Centrosomes in the zebrafish (Danio rerio): a review including the related basal body
© Lessman; licensee BioMed Central Ltd. 2012
Received: 3 February 2012
Accepted: 7 June 2012
Published: 7 June 2012
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© Lessman; licensee BioMed Central Ltd. 2012
Received: 3 February 2012
Accepted: 7 June 2012
Published: 7 June 2012
Ever since Edouard Van Beneden and Theodor Boveri first formally described the centrosome in the late 1800s, it has captivated cell biologists. The name clearly indicated its central importance to cell functioning, even to these early investigators. We now know of its role as a major microtubule-organizing center (MTOC) and of its dynamic roles in cell division, vesicle trafficking and for its relative, the basal body, ciliogenesis. While centrosomes are found in most animal cells, notably it is absent in most oocytes and higher plant cells. Nevertheless, it appears that critical components of the centrosome act as MTOCs in these cells as well. The zebrafish has emerged as an exciting and promising new model organism, primarily due to the pioneering efforts of George Streisinger to use zebrafish in genetic studies and due to Christiane Nusslein-Volhard, Wolfgang Driever and their teams of collaborators, who applied forward genetics to elicit a large number of mutant lines. The transparency and rapid external development of the embryo allow for experiments not easily done in other vertebrates. The ease of producing transgenic lines, often with the use of fluorescent reporters, and gene knockdowns with antisense morpholinos further contributes to the appeal of the model as an experimental system. The added advantage of high-throughput screening of small-molecule libraries, as well as the ease of mass rearing together with low cost, makes the zebrafish a true frontrunner as a model vertebrate organism. The zebrafish has a body plan shared by all vertebrates, including humans. This conservation of body plan provides added significance to the existing lines of zebrafish as human disease models and adds an impetus to the ongoing efforts to develop new models. In this review, the current state of knowledge about the centrosome in the zebrafish model is explored. Also, studies on the related basal body in zebrafish and their relationship to ciliogenesis are reviewed.
According to EB Wilson in his classic text, The Cell in Development and Heredity , Van Beneden first described the "polar corpuscle" in 1876 and Boveri later named it the "centrosome" in 1888. For more than a century, the centrosome has intrigued scientists and continues to do so today. Although much is now known about the centrosome, it remains somewhat mysterious, with many secrets left to reveal about its function and regulation. The zebrafish (Danio rerio), a small tropical freshwater teleost, has emerged as a model for cell and developmental biology because of its high fecundity, short generation time and rapid development of the externally fertilized and translucent embryos  (see also ). As a relatively new model organism, the zebrafish has attracted considerable attention in the scientific community due to its genetic tractability, speed of embryonic development and optical clarity. Many scientists espouse the hope that the advantages of the zebrafish model system will allow solutions to long-standing questions. For example, how is the centrosome regulated? Exactly what does it do in cell division? What is its relationship to basal bodies and ciliogenesis? It is the purpose of this review to summarize and outline the current state of knowledge about the centrosome and its relative, the basal body, in zebrafish.
Centrosomes in animal cells usually consist of γ-tubulin ring complexes (γ-TuRCs), centrioles, pericentriolar material and tubulins, along with a number of other centrosome-associated proteins. A previous proteomic analysis of isolated human centrosomes indicated about 70 protein components and revealed the complexity of the centrosome . The major components are briefly reviewed regarding our knowledge of most animal cells and then those of the zebrafish in particular. Centrosomes and the related basal bodies of cilia are important microtubule (MT)-organizing centers (MTOCs) of animal cells. A number of serious diseases have been linked to their dysfunction (reviewed in ). A major centrosomal-basal body protein, γ-tubulin, a member of the tubulin superfamily, appears to play a central role in MTOC activity; nevertheless, our specific knowledge about γ-tubulin's role is far from complete. Like other tubulins, such as α- and β-tubulin, γ-tubulin exists as both a soluble and a polymer pool, albeit at about 1% the abundance of α- and β-tubulins. Very enlightening work on Drosophila and Xenopus has revealed that the γ-TuRCs are composed of about 13 γ-tubulin subunits, together with several different proteins called "γ-tubulin complex proteins" (γ-tubulin ring-associated protein, or GRIP) [6–8], and are found at the minus ends of microtubules associated with centrosomes. The association of the γ-TuRCs with the centrosome appears to be dynamic, as seen in Caenorhabditis elegans early cleavage embryos expressing γ-tubulin-GFP . Clearly, the centrosome duplication and other dynamic processes are synchronized with the cell cycle, presumably by the cyclin-dependent kinase (CDK) system, but the exact mechanism by which they occur is unclear. How γ-tubulin and its associated proteins are recruited to and docked at the centrosome is also largely unknown. The microtubule cytoskeleton is essential for a variety of cellular processes, including cell movement, organelle transport and cell division and, for primary cilia, sensation and signaling. Moreover, in oocytes and early embryos, microtubules have been implicated in localization of important embryonic determinants such as bicoid mRNA in Drosophila  and Vg1 mRNA in Xenopus , as well as trafficking cell components such as β-catenin to cytokinesis furrows in cleaving zebrafish embryos . Recently, it was reported that nocodazole, a MT inhibitor, abolished translocation of the dorsal determinant Wnt8a in zebrafish embryos, emphasizing the importance of MT in zebrafish development . MTOCs organize MTs by initiating noncovalent assembly of αβ-tubulin heterodimers, anchoring them at their minus ends and facilitating MT extension at the rapidly growing plus ends. These complicated dynamic properties involve the functions of numerous MT-associated proteins. MTOCs thus organize and control the MT network both spatially and temporally, including that of cilia. They also help to define the MT surface lattice . The morphology, subcellular localization and molecular make-up of MTOCs vary across different species and different cell types within single species.
Although the main topic of this review is the centrosome, it is becoming increasingly clear that centrosomes and basal bodies are intimately related, especially in some cell types, such as stem cells containing primary cilia. Basal bodies are derived from centrosome components, especially the mother centriole, which is thought to direct assembly of the axoneme (reviewed in [15–17]). During the cell cycle, the axoneme is disassembled and the basal body components are used to reconstitute the centrosome . Thus the centrosome and basal body are intimately related and composed of many of the same components, and their formation is regulated in concert with the cell cycle.
The centrosome and related basal body are thought to provide sensation, motion and cell division to the eukaryotic cell and thus are ancient structures believed to be found in the very first eukaryotes (reviewed in [19–21]). The centrosome and basal body structures are more than MTOCs  in that they may act as scaffolds for signal cascades and may contribute to disease states such as cancer  and ciliopathies . In most animal cells, centrosomes are composed of a pair of centrioles surrounded by an amorphous cloud of electron-dense material, the pericentriolar material (PCM) . Mass spectrometric analysis of purified human centrosomes has revealed 47 previously known centrosome components in four major protein groups: (1) structural components including tubulins, γ-tubulin complex components, centrins, A-kinase anchoring protein 450 (AKAP450), pericentrin, ninein, PCM-1 and centriole-associated proteins; (2) regulatory molecules, including cyclin/CDK1, protein kinase A (PKA), Plk1, type 1 pyrophosphatase (PPase 1) and PPase 2A; (3) motor proteins, including dynein and dynactins; and (4) heat shock proteins, including Hsp90 and Hsp73 [4, 25]. An additional 64 proteins were newly identified. Many of these were coiled-coil domain proteins. The PCM is largely proteinaceous [26, 27] and consists of a matrixlike structure (also referred to as the "centrosome scaffold") [24, 28, 29]. One characterized PCM protein required for in vivo MT nucleation is γ-tubulin. In Drosophila and Xenopus, γ-tubulin contributes to macromolecular complexes or γ-TuRCs, with a characteristic ring structure (approximately 25 nm in diameter), together with a variety of associated proteins [6, 29–31]. Hundreds of 32S γ-TuRC tether to the centrosome scaffold to serve as the site of origin for MTs [24, 28, 29] and are required for spindle assembly and progression through mitosis [7, 32–34]. Thus the centrosome is the primary site for MT nucleation, and the centrosomal γ-TuRC is thought to be necessary for anchoring of MT minus ends to centrosomes [35–39]. In addition to playing roles in centrosome-dependent MT nucleation , γ-tubulin has also been found to be closely associated with the PCM and centrioles and to be a core component of the centriole . It plays a key role in daughter centriole formation  or in the nucleation and/or stabilization of the centriolar MTs .
In mammalian cells, a significant fraction of the total pool of γ-tubulin is found in cytosolic complexes . Inside the cell, γ-tubulin is located at the centrosome as γTuRCs and is also distributed in the cytoplasm as a soluble pool of inactive smaller complexes that constitute an exchangeable stock of material [43–46]. Recruitment of γ-tubulin to sperm basal bodies is necessary for the assembly of a MT nucleation-competent paternal centrosome [7, 47]. Using immunofluorescence and GFP reporter construct techniques, γ-tubulin was shown to be associated with centrosomes dynamically in mitotic cells, being massively recruited at prophase and released at anaphase-telophase. This accumulation in mitotic centrosomes is dramatic during the first embryonic divisions in C. elegans . Moreover, fluorescence measurements suggest that the amount of antigenic γ-tubulin increases during mitosis and that, in HeLa cells, the total amount of γ-tubulin in the spindle is larger than the amount of γ-tubulin in the spindle poles . The cell-stage distribution of γ-tubulin varies between animal cells of different species and between cells of different tissues within the same species. These observations imply that both the localization and the concentration of γ-tubulin are highly regulated during the cell cycle and that the regulation may vary between cell types and between species . Thus understanding how γ-TuRCs are recruited to and docked at the centrosome is essential for understanding the regulation of MT nucleating activity. The function of the soluble cytoplasmic γ-tubulin remains unclear. Also, very little is known about how the γ-TuRC is assembled and tethered in the centrosome. Moreover, the redistribution of centrosomal proteins to specific sites of the cell is poorly understood, and the mechanisms controlling MT nucleation within the living cell are still unclear. Thus, though considerable progress has been made in the understanding of centrosomes, significant work remains to be done. The zebrafish system shows promise in providing more insight.
Results of an NCBI database search for γ-tubulin complex component proteins in zebrafisha
Cal. MW (Da)
γ-tubulin complex protein 2
79 to 88
γ-tubulin complex protein 3
77 to 79
γ-tubulin complex protein 4
88 to 89
γ-tubulin complex protein 5
72 to 73
1 to 2
HAUS augmin-like protein 6
30 to 48
5 to 8
Mitotic spindle-organizing protein 1
80 to 90
Mitotic spindle-organizing protein 2
52 to 61
5 to 8
Centrioles are cylindrical structures with ninefold radial symmetry and triplets of short MTs forming the outside wall in many species. A pair of centrioles oriented to each other at about a right angle is found in centrosomes and basal bodies . Although centrioles are found in most animal cells, they are notably absent in oocytes and eggs until the latter are fertilized. The sperm provides the initial centriole to reconstitute centrosomes in the zygote . In preparation for cell division, a procentriole, or daughter, forms at the base of each mother centriole by first forming a cartwheel structure, then forming MTs . The cartwheel has a ninefold symmetry composed of spindle assembly abnormal SAS-6 homodimers with coiled-coil domains radiating outermost as spokes . This process of centriole replication or procentriole formation occurs in two ways: (1) the canonical pathway, involving mother-daughter proximity and resulting in exactly one net new centriole pair and (2) the de novo pathway, particularly active in ciliogenesis in ciliated vertebrate epithelial cells, involving multiple procentriole formations from fibrous granules and deuterostomes not necessarily containing existing centrioles . The mother centriole is older and has specializations that include filaments or appendages  and additional proteins such as centriolin . In addition, the mother centriole produces the cilium from the basal body . Questions remain about the underlying factors that produce differences between the mother and daughter centrioles' ability to organize centrosomes or basal bodies and, in turn, to organize a spindle or a cilium, respectively. Other areas of centriole and/or basal body biology that need further investigation include (1) temporal and genetic factors involved in aging and positioning of centrioles relative to the formation of centrosomes versus basal bodies, (2) the role of centriole condition in stem cell maintenance or differentiation and (3) centriole fate and mechanism of loss in oocytes and other cells that lose centrioles normally (reviewed in ).
Centrins are small Ca2+-binding proteins of the EF-hand superfamily related to calmodulin, troponin C and parvalbumin  that are associated with centrioles, but also may be found elsewhere, such as in the nucleus. In human cells, more than 90% of the centrin is not centrosome-associated . In mammalian cells, centrin 2 is required for centriole duplication , and use of GFP-centrin constructs have shown differential behavior of mother and daughter centrioles [82, 83]. Some centrins are usually associated with centrosomes, except in preimplantation porcine embryos , and are also found in basal bodies, including the connecting cilium of photoreceptors . Centrin 2 has been shown to self-assemble into fibers in the presence of Ca2+ reminiscent of those associated with basal bodies . In addition, a Chlamydomonas centrin mutant has acentriolar spindle poles and lacks rhizoplasts that normally join the centrioles to the nucleus and result in increased chromosome loss and genomic instability . Phosphorylation of human centrin at serine 170 during the G2/M phase has been implicated as the signal for centriole separation before centrosome duplication . Another prominent signaling role for centrin is its interaction with transducin within the connecting cilium of photoreceptor cells .
Centrin 2 amino acid sequence comparison among zebrafish, Xenopus and mousea
1 MASNYKKPSLGVTTQRKKPVPKPELTEEQKQEIREAFDLFDTDGAGTIDVKELKVAMRAL 60 XENTR
1 MASNFKKTTMASSAQRKRMSPKPELTEDQKQEIREAFDLFDADGTGTIDIKELKVAMRAL 60 MOUSE
1 MASGFRK-SSASANQRKKAGPKPELTEEQKQEIKEAFDLFDTDGSGTIDVKELKVAMRAL 59 DANRE
61 GFEPKKEEIKKMIADIDKEGTGKISFGDFMSAMTQKMAEKDSKEEIMKAFRLFDDDETGK 120 XENTR
61 GFEPKKEEIKKMISEIDKEGTGKMNFSDFLTVMTQKMSEKDTKEEILKAFKLFDDDETGK 120 MOUSE
60 GFEPKKEEIKKMIADIDKEGSGVIGFSDFLSMMTQKMSEKDSKEEILKAFRLFDDDCTGK 119 DANRE
121 ISFKNLKRVAKELGENLTDEELQEMIDEADRDGDGEVNEQEFLRIMKKTSLY- 172 XENTR
121 ISFKNLKRVAKELGENLTDEELQEMIDEADRDGDGEVNEQEFLRIMKKTSLY- 172 MOUSE
120 ISFKNLKRVAKELGENLTDEELQEMIDEADRDGDGEINEQEFLRIMKKTNLYG 172 DANRE
Antibodies from human scleroderma patients have been used to discover another centrosomal protein, called "pericentrin," with a molecular weight of about 220 kDa . Pericentrin was found to associate with γ-TuRCs and form a lattice that enlarges, then disassembles with the cell cycle . Kendrin, a larger splice variant (about 380 kDa) of the PCNT pericentrin gene, was discovered in human cells and was found to bind calmodulin [94, 95]. In kendrin, also called "pericentrin B," overexpression was found in carcinoma cells known to have centrosomes of abnormal size and number . Some uncertainty exists regarding the number and size of pericentrin isoforms and splice variants . Nevertheless, pericentrin derangement has been implicated in a number of disease states, including Seckel syndrome, microcephalic osteodysplastic primordial dwarfism, cancer, ciliopathies and mental disorders such as schizophrenia . It has been suggested that this diversity of diseases is attributable to the many binding partners of pericentrin, including γ-tubulin, γ-TuRCs, PCM1, AKAP450, DISC-1, Chk1, kinases, intraflagellar transport (IFT) and PC2 . Pericentrin and AKAP450 bind to a γ-tubulin complex binding protein called CDK5RAP2 via a CM-2-like motif that is also conserved in zebrafish .
Zebrafish primary cilia have been described or alluded to in an expanding number of recent reports [105–126]. A particularly informative study using the maternal-zygotic oval (MZovl; ift88) zebrafish mutant that lacks all cilia revealed dampened Hedgehog signaling but normal Wnt signaling . The MZovl mutants had normal basal bodies but failed to localize Smoothened to the cell membrane in association with basal bodies. In addition, left-right patterning was deranged, and the mutants also developed pronephric cysts and pericardial edema indicative of ciliopathies. Thus, though basal bodies are required, they do not appear to be sufficient for ciliogenesis or cilia maintenance. Other gene products reported to be involved in ciliogenesis in zebrafish include the zinc-finger protein iguana , geminin , fused , fibroblast growth factors FGF8 and FGF24 , Smoothened , the fleer gene product , cdc14B phosphatase , Cep70 and Cep131 , Rab11, Rabin8 and transport protein particle II (TRAPPII)  and Nde-1 .
Gene knockdown of the centrosomal protein Cep290 resulted in zebrafish with visual defects and other symptoms of ciliopathies similar to those seen in Leber's congenital amaurosis, Meckel-Gruber syndrome, Joubert syndrome, Senor-Loken syndrome and Bardet-Bledl syndrome . In addition, the visual defect may be rescued by expressing the N-terminal region of the human Cep290 protein in the zebrafish. Cep290 is one of eleven ciliopathy genes that include cc2d2a and involve retinal dystrophy as well as other defects, such as polycystic kidney . The cc2d2a protein localizes to the photoreceptor-connecting cilium transition zone, and mutations result in visual defects as determined on the basis of electroretinograms in zebrafish .
A-kinase anchoring protein
cell division control
cyclin-dependent kinase 5 regulatory subunit-associated protein 2
checkpoint 1 kinase
disrupted in schizophrenia 1
fibroblast growth factor
furrow microtubule array
green fluorescent protein
γ-tubulin ring-associated protein
γ-tubulin ring complex
γ-tubulin small complex
heat shock protein
nuclear distribution protein
platelet-derived growth factor
protein kinase A
spindle assembly abnormal
spindle pole body component
transport protein particle II
yellow fluorescent protein.
I thank current and former members of my laboratory for their help and discussions and Rick Heil-Chapdelaine for help with confocal imaging. I also acknowledge the support of my department. I apologize to those researchers whose work was not covered due to space limitations. I thank two anonymous reviewers for their time and efforts to improve this review.