The yeast gamma-tubulin, Tub4p, forms a 6S complex with the spindle pole body components Spc98p and Spc97p. Purified Tub4p complex contains one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. Studies have been carried out to determine how the Tub4p complex binds to the yeast microtubule organizing center, the spindle pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p from the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. A complex has been isolated containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. This paper describes how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB (Knop, 1997).
The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, temperature-sensitive mutations were created in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the gamma-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. These results strongly argue that Spc110p links the gamma-tubulin complex to the central plaque of the SPB (Sundberg, 1997).
The yeast microtubule organizing center (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules, which are functionally and spatially distinct. The SPB of Saccharomyces cerevisiae offers an example of one MTOC that can organize functionally and spatially distinct classes of microtubules. The S. cerevisiae SPB is embedded in the nuclear envelope during the entire cell cycle. Substructures named the outer, central and inner plaques have been described by electron microscopy. The outer and inner plaques organize the cytoplasmic and nuclear microtubules, respectively. The cytoplasmic microtubules have functions in nuclear positioning and nuclear movement, while the nuclear microtubules are involved in spindle formation and chromosome segregation in mitosis and meiosis. The S. cerevisiae gamma-tubulin, Tub4p, forms a stable complex with two other proteins, Spc98p and Spc97p, and this gamma-tubulin complex (Tub4p complex) is localized at the outer and inner plaques of the SPB. Conditional lethal mutants in SPC98 and SPC97 reveal a role of the encoded proteins in microtubule organization by the SPB (Knop, 1998 and references therein).
Microtubule organization requires the Tub4p complex, which binds to the nuclear side of the SPB at the N-terminal domain of Spc110p. In this paper, the essential SPB component Spc72p is described, whose N-terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. This Tub4p complex-binding domain of Spc72p is essential: temperature-sensitive alleles of SPC72 or overexpression of a binding domain-deleted variant of SPC72 (DeltaN-SPC72) impair cytoplasmic microtubule formation. Consequently, polynucleated and anucleated cells accumulate in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules, as compared with wild-type. Exchange of the Tub4p complex-binding domains of Spc110p and Spc72p establishes that the Spc110p domain, when attached to DeltaN-Spc72p, is functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p, when fused to DeltaN-Spc110p, leads to a dominant-negative effect. These results suggest that different components of MTOCs act as receptors for gamma-tubulin complexes and that they are essential for the function of MTOCs (Knop, 1998).
The components of the yeast Tub4p complex have been localized to the outer and inner plaques, suggesting that they represent universal components of the microtubule organization machinery. In contrast, Spc110p and Spc72p are the first side-specific proteins of the SPB involved in microtubule organization. These data suggest that Spc110p and Spc72p have at least two functionally distinct domains: an N-terminal domain that interacts with the Tub4p complex and a C-terminal domain, which binds to at least one other SPB component. Cell-cycle-dependent modification of the N-terminal domain of Spc110p and Spc72p could in fact modify the microtubule organization properties of the inner and outer plaques. In this respect, it is interesting that Spc110p is a phosphoprotein and that Spc72p was resolved by SDS-PAGE into multiple bands, suggesting that it is subject to modification. The C-terminal domains of Spc110p and Spc72p carry the information as to which side of the SPB the proteins bind. Spc110p has been purified in complex with Spc42p, calmodulin and an SPB component with an apparent mol. wt of 35 kDa, indicating a physical interaction between the four SPB components. How Spc72p is bound to the SPB is still an open question (Knop, 1998 and references therein).
The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. A synthetic lethal screen was undertaken to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98-63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, it has been shown that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, it was shown that the lethal phenotype in spc98-63 spc110-221 cells is caused by the failure of Spc98-63p to interact with Spc110-221p. In contrast, the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p (Nguyen, 1998).
In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. The inner/central plaque is composed of a stable SPB subcomplex, containing the gamma-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication (Elliott, 1999).
During spindle pole body (SPB) duplication, the new SPB is assembled at a distinct site adjacent to the old SPB. Using quantitative fluorescence methods, the assembly and dynamics of the core structural SPB component Spc110p was examined. The SPB core exhibits both exchange and growth in a cell cycle-dependent manner. During G1/S phase, the old SPB exchanges approximately 50% of old Spc110p for new Spc110p. In G2 little Spc110p is exchangeable. Thus, Spc110p is dynamic during G1/S and becomes stable during G2. The SPB incorporates additional Spc110p in late G2 and M phases; this growth is followed by reduction in the next G1. Spc110p addition to the SPBs (growth) also occurs in response to G2 and mitotic arrests but not during a G1 arrest. These results reveal several dynamic features of the SPB core: cell cycle-dependent growth and reduction, growth in response to cell cycle arrests, and exchange of Spc110p during SPB duplication. Moreover, rather than being considered a conservative or dispersive process, the assembly of Spc110p into the SPB is more readily considered in terms of growth and exchange (Yoder, 2003).
The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to one another was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis (Muller, 2005).
The spindle pole body is the microtubule organizing center of Saccharomyces cerevisiae (Jaspersen, 2004). Two SPBs establish the bipolar mitotic spindle, a defining event of mitosis that allows the stable transmission of equivalent genetic material to the mother and daughter cell at the time of cell division. This role of the SPB is carried out by the centrosome in higher eukaryotes (Muller, 2005).
The structure of the SPB is reviewed by Jaspersen (2004). Briefly the ultrastructure observed by electron tomography consists of a series of stacked layers embedded in the nuclear envelope. The inner plaque is the area where the microtubules dock to the SPB; this plaque harbors the gamma-tubulin complex and the N-terminus of Spc110. The central plaque and the IL2 layer are the two core layers. This core is composed of 5 proteins. Spc29 and Cmd1 reside in the central plaque. Spc42 is thought to begin within the central plaque, but terminate in the IL2 layer. The C-terminus of Spc110 is in the central plaque where it binds Cmd1. The C-terminus of Cnm67 lies in the IL2 layer where it binds Spc42 and links the SPB core to the outer plaque. The outer plaque is the cytoplasmic boundary of the SPB where the astral microtubules nucleate from a second region of gamma-tubulin. Based on primarily two-hybrid interactions the SPB core proteins are typically depicted as components lying along a linear path that proceeds from Spc110 to Spc29 to Spc42 to Cnm67 (Muller, 2005).
The ultrastructure of the SPB is clearly quite different from the centrosome. Centrioles are not present and the SPB remains inserted in the nuclear envelope during mitosis. Yet both have in common the gamma-tubulin complex, Spc110/kendrin/AKAP-450, calmodulin, centrin, and Sfi1p. (The latter two proteins are part of the SPB half-bridge, a domain involved in SPB duplication. Despite differences in gross anatomy, the SPB and centrosome likely share an underlying structure. To date the only component of either the SPB or centrosome whose structure is solved at atomic resolution is calmodulin. The paucity of structural information has limited the understanding of the molecular functions performed by individual SPB proteins. Without crystals or well behaved soluble proteins, the available research tools to probe the SPB structure or any large macromolecular complex are few (Muller, 2005).
This study used a hybrid approach that combined in vivo live-cell FRET measurements with previous cryo-EM analysis. CFP and YFP were used as FRET donor and acceptor and attached to the components of the SPB. Initially FRET values were classified as either positive or negative for energy transfer as judged by a comparison to carefully designed controls. This binary classification system allowed mapping of the ends of proteins within the architecture of the SPB. Next the positive values were subdivided into classes. The classification specified a unique geometry for the SPB components that was not only consistent with previous structural and genetic studies, but broadened the understanding of SPB organization (Muller, 2005).
The FRET results suggest that the Il2 layer and central plaque form an integrated meshwork of proteins with Spc42 closely associated with all components of the central plaque. The general features of the core proteins of the IL2 and central plaque, based on the current results and the general literature, are as follows. The N-terminus of Spc42 begins at the inner boundary of the central plaque, forms a coiled-coil domain that defines the spacing of the gap between layers, enters the IL2 layer, and finally loops back to end at the internal face of the IL2 layer. Remarkably, even though the N-terminal domain before the coiled coil is only ~60 amino acids long, the N-terminus is in close proximity to the C-termini of Spc29, Cmd1, and Spc110. Cnm67 begins at the outer plaque, penetrates the IL2 and ends in close proximity to the C-terminus of Spc42. The N- and C-termini of Spc29 both lie on the inner face of the central plaque. Cmd1 is situated near the C-terminal end of Spc110, consistent with in vitro binding experiments, genetic and two-hybrid results. Finally Spc110, which at its N-terminus binds the gamma-tubulin complex extends from the inner plaque through the central plaque and ends in close juxtaposition to the C-terminus of Spc42. All the termini of the central plaque and IL2 layer proteins lie along the internal edges of the IL2 and central plaque layers, facing the space between the two layers (Muller, 2005).
The SPB is organized around a hexagonal lattice of Spc42. The arrangement of Spc42 in the Il2 layer was suggested by analysis of cryoelectron micrographs of both SPB cores and two-dimensional crystals of Spc42 that arise in vivo upon Spc42 overexpression. Because the N-terminus of Spc42 is situated in the central plaque, the arrangement of Spc42 in the IL2 layer necessarily imposes the same organization on the location of Spc42 in the central plaque. Cryoelectron microscopy has not revealed this implied organization of the central plaque. However the visualization of the Spc42 arrangement in the IL2 relied upon the contrast between regions of high protein density and pockets of low or no density. If, as supported by the FRET results, the components of the central plaque are densely packed, a uniform and high protein density would mask the organization in electron micrographs (Muller, 2005).
The Spc42 lattice provided a template that enabled the FRET-based geometry of the core proteins to be taken and and a model to be generated for the organization of the central plaque. The model suggests that Spc42 and Spc29 form the heart of the central plaque. A strong association between Spc29 and Spc42 is well documented. Spc29 has a robust two-hybrid interaction with the N-terminus of Spc42. In an Spc110-226 mutant, Spc29 remains associated with Spc42 under the conditions in which Spc110-226, calmodulin, and the gamma-tubulin complex pull away from the SPB. Finally, Spc29 is seen with Spc42 at the satellite of the SPB. In this model Spc29 lies along the path of Spc42 and together they form a ring of protein around the center of the hexagonal unit in the central plaque (Muller, 2005).
At the center of the hexagonal unit is placed a trimer of Spc110 dimers as they unravel from their coiled coil motif. In the model Spc110 enters the central plaque through the ring of Spc29 and Spc42. Two-hybrid analysis suggested that Spc29 binds to Spc110 between the end of the coiled coil and the start of the Cmd1-binding domain, from positions 811 to 898. This region overlaps Region II of Spc110 (position 772-836), a domain that plays a role in locking Spc110 in place during mitosis. The FRET model is consistent with Spc29 and Spc42 acting as a clasp to surround and lock Spc110 in place. However the central plaque must not only lock Spc110 in place to withstand the push and pull of mitosis, but also must be organized in a way that facilitates the remodeling of the SPB during G1/S-phase when 50% of Spc110 turns over. Therefore any locking mechanism must be reversible and the interaction between Spc110 and Spc29 must be dynamic (Muller, 2005).
Calmodulin and the C-terminal domain of Spc110 are positioned to reinforce lateral stability of the central plaque. This is evident when the hexagonal unit is tessellated to form a mosaic lattice of the central plaque components. Calmodulin and the C-terminal domain of Spc110 from one hexagonal unit are juxtaposed with their counterparts in the adjoining hexagonal units. The dimerization of the C-terminal Spc110/Cmd1 domain was confirmed in vitro. Surprisingly even though calmodulin is a highly conserved component of the SPB, it is not required. An SPC110-407 mutant of S. cerevisiae that lacks the calmodulin-binding domain is still viable. One explanation is that the integrity of the SPB is maintained through structurally redundant lateral connections in IL2 layer and central plaque (Muller, 2005).
The tessellation of the repeat unit prompts the question of what determines the lateral limits of the SPB. How is the repeat symmetry broken and the boundary with the nuclear envelope established? One clue may come from a comparison of the dimensions of the SPB with the cluster of nuclear microtubules that originate at the SPB. The SPB is circular with an average diameter of ~165 nm for the central plaque from a diploid and therefore an area of ~2.1 x 106Å2. A diploid would have ~35 microtubules emanating from the SPB (32 kinetochore microtubules and a three pole-to-pole microtubules. Microtubules have a cross-sectional diameter of 25 nm, so the minimal total area occupied by 35 microtubules (hexagonal packing with a packing density of 91% is 1.9 x 106Å2. Even assuming some spread at the inner plaque, the SPB has almost the minimal area required to attach the nuclear microtubules. One mechanism that could minimize both the size of the SPB and the size of the bundle of microtubules would be feedback between microtubule attachment and Spc110 turnover. A removal of Spc110 molecules that are not nucleating microtubules would break the lattice symmetry, leaving Spc42 and Spc29 to interact with other proteins of the nuclear envelope. Spc110 is only added to the SPB after the insertion of Spc42 and Spc29 into the nuclear envelope, so the edge of the SPB does not require Spc110. The mechanism and role of Spc110 turnover is an area of continued research (Muller, 2005).
The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. A mutant allele spc110-226 causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, it has been shown that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to form a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. It is suggested that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. These results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle (Yoder, 2005).
Antisera from scleroderma patients that react widely with centrosomes in plants and animals were used to isolate cDNAs encoding a novel centrosomal protein. The nucleotide sequence is consistent with a 7 kb mRNA and contains an open reading frame encoding a protein with a putative large coiled-coil domain flanked by noncoiled ends. Antisera recognize a 220 kd protein and stain centrosomes and acentriolar microtubule-organizing centers, where the protein is localized to the pericentriolar material (hence, the name pericentrin). Anti-pericentrin antibodies disrupt mitotic and meiotic divisions in vivo and block microtubule aster formation in Xenopus extracts, but do not block gamma-tubulin assembly or microtubule nucleation from mature centrosomes. These results suggest that pericentrin is a conserved integral component of the filamentous matrix of the centrosome involved in the initial establishment of organized microtubule arrays (Doxsey, 1994).
A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacts with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly binds to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunits of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacts with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP localizes to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha accumulates. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates (Takahashi, 1999).
Eukaryotic chromosome segregation depends on the mitotic spindle apparatus, a bipolar array of microtubules nucleated from centrosomes. Centrosomal microtubule nucleation requires attachment of gamma-tubulin ring complexes to a salt-insoluble centrosomal core, but the factor(s) underlying this attachment remains unknown. In budding yeast, this attachment is provided by the coiled-coil protein Spc110p, which links the yeast gamma-tubulin complex to the core of the yeast centrosome. The large coiled-coil protein kendrin is a human ortholog of Spc110p. Kendrin was identified by its C-terminal calmodulin-binding site, which shares homology with the Spc110p calmodulin-binding site. Kendrin localizes specifically to centrosomes throughout the cell cycle. N-terminal regions of kendrin share significant sequence homology with pericentrin, a previously identified murine centrosome component known to interact with gamma-tubulin. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with spindle microtubules (Flory, 2000).
Recently, a similar role has been suggested for the Drosophila melanogaster abnormal spindle protein (Asp). Asp, a centrosomal protein containing potential calmodulin-binding sites, appears to regulate the mitotic spindle apparatus by tethering gamma-TURCs together. Despite the similarities between Asp and kendrin, the functions of these two proteins are likely distinct. Kendrin and Asp share no homology with one another, whereas kendrin is clearly related to pericentrin, which interacts with gamma-tubulin. The predicted structure of kendrin, like that of Spc110p, contains long central coiled-coil domains flanked by noncoiled ends, whereas the secondary structure of Asp is predicted to be primarily gamma-helical with short stretches of coiled-coil near its C terminus. Additionally, Asp is predicted to contain an actin-binding domain, a feature found in neither kendrin nor Spc110p. The calmodulin-binding site of kendrin is similar to that of S. cerevisiae Spc110p and of the Spc110p homologs identified in A. nidulans and S. pombe, whereas the IQ-type calmodulin-binding site of Asp is more similar to those found in myosins. Finally, Asp localizes to both the centrosome and the spindle and was initially purified as a microtubule-associated protein, whereas kendrin is restricted to the centrosome, as is Spc110p. These differences indicate that the activities of kendrin may be more similar to those of Spc110p than to those of Asp. Further analysis of the functional relationships among kendrin, pericentrin, gamma-tubulin, and Asp will shed light on the mechanisms controlling the complex process of mitotic spindle formation and should aid in the understanding of centrosomal abnormalities that accompany cancerous growth (Flory, 2000 and references therein).
Pericentrin, a critical centrosome component first identified in mouse, recruits factors required for assembly of the mitotic spindle apparatus. A similar yet larger human protein named kendrin was recently identified, but its relationship to pericentrin was not clear. Extensive sequence homology between the mouse chromosome 10 region encoding pericentrin and the human chromosome 21 region encoding kendrin indicates that these proteins are encoded by syntenic loci. However, comparison of the published mouse pericentrin cDNA sequence to mouse genomic DNA sequences revealed two important differences: the stop codon present in the published mouse pericentrin cDNA is not found in the mouse genomic sequence, and the 3' end of the published mouse pericentrin cDNA is a fragment from a different mouse chromosome. To resolve these discrepancies, a mouse expressed sequence tag (EST) was sequenced that corresponds to the 3' end for a 7.1-kb mouse pericentrin RNA encoded on chromosome 10. Extensive northern blot analysis revealed that the pericentrin gene displays a complex expression pattern in both mouse and human: a 10-kb kendrin transcript is found in most tissues, whereas smaller transcripts are detected in a limited subset of tissues. These analyses demonstrate that pericentrin and kendrin are encoded by one gene, correct the previously published pericentrin cDNA sequence, and describe the complex expression pattern for a gene important for centrosome function in normal and transformed cells (Flory, 2003).
Centrosomes provide docking sites for regulatory molecules involved in the control of the cell division cycle. The centrosomal matrix contains several proteins thatanchor kinases and phosphatases. The large A-Kinase Anchoring Protein AKAP450 is acting as a scaffolding protein for other components of the cell signaling machinery. The centrosome was selectively perturbed by modifying the cellular localization of AKAP450. The expression in HeLa cells of the C terminus of AKAP450, which contains the centrosome-targeting domain of AKAP450 but not its coiled-coil domains or binding sites for signaling molecules, leads to the displacement of the endogenous centrosomal AKAP450 without removing centriolar or pericentrosomal components such as centrin, gamma-tubulin, or pericentrin. The centrosomal protein kinase A type II alpha was delocalized upon expression of the C terminus of AKAP450. This expression impairs cytokinesis and increases ploidy in HeLa cells, whereas it arrests diploid RPE1 fibroblasts in G1, thus further establishing a role for the centrosome in the regulation of the cell division cycle. Moreover, centriole duplication is interrupted. These data show that the association between centrioles and the centrosomal matrix protein AKAP450 is critical for the integrity of the centrosome and for its reproduction (Keryer, 2003a).
The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. This study shows, by immunofluorescence, immunoelectron microscopy, and biochemical analysis, that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. Centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome (Keryer, 2003b).
AKAP450 (also known as AKAP350, CG-NAP or Hyperion) and pericentrin are large coiled-coil proteins found in mammalian centrosomes that serve to recruit structural and regulatory components including dynein and protein kinase A. These proteins share a well conserved 90 amino acid domain near their C-termini that is also found in coiled-coil proteins of unknown function from Drosophila and fission yeast. Fusion of the C-terminal region from either protein to a reporter protein confers a centrosomal localization, and overexpression of the domain from AKAP450 displaces endogenous pericentrin, suggesting recruitment to a shared site. When isolated from transfected cells the C-terminal domain of AKAP450 was associated with calmodulin, suggesting that this protein could contribute to centrosome assembly (Gillingman, 2000)
AKAP450 and pericentrin are both very large proteins predicted to form a coiled-coil over most of their length except for N- and C-terminal regions of ∼200 amino acids. Searching the database with the C-terminal region of AKAP450 revealed a strong homology to the equivalent region of pericentrin, and also with the C-terminus of a putative coiled-coil protein of Drosophila that has not been previously characterized. Using the iterative search program PSI-BLAST, with the region shared by these proteins, identified a single further match to the C-terminus of an uncharacterized coiled-coil protein from S. pombe (significance score of 1 × e–5). These sequences show homology over a region of ∼90 amino acids, divided into two more highly conserved blocks. Three of the proteins extend a further c120 residues C-terminal but this region is less well conserved and relatively proline rich (Gillingman, 2000)
To investigate the function of this conserved C-terminal domain, the last 266 amino acids of AKAP450 were attached to the C-terminus of GFP, and expressed in COS cells. Examination of the transfected cells revealed bright GFP fluorescence in perinuclear spots characteristic of the centrosome. At moderate expression levels these were the only structures visible, while at higher levels diffuse fluorescence was also present throughout the cell. The structures labeled with the GFP chimera were also recognized by antibodies against pericentrin and γ-tubulin, confirming that they were centrosomes. The antisera against pericentrin were raised against part of the coiled-coil region of the protein, and hence should not cross react with the region of AKAP450 present in the GFP fusion. No staining of the Golgi apparatus was observed, which is consistent with the notion that AKAP450 is found only on the centrosome. The targeting of the GFP chimera to the centrosome was not affected when microtubules were depolymerized with nocodazole, indicating that the centrosomal location is not simply a reflection of an association with a minus-end-directed motor that concentrates the material near the microtubule organizing center. The C-terminal 226 residues of Drosophila protein CG6735 also targeted GFP to the centrosome in COS cells, indicating that the binding site of the domain has been well conserved in evolution (Gillingman, 2000)
The fusion between GFP and the AKAP450 C-terminus colocalizes with pericentrin in transfected cells. However, in cells expressing high levels of the AKAP fusion the centrosomal staining with antibodies against endogenous pericentrin was substantially reduced. The focused GFP staining and the relative lack of an effect on γ-tubulin, indicate that some centrosomal structure remained in these cells. This suggests that the C-terminal domain of AKAP450 was competing with the related domain in endogenous pericentrin for a binding site in the centrosome. Indeed a fusion of the C-terminal 241 amino acids of human pericentrin to red fluorescent protein (RFP) also accumulates in centrosomes, colocalizing with both anti-pericentrin antibodies and the GFP-AKAP450 chimera, and also reducing the staining of endogenous pericentrin when overexpressed. Taken together these results suggest that the C-terminal domains of AKAP450 and pericentrin share a binding site in the centrosome (Gillingman, 2000)
To examine the contribution of the conserved regions in the AKAP450 C-terminus to centrosomal targeting, truncated versions of this region were expressed in COS cells as fusions to GFP. Protein blotting revealed bands with apparent sizes matching those predicted for these truncated versions. Removal of the 100 amino acids C-terminal of the conserved region did not affect centrosomal localization, indicating that the remaining conserved region contains the targeting activity. Indeed a 91 residue section covering this core conserved region is sufficient for centrosomal targeting. This region contains two highly conserved sections separated by a more variable stretch. When the C-terminus from AKAP450 was divided in its variable stretch both halves showed greatly reduced centrosomal targeting, but nonetheless centrosomal accumulation was not completely absent for either construct. This suggests that efficient recruitment of the domain to the centrosome does not involve binding through a single short motif, but rather requires an extended interaction of multiple motifs or a single large folded structure (Gillingman, 2000)
To identify proteins involved in the centrosomal targeting of AKAP450, the C-terminal domain was expressed in COS cells as a fusion to protein A [this chimera also localized to centrosomes. Transfected cells were lysed, the 'ZZ-AKAP' fusion isolated on immunoglobulin beads, and an eluate from the beads examined for coprecipitating proteins. The domain specifically coprecipitated with an ∼17 kDa protein. The isolation was repeated on a larger scale, and tryptic peptides analysed by mass spectrometry. The fragment masses gave a match to calmodulin, and this identification was confirmed in two ways: (1) the mobility of the band upon electrophoresis increased in the presence of calcium, characteristic of calmodulin, and (2) anti-calmodulin antibodies detected a band of the appropriate size only in GFP-AKAP precipitates (Gillingman, 2000)
To investigate the requirements for this calmodulin interaction, the GFP–AKAP fusions described above were immunoprecipitated from transfected cells, and protein blots of the precipitated material probed for the fusion proteins and calmodulin. Although the different versions of the domain showed varying degrees of calmodulin binding, association of calmodulin with the complete C-terminal domain was calcium independent, and this association remained after removal of the poorly conserved C-terminal region. More surprisingly, removal of the first of the two conserved blocks resulted in the calmodulin binding becoming calcium dependent, although this first block did not bind calmodulin by itself. The core region binds calmodulin, but in a calcium sensitive fashion. A simple interpretation of these results is that, as with some other calmodulin binding proteins, there are at least two independent calmodulin-binding sites of distinct calcium sensitivity, and that these sites act synergistically to allow calmodulin binding that is insensitive to calcium levels. Indeed, the second of the two conserved regions resembles calcium-dependent calmodulin binding sites, being rich in basic and hydrophobic residues, and when this region was deleted calmodulin binding was abolished. Interestingly, this construct still showed centrosomal targeting, but at high levels of expression additional punctate staining was seen in the cytosol, rather than the diffuse staining seen with the other constructs, suggesting that its solubility had been compromised (Gillingman, 2000)
Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. The relationship between these proteins was studied in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins are part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex is distinct from the previously described gamma-tubulin ring complex (gamma-TuRC), because purified gamma-TuRC fractions do not contain detectable pericentrin. When assembled at the centrosome, the two proteins remain in close proximity, as shown by fluorescence resonance energy transfer. The three-dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis reveals a novel reticular lattice that is conserved from mammals to amphibians, and is organized independent of centrioles. The lattice changes dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appear to contact the minus ends of nucleated microtubules. These results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome (Dictenberg, 1998).
Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), has been localized to the centrosome via the carboxyl-terminal region. This region interacts with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of kendrin, another centrosomal coiled-coil protein. The amino-terminal region of either CG-NAP or kendrin indirectly associates with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin coimmunoprecipitate with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins localize to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibits the microtubule nucleation; moreover, the combination of these antibodies results in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC (Takahashi, 2002).
Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. This study, carried out with Xenopus extracts, shows that the centrosomal coiled-coil protein pericentrin anchors gammaTuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupts gamma tubulin localization and spindle organization in mitosis but has no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupts the endogenous pericentrin-gammaTuRC interaction and perturbs astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncouples gammaTuRCs from centrosomes, inhibits microtubule aster assembly, and induces rapid disassembly of preassembled asters. All phenotypes are significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and are specific for mitotic centrosomal asters, since little effect was observed on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induces G2/antephase arrest followed by apoptosis in many but not all cell types. It is concluded that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death (Zimmerman, 2004).
In the human lymphoblastic cell line KE 37, Northern blot analysis with cDNA probes for human regulatory subunits RII alpha RII beta of the cAMP-dependent protein kinase (A-kinase) type II and immunoblotting or immunoprecipitation studies with several antibodies directed against RII alpha and RII beta show that these two isoforms are expressed. The major isoform alpha is mostly cytosolic, whereas the beta isoform appears concentrated in the Golgi-centrosomal area, as judged by immunofluorescence and cell fractionation. Using a 32P-labelled RII overlay on Western blots, a 350-kDa RII-binding protein (AKAP 350) was specifically identified in centrosomes isolated from this cell line, whereas a Golgi fraction had previously been demonstrated to contain an 85-kDa RII-binding protein (AKAP 85). AKAP 350 is highly insoluble and can partially be extracted from centrosomes as a complex of AKAP 350 and RII subunit. AKAP 350 was identified as a specific centrosomal protein previously demonstrated in the pericentriolar material. The potential significance of a specific subcellular distribution for different RII-binding proteins in nonneuronal cells is discussed (Keryer, 1993).
Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA). Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs). AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. Both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo (Diviani, 2000).
Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, centrosomal protein pericentrin has been identified as a scaffold that tethers PKC betaII to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593. Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes (Chen, 2004).
Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, the protein was overexpressed in somatic cells and changes in the composition and function of mitotic spindles and spindle poles were examined. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. Levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitates with overexpressed pericentrin, suggesting that the motor is sequestered in the cytoplasm and is prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150(Glued) subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin-dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles (Purohit, 1999).
The light intermediate chains (LICs) of cytoplasmic dynein consist of multiple isoforms, which undergo post-translational modification to produce a large number of species separable by two-dimensional electrophoresis and which have been proposed to represent at least two gene products. The first known function for the LICs has been demonstrated: binding to the centrosomal protein, pericentrin, which represents a novel, non-dynactin-based cargo-binding mechanism. Rat LIC1, which is approximately 75% homologous to rat LIC2 and also contains a P-loop consensus sequence, has been cloned. LIC1 and LIC2 were compared for the ability to interact with pericentrin, and it was found that only LIC1 will bind. A functional P-loop sequence is not required for this interaction. The interaction maps to the central region of both LIC1 and pericentrin. Using recombinant LICs, it was found that they form homo-oligomers, but not hetero-oligomers, and exhibit mutually exclusive binding to the heavy chain. Additionally, overexpressed pericentrin is seen to interact with endogenous LIC1 exclusively. Together these results demonstrate the existence of two subclasses of cytoplasmic dynein: LIC1-containing dynein, and LIC2-containing dynein, only the former of which is involved in pericentrin association with dynein (Tynan, 2000).
Cytoplasmic dynein mediates assembly of pericentrin and gamma tubulin onto centrosomes. Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. In somatic cells, centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies it is concluded that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation (Young, 2000).
Based on this data, a model is proposed for the assembly of microtubule nucleating proteins. In this model, pericentrin binds to dynein through the light intermediate chain and to the gamma tubulin ring complex (gamma TuRC) through specific subunits of this complex. Dynein would mediate binding of the large pericentrin-gamma TuRC complex to microtubules and direct transport of the complex to centrosomes. At the centrosome, pericentrin-gamma TuRC complexes would be anchored, whereas dynein could be released for additional rounds of transport or anchored to perform additional roles. Dynactin may facilitate microtubule association or processivity of dynein and may contribute to centrosomal anchoring of gamma tubulin. This work raises the possibility that pericentrin mediates centrosome and spindle function through dynein-dependent assembly of microtubule nucleating complexes and other activities (Young, 2000).
There is now good evidence for microtubule-dependent and microtubule-independent mechanisms for recruitment of proteins onto centrosomes. These studies support the idea that dynein-mediated and passive diffusion mechanisms represent parallel pathways for centrosome assembly. It is possible that one pathway predominates over the other in certain biological systems or at different stages of the cell cycle. In embryonic systems, for example, high levels of centrosome proteins may be sufficient to drive the initial stages of microtubule-independent recruitment onto centrioles, although dynein-mediated transport becomes a major contributor at later times. Alternative mechanisms could also account for centrosome protein recruitment. Spontaneously assembled microtubules could be capped by gamma tubulin (and pericentrin) complexes, and these small microtubule fragments could be transported toward the minus ends of microtubules by dynein as described during spindle assembly in Xenopus extracts. These data do not distinguish between this microtubule fragment mechanism and the model presented in this paper in which presumably inactive centrosome proteins are transported to centrosomes and become active for microtubule nucleating activity. Another possibility is that centrosome-nucleated microtubules are released but remain tethered to the centrosome, perhaps through an interaction with dynactin, and they provide new minus ends for binding of gamma tubulin-pericentrin complexes after passive diffusion to these sites. Although this mechanism could account for the microtubule dependency of centrosome protein recruitment, it is inconsistent with kinetic data showing directed movement of GFP-pericentrin toward centrosomes (Young, 2000).
Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits distinctive and important pericellular cleavage functions. MT1-MMP is trafficked to the centrosomes in the course of endocytosis. The data suggested that the functionally important, integral, centrosomal protein, pericentrin-2, is a cleavage target of MT1-MMP in human and in canine cells and that the sequence of the cleavage sites are ALRRLLG1156->L1157FG and ALRRLLS2068->FG, respectively. The presence of Asp-948 at the P1 position inactivates the corresponding site (ALRRLLD948-L949FGD) in murine pericentrin. To confirm that MT1-MMP itself cleaves pericentrin directly, rather than indirectly, the cleavage of the peptides were analyzed that span the MT1-MMP cleavage site. In addition, glioma U251 cells, which co-expressed MT1-MMP with the wild type murine pericentrin and the D948G mutant, were analyzed. The D948G mutant that exhibits the cleavage sequence of human pericentrin is sensitive to MT1-MMP, whereas unmodified murine pericentrin is resistant to proteolysis. Taken together, these results confirm that MT1-MMP cleaves pericentrin-2 in humans but not in mice and that mouse models of cancer probably cannot be used to critically examine MT1-MMP functionality (Golubkov, 2005).
Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. The centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. It is concluded that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella (Jurczyk, 2004).
Factors that determine the biological and clinical behavior of prostate cancer are largely unknown. Prostate tumor progression is characterized by changes in cellular architecture, glandular organization, and genomic composition. These features are reflected in the Gleason grade of the tumor and in the development of aneuploidy. Cellular architecture and genomic stability are controlled in part by centrosomes, organelles that organize microtubule arrays including mitotic spindles. Centrosomes are structurally and numerically abnormal in the majority of prostate carcinomas. Centrosome abnormalities increase with increasing Gleason grade and with increasing levels of genomic instability. Selective induction of centrosome abnormalities by elevating levels of the centrosome protein pericentrin in prostate epithelial cell lines reproduces many of the phenotypic characteristics of high-grade prostate carcinoma. Cells that transiently or permanently express pericentrin exhibit severe centrosome and spindle defects, cellular disorganization, genomic instability, and enhanced growth in soft agar. On the basis of these observations, a model is proposed in which centrosome dysfunction contributes to the progressive loss of cellular and glandular architecture and increasing genomic instability that accompany prostate cancer progression, dissemination, and lethality (Pihan, 2001).
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