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Centrosomes: Coiled-Coils Organize the Cell Center
Current Biology, Vol. 13, R88–R90, February 4, 2003, ©2003 Elsevier Science Ltd. All rights reserved.
Centrosomes: Coiled-coils Organize the Cell Center Jeffrey L. Salisbury
The centrosome serves as a structural context for cytoplasmic organization. Recent studies on mutants of the nematode worm Caenorhabditis elegans have provided new insight into the framework to which microtubules and key regulators of centrosome behavior are anchored.
The centrosome is a fascinating organelle that resides near the cell center — hence its name [1]. It functions in the maintenance of cytoplasmic architecture through the nucleation and organization of microtubule arrays in interphase and mitotic cells. Centrosome defects have been implicated in disease processes, particularly in the origin of mitotic abnormalities and the development of aneuploidy in cancer [2]. In addition to its fundamental role in microtubule organization, the centrosome may also provide an important structural context for coordinating cell-cycle regulation. An understanding of the molecular basis of these diverse cellular functions is beginning to emerge through the careful analysis of centrosome structure and organization in early embryo development and somatic cells. In a recent study which exploited the power of genetics and gene silencing in the nematode Caenorhabditis elegans, Hamill et al. [3] have shed new light on a class of proteins with multiple coiled-coil domains that provide a scaffold onto which key players in centrosome function are attached. The centrosome consists of a pair of centrioles and a surrounding halo of pericentriolar material that harbors, among other proteins, the γ-tubulin protein complexes that nucleate microtubules [4]. In most animals, centrioles are eliminated during oogenesis, while the egg cytoplasm remains enriched for centrosome precursor proteins that are used during early embryonic development. Conversely, during spermatogenesis the sperm centrosome is reduced to its core centrosome components — a pair of centrioles — and, at the time of fertilization, the sperm delivers this stripped-down version of the somatic centrosome and a haploid (pro)nucleus to the egg [5]. In the zygote, the sperm centrioles undergo functional maturation by recruiting egg proteins to form the pericentriolar material of the zygotic centrosome [6,7]. Through this maturation process, the zygotic centrosome acquires the microtubule organizing activity required to orchestrate pronuclear migration and union (karyogomy), and its duplication generates the two centrosomes that organize the first mitotic spindle and define the plane of cleavage. Tumor Biology Program, Mayo Clinic, Rochester, Minnesota 55905, USA. E-mail: [email protected]
PII S0960-9822(03)00033-2
Dispatch
Hamill et al. [3] employed an effective strategy to identify essential components of maternally derived pericentriolar material by screening conditional mutants in C. elegans for defective mitotic spindle function during early development, when egg reserves of centrosome precursors are used for centrosome maturation. C. elegans is a particularly useful model system for this purpose because the behavior of the large and accessible embryo is relatively easy to observe and follow. The authors screened mutagenized populations of nematodes at the non-permissive temperature and identified a handful of alleles required for spindle assembly or stability. Several of the alleles identified by Hamill et al. [3] turned out to be genes that encode proteins already known to play a role in centrosome or mitotic spindle function, validating their methods. But one of the identified genes, spindle defective-5 (spd-5), turned out to encode a novel component of zygotic pericentriolar material. Temperature-sensitive conditional mutants in spd-5 were found to be fully recessive at the non-permissive temperature, exhibiting all the hallmarks of a mutation that results in defective centrosome function. The fertilized eggs of spd-5 mutant worms failed to undergo pronuclear migration or to form mitotic spindles, and frequently zygotes aborted the final stages of cell division resulting in an uncleaved blastomere. Live cell microscopy of early development in transgenic spd-5 worms making fusion proteins of tubulin or histone linked to the green fluorescent protein (GFP) confirmed that the mutants have a defect in sperm aster formation following fertilization and fail to form functional bipolar mitotic spindles to to segregate chromosomes (to view the movies go to http://www.developmentalcell.com/cgi/content/full/3/ 5/673/DC1). Other aspects of cell-cycle progression, including the timing of nuclear envelope breakdown and initiation of the membrane invagination that marks the onset of cytokinesis, appeared normal. Interestingly, the spd-5 mutant showed its conditional phenotype only during early development; its essential centrosome function was either compensated by other gene products at later stages of development or otherwise is restricted to the large early embryonic cells. That the mutant behavior was a consequence of defective SPD-5 protein function was confirmed using RNA interference (RNAi) to ablate wild type spd-5 mRNA, which faithfully phenocopied the characteristics of the spd-5 mutants. In both wild-type and spd-5 mutant embryos, immunofluorescence microscopy revealed centrosomal labeling of SPD-5 protein at the sperm aster, with an increased level of staining as the two mitotic spindle poles formed. Accumulation of centrosomal SPD-5 prior to mitosis was diminished in embryos carrying a mutation in another spindle defective gene, spd-2, which had been previously implicated in centrosome maturation
Current Biology R89
and the formation of anterior-posterior polarity in the developing zygote [8]. Importantly, accumulation of SPD-5 at the centrosome did not depend on intact microtubules or on other centrosome proteins whose activities more directly influence microtubule behavior. In order to elucidate the mechanism of SPD-5 action, Hamill et al. [3] determined the status of other centrosome proteins in the early C. elegans embryo. In wild-type embryos, γ-tubulin, which is responsible for microtubule nucleation, was readily seen to accumulate at the sperm aster (centrosome) and at the poles of mitotic spindles. In the spd-5 mutants, however, γtubulin failed to accumulate into discrete centrosomal foci, explaining the loss of key centrosome microtubule organizing function in the embryo. Two other proteins that regulate important aspects of centrosome behavior, aurora A-like kinase (AIR-1), which is required for spindle assembly, and the microtubule stabilizing protein ZYG-9, also failed to accumulate at the centrosome in mutant embryos. Taken together, these observations implicate SPD-5 as a crucial structural component of pericentriolar material required for centrosome maturation in early embryos, and suggest that a hierarchy of interactions involving SPD-2 and SPD5 may be important in this process. Working further with the spd-5 mutant, Hamill et al. [3] also demonstrated a genetic interaction between spd-5 and dhc-1, the gene encoding the heavy chain of cytoplasmic dynein, a minus-end directed microtubule motor protein. Cytoplasmic dynein has earlier been shown to transport cargo along microtubules toward the centrosome, and while the available data do not allow conclusive interpretation on this issue, it is possible that dynein and SPD-5 may interact directly at the cell center to mediate the accumulation of additional proteins into pericentriolar material during centrosome maturation. Genetic mapping and sequencing showed that spd5 encodes a 1198 residue protein of predicted molecular weight 135 kDa [3]. A distinguishing feature of SPD-5 is 11 predicted short coiled-coil domains, ranging from roughly 30 to 60 amino acids in length (Figure 1). The original spd-5 mutation turns out to be a single base change that results in substitution of arginine 593 by lysine in the central coiled-coil domain. The coiled-coil protein motif is an evolutionarily conserved structure tailored to mediate protein–protein interactions. Coiled-coils consist of intertwined parallel right-handed α helices characterized by a repeating pattern of hydrophilic and hydrophobic amino acids [9,10]. Classic examples of protein–protein interaction through coiled-coil domains include the fiber-forming proteins tropomyosin and the keratins, and examples of globular proteins with coiled-coil domains that are important for protein–protein interactions include α- and β-tubulin and many DNA binding proteins containing zipper-like coiled-coil motifs. Given these properties, Hamill et al. [3] propose that the multiple coiled-coil domains of SPD-5 mediate interactions between molecules to form a structural lattice or matrix of pericentriolar material at the centrosome of early embryos. SPD-5 may also act in the
SPD-5 * Pericentrin
Ninein Cep135
Current Biology
Figure 1. A schematic diagram of coiled-coil proteins found in pericentriolar material. SPD-5 of C. elegans early embryo pericentriolar material: the spd-5 conditional mutation leads to substitution of an arginine by a lysine in the central coiled-coil domain, indicated by the asterisk. Pericentrin, ninein and Cep135 are found in vertebrate pericentriolar material. The predicted coiled-coil domains are illustrated in blue.
recruitment of other proteins — including the γ-tubulin complex, AIR-1 and ZYG-9 — to the centrosome via coiled-coil protein–protein interaction domains. While the properties of SPD-5 and the spd-5 mutant are consistent with this proposal, SPD-5 function may be restricted to centrosomes in early C. elegans embryos. Given the operative diversity of centrosomes, the pericentriolar material of higher eukaryotic centrosomes is likely to be a structurally complex matrix and involve multiple coiled-coil proteins. Several centrosome proteins of vertebrate cells share properties of SPD-5. For example, pericentrin [11,12], Cep135 [13] and ninein [14] are multiple coiled-coil domain proteins that are integral components of pericentriolar material. Like SPD-5, their centrosomal localization is independent of microtubule integrity, and their depletion using blocking antibodies or by gene silencing leads to disruption of centrosome structure and microtubule organizing function. Conversely, overproduction of these proteins in cultured cells results in excessive accumulation of pericentriolar material, the formation of disorganized microtubule arrays and mitotic spindle abnormalities. Pericentrin is also similar to SPD-5 in its ability to interact with cytoplasmic dynein and its requirement for centrosomal targeting of γ-tubulin [12,15]. Pericentrin, Cep135 and ninein are very likely just a subset of the coiled-coil-containing elements of the pericentriolar matrix. Looking to yeast for guidance is helpful, but not completely satisfying. The Saccharomyces cerevisiae spindle pole body — a highly abridged version of the centrosome — is the ultimate paradigm for studying protein–protein interactions critical to centrosome structure–function relationships [16,17]. Most spindle pole body components are coiled-coil proteins; these include a half dozen or so stable core components and about 15 loosely associated proteins, several of which have homologous counterparts that function in the higher eukaryotic centrosome. It is difficult, however, to directly translate the structural features of the extremely reduced and compact yeast spindle pole body into that seen in higher eukaryotic
Dispatch R90
Figure 2. The centrosome is a tripartite structure, consisting of core centrioles, a matrix of pericentriolar material and microtubule nucleating sites. An electron micrograph (A) and a schematic representation (B) of a vertebrate cell centrosome and surrounding cytoplasm. Near the center is a cross section of a centriole (a second centriole is out of the plane of the section). A fine matrix of pericentriolar material extends outward from the centriole in an irregular fashion; its limits are operationally defined as a zone excluding most other organelles (membranes and ribosomes). Several darkly staining pericentriolar ‘satellites’ are scattered throughout the matrix. Microtubules emanate from the pericentriolar material.
centrosomes. Nonetheless, a tripartite model for the structural complexity of the higher eukaryotic centrosome that is consistent with what is currently known is illustrated in Figure 2. The notion that pericentriolar material consists of a ‘sticky’ lattice-forming protein matrix is attractive because, at the electron microscope level, that is what pericentriolar material looks like — it has been variously described as ‘featureless’, an ‘amorphous cloud of electron dense material’, a ‘pericentriolar matrix or lattice’ and a ‘centromatrix’. At last, as the characteristic coiled-coil features of integral pericentriolar material proteins come into focus, a clearer picture of the structural nature of the elusive cloud surrounding the centrioles is beginning to emerge. References 1. Wilson, E.B. (1925). The Cell in Development and Heredity, 3rd Edn (Macmillan Company, New York). 2. Brinkley, B.R. (2001). Managing the centrosome numbers game: from chaos to stability in cancer cell division. Trends Cell Biol. 11, 18–21. 3. Hamill, D.R., Severson, A.F., Carter, J.C. and Bowerman, B. (2002). Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains. Dev. Cell 3, 673–684. 4. Palazzo, R., Vaisberg, E., Cole, R. and Rieder, C. (1992). Centriole duplication in lysates of Spisula solidissma oocytes. Science 256, 219–221. 5. Manandhar, G. and Schatten, G. (2000). Centrosome reduction during rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration. Mol. Reprod. Dev 56, 502–511. 6. Holy, J. and Schatten, G. (1991). Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores. Dev. Biol. 147, 343–353. 7. Stearns, T. and Kirschner, M. (1994). In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 76, 623–637. 8. O’Connell, K.F., Maxwell, K.N. and White, J.G. (2000). The spd-2 gene is required for polarization of the anteroposterior axis and formation of the sperm asters in the Caenorhabditis elegans zygote. Dev. Biol. 222, 55–70. 9. Berger, B., Wilson, D.B., Wolf, E., Tonchev, T., Milla, M. and Kim, P.S. (1995). Predicting coiled coils by use of pairwise residue correlations. Proc. Natl. Acad. Sci. U.S.A. 92, 8259–8263. 10. Lupas, A., Van Dyke, M. and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1162–1164. 11. Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D. and Kirschner, M. (1994). Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell 76, 639–650. 12. Dictenberg, J.B., Zimmerman, W., Sparks, C.A., Young, A., Vidair, C., Zheng, Y., Carrington, W., Fay, F.S. and Doxsey, S.J. (1998). Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell Biol. 141, 163–174.
13.
14.
15.
16.
17.
Ohta, T., Essner, R., Ryu, J.-H., Palazzo, R.E., Uetake, Y. and Kuriyama, R. (2002). Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells. J. Cell Biol. 156, 87–100. Bouckson-Castaing, V., Moudjou, M., Ferguson, D.J., Mucklow, S., Belkaid, Y., Milon, G. and Crocker, P.R. (1996). Molecular characterization of ninein, a new coiled-coil protein of the centrosome. J. Cell Sci. 109, 179–190. Young, A., Dictenberg, J.B., Purohit, A., Tuft, R. and Doxsey, S.J. (2000). Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes. Mol. Biol. Cell 11, 2047–2056. Nguyen, T., Vinh, D.B.N., Crawford, D.K. and Davis, T.N. (1998). A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gammatubulin complex. Mol. Biol. Cell 9, 2201–2216. Wigge, P.A., Jensen, O.N., Holmes, S., Soues, S., Mann, M. and Kilmartin, J.V. (1998). Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. J Cell Biol. 141, 967–977.
Centrosomes: Coiled-coils Organize the Cell Center Jeffrey L. Salisbury
The centrosome serves as a structural context for cytoplasmic organization. Recent studies on mutants of the nematode worm Caenorhabditis elegans have provided new insight into the framework to which microtubules and key regulators of centrosome behavior are anchored.
The centrosome is a fascinating organelle that resides near the cell center — hence its name [1]. It functions in the maintenance of cytoplasmic architecture through the nucleation and organization of microtubule arrays in interphase and mitotic cells. Centrosome defects have been implicated in disease processes, particularly in the origin of mitotic abnormalities and the development of aneuploidy in cancer [2]. In addition to its fundamental role in microtubule organization, the centrosome may also provide an important structural context for coordinating cell-cycle regulation. An understanding of the molecular basis of these diverse cellular functions is beginning to emerge through the careful analysis of centrosome structure and organization in early embryo development and somatic cells. In a recent study which exploited the power of genetics and gene silencing in the nematode Caenorhabditis elegans, Hamill et al. [3] have shed new light on a class of proteins with multiple coiled-coil domains that provide a scaffold onto which key players in centrosome function are attached. The centrosome consists of a pair of centrioles and a surrounding halo of pericentriolar material that harbors, among other proteins, the γ-tubulin protein complexes that nucleate microtubules [4]. In most animals, centrioles are eliminated during oogenesis, while the egg cytoplasm remains enriched for centrosome precursor proteins that are used during early embryonic development. Conversely, during spermatogenesis the sperm centrosome is reduced to its core centrosome components — a pair of centrioles — and, at the time of fertilization, the sperm delivers this stripped-down version of the somatic centrosome and a haploid (pro)nucleus to the egg [5]. In the zygote, the sperm centrioles undergo functional maturation by recruiting egg proteins to form the pericentriolar material of the zygotic centrosome [6,7]. Through this maturation process, the zygotic centrosome acquires the microtubule organizing activity required to orchestrate pronuclear migration and union (karyogomy), and its duplication generates the two centrosomes that organize the first mitotic spindle and define the plane of cleavage. Tumor Biology Program, Mayo Clinic, Rochester, Minnesota 55905, USA. E-mail: [email protected]
PII S0960-9822(03)00033-2
Dispatch
Hamill et al. [3] employed an effective strategy to identify essential components of maternally derived pericentriolar material by screening conditional mutants in C. elegans for defective mitotic spindle function during early development, when egg reserves of centrosome precursors are used for centrosome maturation. C. elegans is a particularly useful model system for this purpose because the behavior of the large and accessible embryo is relatively easy to observe and follow. The authors screened mutagenized populations of nematodes at the non-permissive temperature and identified a handful of alleles required for spindle assembly or stability. Several of the alleles identified by Hamill et al. [3] turned out to be genes that encode proteins already known to play a role in centrosome or mitotic spindle function, validating their methods. But one of the identified genes, spindle defective-5 (spd-5), turned out to encode a novel component of zygotic pericentriolar material. Temperature-sensitive conditional mutants in spd-5 were found to be fully recessive at the non-permissive temperature, exhibiting all the hallmarks of a mutation that results in defective centrosome function. The fertilized eggs of spd-5 mutant worms failed to undergo pronuclear migration or to form mitotic spindles, and frequently zygotes aborted the final stages of cell division resulting in an uncleaved blastomere. Live cell microscopy of early development in transgenic spd-5 worms making fusion proteins of tubulin or histone linked to the green fluorescent protein (GFP) confirmed that the mutants have a defect in sperm aster formation following fertilization and fail to form functional bipolar mitotic spindles to to segregate chromosomes (to view the movies go to http://www.developmentalcell.com/cgi/content/full/3/ 5/673/DC1). Other aspects of cell-cycle progression, including the timing of nuclear envelope breakdown and initiation of the membrane invagination that marks the onset of cytokinesis, appeared normal. Interestingly, the spd-5 mutant showed its conditional phenotype only during early development; its essential centrosome function was either compensated by other gene products at later stages of development or otherwise is restricted to the large early embryonic cells. That the mutant behavior was a consequence of defective SPD-5 protein function was confirmed using RNA interference (RNAi) to ablate wild type spd-5 mRNA, which faithfully phenocopied the characteristics of the spd-5 mutants. In both wild-type and spd-5 mutant embryos, immunofluorescence microscopy revealed centrosomal labeling of SPD-5 protein at the sperm aster, with an increased level of staining as the two mitotic spindle poles formed. Accumulation of centrosomal SPD-5 prior to mitosis was diminished in embryos carrying a mutation in another spindle defective gene, spd-2, which had been previously implicated in centrosome maturation
Current Biology R89
and the formation of anterior-posterior polarity in the developing zygote [8]. Importantly, accumulation of SPD-5 at the centrosome did not depend on intact microtubules or on other centrosome proteins whose activities more directly influence microtubule behavior. In order to elucidate the mechanism of SPD-5 action, Hamill et al. [3] determined the status of other centrosome proteins in the early C. elegans embryo. In wild-type embryos, γ-tubulin, which is responsible for microtubule nucleation, was readily seen to accumulate at the sperm aster (centrosome) and at the poles of mitotic spindles. In the spd-5 mutants, however, γtubulin failed to accumulate into discrete centrosomal foci, explaining the loss of key centrosome microtubule organizing function in the embryo. Two other proteins that regulate important aspects of centrosome behavior, aurora A-like kinase (AIR-1), which is required for spindle assembly, and the microtubule stabilizing protein ZYG-9, also failed to accumulate at the centrosome in mutant embryos. Taken together, these observations implicate SPD-5 as a crucial structural component of pericentriolar material required for centrosome maturation in early embryos, and suggest that a hierarchy of interactions involving SPD-2 and SPD5 may be important in this process. Working further with the spd-5 mutant, Hamill et al. [3] also demonstrated a genetic interaction between spd-5 and dhc-1, the gene encoding the heavy chain of cytoplasmic dynein, a minus-end directed microtubule motor protein. Cytoplasmic dynein has earlier been shown to transport cargo along microtubules toward the centrosome, and while the available data do not allow conclusive interpretation on this issue, it is possible that dynein and SPD-5 may interact directly at the cell center to mediate the accumulation of additional proteins into pericentriolar material during centrosome maturation. Genetic mapping and sequencing showed that spd5 encodes a 1198 residue protein of predicted molecular weight 135 kDa [3]. A distinguishing feature of SPD-5 is 11 predicted short coiled-coil domains, ranging from roughly 30 to 60 amino acids in length (Figure 1). The original spd-5 mutation turns out to be a single base change that results in substitution of arginine 593 by lysine in the central coiled-coil domain. The coiled-coil protein motif is an evolutionarily conserved structure tailored to mediate protein–protein interactions. Coiled-coils consist of intertwined parallel right-handed α helices characterized by a repeating pattern of hydrophilic and hydrophobic amino acids [9,10]. Classic examples of protein–protein interaction through coiled-coil domains include the fiber-forming proteins tropomyosin and the keratins, and examples of globular proteins with coiled-coil domains that are important for protein–protein interactions include α- and β-tubulin and many DNA binding proteins containing zipper-like coiled-coil motifs. Given these properties, Hamill et al. [3] propose that the multiple coiled-coil domains of SPD-5 mediate interactions between molecules to form a structural lattice or matrix of pericentriolar material at the centrosome of early embryos. SPD-5 may also act in the
SPD-5 * Pericentrin
Ninein Cep135
Current Biology
Figure 1. A schematic diagram of coiled-coil proteins found in pericentriolar material. SPD-5 of C. elegans early embryo pericentriolar material: the spd-5 conditional mutation leads to substitution of an arginine by a lysine in the central coiled-coil domain, indicated by the asterisk. Pericentrin, ninein and Cep135 are found in vertebrate pericentriolar material. The predicted coiled-coil domains are illustrated in blue.
recruitment of other proteins — including the γ-tubulin complex, AIR-1 and ZYG-9 — to the centrosome via coiled-coil protein–protein interaction domains. While the properties of SPD-5 and the spd-5 mutant are consistent with this proposal, SPD-5 function may be restricted to centrosomes in early C. elegans embryos. Given the operative diversity of centrosomes, the pericentriolar material of higher eukaryotic centrosomes is likely to be a structurally complex matrix and involve multiple coiled-coil proteins. Several centrosome proteins of vertebrate cells share properties of SPD-5. For example, pericentrin [11,12], Cep135 [13] and ninein [14] are multiple coiled-coil domain proteins that are integral components of pericentriolar material. Like SPD-5, their centrosomal localization is independent of microtubule integrity, and their depletion using blocking antibodies or by gene silencing leads to disruption of centrosome structure and microtubule organizing function. Conversely, overproduction of these proteins in cultured cells results in excessive accumulation of pericentriolar material, the formation of disorganized microtubule arrays and mitotic spindle abnormalities. Pericentrin is also similar to SPD-5 in its ability to interact with cytoplasmic dynein and its requirement for centrosomal targeting of γ-tubulin [12,15]. Pericentrin, Cep135 and ninein are very likely just a subset of the coiled-coil-containing elements of the pericentriolar matrix. Looking to yeast for guidance is helpful, but not completely satisfying. The Saccharomyces cerevisiae spindle pole body — a highly abridged version of the centrosome — is the ultimate paradigm for studying protein–protein interactions critical to centrosome structure–function relationships [16,17]. Most spindle pole body components are coiled-coil proteins; these include a half dozen or so stable core components and about 15 loosely associated proteins, several of which have homologous counterparts that function in the higher eukaryotic centrosome. It is difficult, however, to directly translate the structural features of the extremely reduced and compact yeast spindle pole body into that seen in higher eukaryotic
Dispatch R90
Figure 2. The centrosome is a tripartite structure, consisting of core centrioles, a matrix of pericentriolar material and microtubule nucleating sites. An electron micrograph (A) and a schematic representation (B) of a vertebrate cell centrosome and surrounding cytoplasm. Near the center is a cross section of a centriole (a second centriole is out of the plane of the section). A fine matrix of pericentriolar material extends outward from the centriole in an irregular fashion; its limits are operationally defined as a zone excluding most other organelles (membranes and ribosomes). Several darkly staining pericentriolar ‘satellites’ are scattered throughout the matrix. Microtubules emanate from the pericentriolar material.
centrosomes. Nonetheless, a tripartite model for the structural complexity of the higher eukaryotic centrosome that is consistent with what is currently known is illustrated in Figure 2. The notion that pericentriolar material consists of a ‘sticky’ lattice-forming protein matrix is attractive because, at the electron microscope level, that is what pericentriolar material looks like — it has been variously described as ‘featureless’, an ‘amorphous cloud of electron dense material’, a ‘pericentriolar matrix or lattice’ and a ‘centromatrix’. At last, as the characteristic coiled-coil features of integral pericentriolar material proteins come into focus, a clearer picture of the structural nature of the elusive cloud surrounding the centrioles is beginning to emerge. References 1. Wilson, E.B. (1925). The Cell in Development and Heredity, 3rd Edn (Macmillan Company, New York). 2. Brinkley, B.R. (2001). Managing the centrosome numbers game: from chaos to stability in cancer cell division. Trends Cell Biol. 11, 18–21. 3. Hamill, D.R., Severson, A.F., Carter, J.C. and Bowerman, B. (2002). Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains. Dev. Cell 3, 673–684. 4. Palazzo, R., Vaisberg, E., Cole, R. and Rieder, C. (1992). Centriole duplication in lysates of Spisula solidissma oocytes. Science 256, 219–221. 5. Manandhar, G. and Schatten, G. (2000). Centrosome reduction during rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration. Mol. Reprod. Dev 56, 502–511. 6. Holy, J. and Schatten, G. (1991). Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores. Dev. Biol. 147, 343–353. 7. Stearns, T. and Kirschner, M. (1994). In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 76, 623–637. 8. O’Connell, K.F., Maxwell, K.N. and White, J.G. (2000). The spd-2 gene is required for polarization of the anteroposterior axis and formation of the sperm asters in the Caenorhabditis elegans zygote. Dev. Biol. 222, 55–70. 9. Berger, B., Wilson, D.B., Wolf, E., Tonchev, T., Milla, M. and Kim, P.S. (1995). Predicting coiled coils by use of pairwise residue correlations. Proc. Natl. Acad. Sci. U.S.A. 92, 8259–8263. 10. Lupas, A., Van Dyke, M. and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1162–1164. 11. Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D. and Kirschner, M. (1994). Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell 76, 639–650. 12. Dictenberg, J.B., Zimmerman, W., Sparks, C.A., Young, A., Vidair, C., Zheng, Y., Carrington, W., Fay, F.S. and Doxsey, S.J. (1998). Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell Biol. 141, 163–174.
13.
14.
15.
16.
17.
Ohta, T., Essner, R., Ryu, J.-H., Palazzo, R.E., Uetake, Y. and Kuriyama, R. (2002). Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells. J. Cell Biol. 156, 87–100. Bouckson-Castaing, V., Moudjou, M., Ferguson, D.J., Mucklow, S., Belkaid, Y., Milon, G. and Crocker, P.R. (1996). Molecular characterization of ninein, a new coiled-coil protein of the centrosome. J. Cell Sci. 109, 179–190. Young, A., Dictenberg, J.B., Purohit, A., Tuft, R. and Doxsey, S.J. (2000). Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes. Mol. Biol. Cell 11, 2047–2056. Nguyen, T., Vinh, D.B.N., Crawford, D.K. and Davis, T.N. (1998). A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gammatubulin complex. Mol. Biol. Cell 9, 2201–2216. Wigge, P.A., Jensen, O.N., Holmes, S., Soues, S., Mann, M. and Kilmartin, J.V. (1998). Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. J Cell Biol. 141, 967–977.