacent centrioles despite the degeneration of the distal centriole. However, immuno-EM studies revealed decoration of only the remaining proximal centriole (Simerly et al., 1999). These results suggest that the absence of a distinct centriolar structure does not mean that all centrosomal components are missing as well. The mature sperm nevertheless might retain some centrosomal components that are transmitted to the oocyte at fertilization to enable reconstitution of the zygotic centrosome.
In contrast to most mammals (including human), both distal and proximal centrioles degenerate during spermiogenesis in mice and other rodents. Additionally, mouse sperm are devoid of key centrosomal components and do not have microtubule nucleation capacity anymore. The MT nucleation capacity can be monitored by the formation of a monastral aster adjacent to the incorporated sperm nucleus in fertilized oocytes (Schatten et al., 1986). Loss of key PCM proteins and degeneration of centrioles are consecutive events. Round spermatids as well as elongating spermatids display distal and proximal centrioles along with γ-tubulin and centrin containing foci. In elongating spermatids γ-tubulin is mainly found around the centriolar adjunct which nucleates microtubules. At the time of spermiation, γ-tubulin is lost from the neck region and shed along with the residual body. The distal centriole disintegrates during testicular stage, and the proximal centriole degenerates in the epididymis. Mature mouse sperm neither contain centrioles nor γ-tubulin or centrin (Manandhar et al., 1998; Manandhar et al., 1999). Partial versus complete degeneration of centrioles and associated centrosomal proteins in human and mouse spermatozoa, respectively, is also supported by the species-specific distribution of centrosomal proteins (e.g. TSKS, substrate of testis-specific serine kinases 1 and 2; Xu et al., 2008).
The proper function of the centrosome is vitally important for the unification of male and female pronuclei after fertilization and ongoing development. In most animals, the centrosome of the spermatozoon organizes the sperm aster, which brings the parental genomes together during fertilization. Improper centrosomal inheritance or dysfunction of the sperm centriole might be associated with cleavage irregularities of the fertilized oocyte and abnormal embryonic development thus linking centrosomes to infertility (Simerly et al., 1995; Palermo et al., 1997; Nagy, 2000; Chatzimeletiou et al., 2008; reviewed in: Schatten and Sun, 2009a). It has been shown in bull that naturally occurring variations in their centrosomes correlate with reproductive success (Navara et al., 1996). Likewise, centrosome dysfunction might be responsible for low efficiency rate of cloning mammals by somatic cell nuclear transfer (SCNT), and might even affect the success of intracytoplasmic sperm injection (ICSI) in assisted reproduction techniques (Chemes et al., 1999; Zhong et al., 2005; Dai et al., 2006; Zhong et al., 2007; Terada, 2007).
The centrosome during oogenesis and in oocytes
Oogonia and oocytes up to the pachytene stage of hamsters, mice, rats, gerbils, and man contain centrioles, but in subsequent meiotic stages, centrioles are absent. At the time of germinal vesicle breakdown microtubule polymerization focuses on several electron-dense aggregates inside the ooplasm (Szöllösi et al., 1972; Sathananthan et al., 2006). Mature oocytes of rabbits, cows, and sheep are likewise devoid of centrioles (Sathananthan et al., 1997; Crozet et al., 2000). However, as has been revealed by immunolocalization studies mostly during mouse oogenesis, PCM proteins, including γ-tubulin, pericentrin, and NuMA are not eliminated during meiotic stages but show a dynamic redistribution. NuMA is a nuclear matrix protein which plays an important role in establishment and maintenance of the bipolar spindle apparatus (Maro et al., 1985; Gueth-Halonet et al., 1993; Calarco, 2000; Carabatsos et al., 2000; Lee et al., 2000; Can et al., 2003; Meng et al., 2004; Tang et al., 2004; Sedo et al., 2011). 4-D imaging in live maturing mouse oocytes revealed, that numerous acentrosomal MTOCs, spread in the ooplasm, then assemble into the functional acentrosomal meiotic spindle (Schuh and Ellenberg, 2007). Thus, centrosomal material is stored in the oocyte albeit in a somehow unfocussed manner, that is then attracted by the sperm after fertilization to generate a functional centrosome. Studies of aster formation in the cow suggested that the centrosome generated after fertilization is a blending of paternal and maternal components (Navara et al., 1994).
2. The centrosome during early development
In humans, the sperm contributes the proximal centriole and the mid-piece of the sperm tail to the acentriolar oocyte at fertilization. Proximal centriole and sperm mid-piece are closely associated with the decondensing sperm nucleus that gradually develops into a male pronucleus. Shortly after sperm incorporation into the oocyte, a sperm aster is formed from the proximal centriole. The aster is build up of microtubules required for the apposition of the male and female pronuclei - an essential prerequisite for merging of paternal and maternal genomes and successful fertilization. In prometaphase, when male and female pronuclei breakdown, the centrosome is separated to form the two poles of the mitotic spindle. Transmission electron microscopy revealed single or double centrioles within centrosomes at one pole of the first cleavage spindle (Sathananthan et al., 1991). Most likely due to the difficulties to gather both centrioles of one centrosome or even both centrosomes at the same section plane in the voluminous ooplasm, double centrioles are not found at each pole of the first cleavage spindle. But afterwards, centrioles are then found at every stage of early embryo development (Sathananthan et al., 1996; Sathananthan, 1997).
In bovine, centrioles are likewise paternally inherited, and organize the sperm aster after fertilization (Sathananthan et al., 1997). Zygotes from humans, sheep and cows thus display centrioles at the spindle poles at first mitosis, although extensive electron microscopic studies have failed to find duplex centrioles in all spindle poles during the first cleavage of human zygotes (Sathananthan et al., 1991; Sathananthan et al., 1996; Santhananthan et al., 1997; Le Guen and Crozet, 1989). In sheep zygotes, the first cleavage spindle possesses two centrioles at one pole and only one centriole at the other pole, probably owing to difficulties to gather all centrioles at the same section plane (Crozet et al., 2000). However, in late-stage embryonic cells centriolar duplexes are invariably found in all cells (Sathananthan, 1997).
The paternal centrosomal constituents of human and bovine mature sperm are subject to phosphorylation in vitro after exposure to X. laevis cell-free egg extracts as well as in vivo after oocyte penetration as visualized by the phosphoprotein specific antibody MPM-2 (Simerly et al., 1999). This suggested that paternal centrosomal proteins might be intensively modified post-translationally after exposure to ooplasm at fertilization. After fertilization, the sperm centrosome recruits γ-tubulin from the ooplasm resulting in an accumulation of mostly maternal γ-tubulin at the zygotic centrosome (Simerly et al., 1999; Shin and Kim, 2003).
Mature oocytes are devoid of centrioles but contain PCM proteins. Moreover, in mice and other rodents, the sperm does not contribute a centriole to the oocyte at fertilization. The microtubules are therefore organized after fertilization by components stored in the oocyte but not by the incorporated sperm nucleus. During early mouse development important changes in composition and structure of MTOCs take place, reflected by the varying number of PCM foci as well as by their protein composition (Gueth-Hallonet et al., 1993). Though, first and second cleavage stage mouse embryos are devoid of centrioles, centrioles arise during the blastocyst stage by de novo synthesis (Szöllösi et al., 1972). De novo formation of centrioles has also been observed in rabbit oocytes parthenogenetically activated (Szöllösi and Ozil, 1991). Centriole precursors are similar to deuterosomes, intermediate structures of de novo formation of basal bodies in multi-ciliated cells, or procentrioles. Procentriole-like bodies may first form solitary centrioles that replicate in an orthogonal manner afterwards like in parthenogenetically activated sea urchin eggs (Kallenbach and Mazia, 1982). Centrioles with the usual morphology could first be found in the 16-cell stage by electron microscopy (Szöllösi et al., 1972; Gueth-Hallonet et al., 1993). These observations suggest once more that centrioles are not essential for formation of bipolar spindles in animals, and that centrioles are generated de novo in the early mouse embryo.
Summary: From germ cells to zygotes
In mammalian species (including human), a gradual degeneration of the centrosome during spermiogenesis is observed. Loss of key PCM proteins follows loss of microtubule nucleation capacity and eventually results in partial or complete degeneration of centrioles. In most mammals (including human), the sperm retains the proximal centriole but degenerates the distal one. In contrast, oogonia originally contain a pair of well-defined centrioles that are lost during oogenesis, resulting in the mature oocyte being devoid of centrioles. However, PCM is retained albeit dispersed throughout the egg cytoplasm. As in most mammals the human oocyte has no granular centrosomal material at meiotic spindle poles which is a remarkable difference to mouse oocytes that have a dominant maternal centrosome. Upon fertilization, a functional centrosome is restored in the zygote. In mouse and other rodents, in which the sperm does not contribute a centriole, the zygotic centrosome is constituted from maternal material, whereas in most other mammals (including human) the zygotic centrosome is restored by the proximal centriole submitted by the sperm and maternal PCM components that are attracted by the sperm (Schatten and Sun, 2009b). Restoration of the centrosome after fertilization thus occurs in most mammals (with the exception of rodents) by the sperm supplying the centriole and the PCM supplied by the egg cytoplasm.
3. The centrosome in stem cells
The formation of a bipolar mitotic spindle is a crucial event for proper cell division ensuring equal distribution of chromosomes as well as equal amounts of cytoplasmic components to daughter cells. Concurrently, centrosomes likewise may be equally distributed to daughter cells. However, this might not always be the case. Unequal centrosome distribution to daughter cells may set up the pattern for differentiation. A key feature in stem cell biology is asymmetric cell division. Asymmetric cell division results in two unequal daughter cells that receive different amounts of cytoplasmic material and organelles and possibly also centrosomal components. One of the daughter cells maintains stem cell characteristics by remaining at the stem cell niche and receiving signals from the hub (reviewed in: Morrison and Spradling, 2008). The sibling cell is no longer in contact with the hub and undergoes differentiation. This kind of cell division which results in two sibling cells undergoing different cell fates requires a specific arrangement of the mitotic spindle that has to be perpendicular to the hub. Disturbances of spindle position therefore affect the fate of the daughter cells. If both sibling cells will remain in contact with the hub, two stem cells instead of one stem cell and one differentiating cell will be the result. Disturbances in spindle position therefore may derange the balance between stem cells and differentiated cells and result in uncontrolled proliferation and cancer (reviewed in: Pease and Tirnauer, 2011). However, mammals are not a good model system to study the mode of cell division in stem cell maintenance and the contributions the centrosome provides to cell differentiation and development.
Our knowledge on asymmetric stem cell division and stem cell centrosomes is mainly based on studies in the fruit fly Drosophila, and the nematode Caenorhabditis elegans (reviewed in: Yamashita and Fuller, 2008; Gönczy, 2008; Neumüller and Knoblich, 2009). In Drosophila males, germline stem cells (GSC) preferentially inherit and maintain the mature centrosome (comprising the mother centriole) as long as they remain in the niche. The daughter centrosome is inherited by the daughter cell fated to differentiate. Asymmetric distribution of centrosomes is determined by the differential microtubule nucleation capacity of mother and daughter centrosomes affecting spindle orientation (Yamashita et al., 2003; Yamashita et al., 2007). Misorientation of centrosomes during ageing affects stem cell division (Cheng et al., 2008). Unequal centrosome distribution has also been reported in Drosophila neurogenesis. Larval neural stem cells called neuroblasts divide asymmetrically to produce a larger self-renewing neuroblast and a smaller larval basal ganglion mother cell that pursues neural differentiation. One neuroblast centrosome remains associated with the neuroblast cortex while the other centriole pair moves away (Rebello et al., 2007; Rusan and Pfeifer, 2007). However, in this case the mother centrosome is inherited by the differentiating daughter cell (Januschke et al., 2011). In the female Drosophila germline centrosomes segregate randomly (Stevens et al., 2007). Thus, asymmetric centrosome inheritance is not a general feature of stem cell lineages.
In mammals, the best studied example of asymmetric cell division is provided by the developing mouse forebrain. Early in development, progenitor cells in the ventricular zone divide symmetrically to increase the progenitor cell pool. From day 10 of embryonic development, progenitor cells, now called radial glia progenitors, divide asymmetrically allowing more differentiated cells to be produced (Götz and Huttner, 2005). Asymmetric division of radial glia progenitors produce a self-renewing radial glia and a differentiating cell that leaves the ventricular zone to become a neuron. Radial glia progenitors preferentially inherit the older centrosome, whereas the cell that leaves the ventricular zone to become a neuron preferentially inherits the daughter centrosome. By removal of ninein that is associated with the mother centriole, centrosome asymmetry may be impaired leading to disruption of asymmetric centrosome inheritance and depletion of progenitor cells from the ventricular zone (Wang et al., 2009). Asymmetric centrosome inheritance thus maintains neural progenitor cells in the neocortex of the mouse. The importance of the mature centrosome to maintain the progenitor pool in the ventricular zone is further substantiated by silencing of genes encoding centrosomal proteins. Likewise, the human disease microcephaly, characterised by a severely decreased brain size, is caused by loss of function of centrosome-related proteins (reviewed in: Lesage et al., 2010).
Symmetric cell division, as apparently seen in cells in culture, does by no means demonstrate equal distribution of cellular components to daughter cells (Fuentealba et al., 2008). As shown by Fuentealba et al. in mammalian cultured cell lines, including human ES cells, and in vivo in Drosophila embryos, both centrosomes of a mitotic cell can differ in composition. Daughter cells therefore receive a differential protein pool.
4. The centrosome in cancer
Although centrosome duplication is tightly linked to the progression of the cell cycle under normal conditions, surprisingly faulty centrosome duplication did not cause cell cycle arrest (Uetake and Sluder, 2004; Wong and Stearns, 2005). In almost all types of solid tumors as well as in hematological malignancies like lymphoma and leukemia abnormal centrosome amplification is common. Centrosome amplification together with aneuploidy are hallmarks of many cancers (reviewed in: Salisbury et al., 1999; Nigg, 2002; Wang et al., 2004; Sagona and Stenmark, 2010). Although typical centrosomes with two centrioles have been identified by electron microscopy in undifferentiated human ES cells, a very high percentage of cultured human embryonic stem cells display numerical centrosome abnormalities that may be responsible for the observed chromosome instabilities in these cells (Sathananthan et al., 2002; Holubcova et al., 2011). The question whether centrosome amplification causes aneuploidy, or vice versa, is still a matter of debate. It has been demonstrated that cells with supernumerary centrosomes and tetraploid genomes can continue to cycle eventually resulting in aneuploid cells. Moreover, tetraploid cells are more likely to be tumorigenic than their diploid counterparts (reviewed in: Sankaran and Parvin, 2006). A first direct causative link between centrosome amplification and tumorigenesis has been provided by studies in the fruit fly Drosophila melanogaster. Larval brain from animals possessing supernumerary centrosomes due to Plk4 overexpression was able to initiate tumorigenesis in wild type host flies when transplanted (Basto et al., 2008; Castellanos et al., 2008). Surprisingly, in mouse fibroblasts Plk4 heterozygosity impairs cytokinesis leading to tetraploid daughter cells with amplified centrosomes. Moreover, aberrant Plk4 activity seems to be involved in tumor formation in mice and in humans (Macmillan et al., 2001; Ko et al., 2005; Rosario et al., 2010). Tight control of Plk4 level and activity is therefore mandatory. Plk4 stability is regulated by autophosphorylation and subsequent ubiquitin-dependent degradation (Holland et al., 2010).
Besides polo-like kinase Plk4, various other proteins are dysregulated in a lot of tumors which concurrently exhibit centrosome aberrations. In breast and colorectal tumors the Aurora-A gene is frequently amplified. Aurora-A belongs to the family of aurora protein kinases which consists of three members in mammals. Aurora-A is a mitotic kinase which localizes to centrosomes. Apart from gene amplification, Aurora-A overexpression is found in many tumors. In cultured cells, overexpression of Aurora-A leads to centrosome amplification (Sen et al., 1997; Bischoff et al., 1998; Zhou et al., 1998; Meraldi et al., 2002; reviewed in: Giet et al., 2005). Aurora-A phosphorylates the E3-ubiquitin ligase BRCA1 to diminish its activity. Decreased ubiquitin-ligase activity inhibits γ-tubulin degradation leading to centrosome reduplication. Breast and ovary tumors are frequently associated with loss of the E3-ubiquitin ligase BRCA1 (Sankaran and Parvin, 2006). An important tumor suppressor is p53, and point mutations affecting p53 are common in many forms of human cancer (Hainaut et al., 1997). That p53 affects centrosome duplication has been shown in embryonic fibroblasts generated from p53-/- -mice. P53 deficient cells display multiple copies of functional centrosomes (Fukusawa et al., 1996), most likely due to tetraploidization caused by Aurora-A overexpression (Meraldi et al., 2002).
Centrosome amplification and chromosome instability are common hallmarks of cancer. Up to now, most results favour the view that overduplication of centrosomes is the primary cause of aneuploidy. Apart from centrosome amplification, structural and functional abnormalities of the centrosome have been observed in tumor cells as well. Moreover, centrosome dysfunction has been implicated in a wide variety of human diseases (reviewed in: Badano et al., 2005).
The centrosomes of stem cells are a relatively unexplored field. There are some hints, that centrosomes may be important for stem cell maintenance, and that mature and daughter centrosome differ from each other. But it is not known whether centrosomes from stem cells differ in composition from somatic centrosomes. Likewise, their functional contributions to stem cell maintenance or cell differentiation remain largely in the dark. Numerical, structural, and functional aberrations of the centrosome are associated with a wide variety of human diseases including cancer. Centrosome amplification in tumor cells may pave the way for chromosome instabilities leading to cancer progression.