Nedd1 expression as a marker of dynamic centrosomal localization during mouse embryonic development

As the primary microtubule-organizing centre of the mammalian cell, the centrosome plays many important roles during cell growth and organization. This is evident across a broad range of cell types and processes, such as the proliferation, differentiation and polarity of neural cells. Additionally, given its localization and function, there are likely to be many more processes that rely on the centrosome that have not yet been characterized. Currently, little is known about centrosomal dynamics during mammalian development. In this study, we have analyzed Nedd1 protein expression to characterize the localization of the centrosome during some aspects of mouse embryogenesis. Using a Nedd1 antibody we have demonstrated the colocalization of Nedd1 with centrosomal markers. We found strong expression of Nedd1, and therefore the centrosome, in highly proliferating cells during neural development. Additionally, Nedd1 was found to have high expression in the cytoplasm of a subset of cells in the dorsal root ganglia. We have also shown a distinct, polarized centrosomal localization of Nedd1 in the developing lens, retina and other polarized tissues. This study reveals the localization of Nedd1 and the centrosome during important processes in mouse embryogenesis and provides a basis for further study into its role in development.     

Cohesin protein SMC1 is a centrosomal protein

Structural maintenance of chromosome protein 1 (SMC1) is well known for its roles in sister chromatid cohesion and DNA repair. In this study, we report a novel centrosomal localization of SMC1 within the cytoplasm in a variety of mammalian cell lines. We showed that SMC1 localized to centrosomes throughout the cell cycle in a microtubule-independent manner. Biochemically, SMC1 was cofractionated with the centrosomal protein γ-tubulin in centrosomal preparation. Immunohistochemistry and immunoelectron microscopy performed on mouse tissue sections revealed that SMC1 antibody strongly labeled the base of cilia in ciliated epithelia, where basal bodies were located. Furthermore, we showed that SMC1 was associated with both centrioles of a centrosome at G0/G1 stage of the cell cycle. These results demonstrate that SMC1 is a centrosomal protein, suggesting possible involvement of SMC1 in centrosome/basal body-related functions, such as organization of dynamic arrays of microtubules and ciliary formation.     

Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world

In characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables. The speed of this sliding movement is the fastest among many molecular motors known so far. We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin. The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive alpha-helical coiled-coil domain, and a C-terminal globular domain. Phylogenetic analysis suggested that this myosin belongs to class XI.     

The centrosomal protein CP190 regulates myosin function during early Drosophila development

Centrosomes are the main microtubule (MT)-organizing centers in animal cells, but they also influence the actin/myosin cytoskeleton. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis, and binds to MTs directly in vitro. CP190 has an essential function in the nucleus as a chromatin insulator, but centrosomes and MTs appear unperturbed in Cp190 mutants. Thus, the centrosomal function of CP190, if any, is unclear. Here, we examine the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos.     

中心体蛋白Centlein的敲除促进细胞周期进程

中心体是大部分动物细胞的微管组织中心,它确保了有序的细胞周期进程以及染色体的精确分离,我们之前报道了中心体蛋白Centlein作为一个分子连接,与C-Nap1和Cep68一起形成复合物维持中心体的连接. 然而,关于Centlein的其他功能我们还知之甚少. 在本研究中,建立了Centlein的敲除细胞系,并且运用RNA-seq技术分析了敲除细胞系和正常野生型细胞系之间转录水平的差异. 发现Centlein敲除细胞系中细胞周期相关基因PLK1、CCNB1、CCNA2和CDC20的表达量上调,流式结果又表明Centlein的敲除促进了细胞周期进程. 同时发现Centlein与PLK1之间存在细胞内相互作用,于是我们提出了Centlein通过与PLK1的作用参与细胞周期进程.     

Cytoplasmic myosin from Drosophila melanogaster

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.     

PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

The microtubule-organizing activity of the centrosome oscillates during the cell cycle, reaching its highest level at mitosis. At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar matrix (PCM) and a robust increase in microtubule-organizing activity. It is known that PLK1 is critical for the initiation of centrosome maturation. In this paper, we report that pericentrin (PCNT), a PCM protein, was specifically phosphorylated by PLK1 during mitosis. Phosphoresistant point mutants of PCNT did not recruit centrosomal proteins, such as CEP192, GCP-WD (γ-complex protein with WD repeats), γ-tubulin, Aurora A, and PLK1, into the centrosome during mitosis. However, centrosomal recruitment of CEP215 depended on PCNT irrespective of its phosphorylation status. Furthermore, ectopic expression of PLK1-PCNT fusion proteins induced the centrosomal accumulation of CEP192, GCP-WD, and γ-tubulin even in interphase cells, mimicking centrosome maturation. Based on these results, we propose that PLK1-mediated phosphorylation of PCNT initiates centrosome maturation by organizing the spindle pole-specific PCM lattice.     

A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication

The centrosome plays a fundamental role in organizing the interphase cytoskeleton and the mitotic spindle, and its protein complexity is modulated to support these functions. The centrosome must also duplicate itself once during each cell cycle, thus ensuring the formation of a bipolar spindle and its continuity through successive cell divisions. In this study, we have used a battery of antibodies directed against centrosomal components to study the general organization of the centrosome during the cell cycle and during the centrosome duplication process. We demonstrate that a subset of centrosomal proteins are arranged together to form a tubular pattern within the centrosome. The tubular conformation defined by these proteins has a polarity and is closed at one end. The centriole complement of the centrosome is normally placed near this end. We show that the "wall" of the tube is enriched in proteins such as CDC2, ninein, and pericentrin as well as gamma-tubulin. In addition, a subset of gamma-tubulin is localized to the "lumen" of the tube. We also demonstrate, for the first time, that antibody staining can be used to detect centrosome duplication allowing the identification of duplication intermediates. We show that one product of centrosome duplication is the replication of the tubular structure found within the centrosome. The position of the centriole duplexes prior to and during centrosome duplication is documented and a model of the morphogenesis of the centrosome during the duplication process is proposed.     

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.     

CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly

Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.     

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show 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. We also demonstrate that 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.     

Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidates--currently subject of further investigations--for the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome.     

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.     

CP110, a cell cycle-dependent CDK substrate,regulates centrosome duplication in human cells

Centrosome duplication and separation are linked inextricably to certain cell cycle events, in particular activation of cyclin-dependent kinases (CDKs). However, relatively few CDK targets driving these events have been uncovered. Here, we have performed a screen for CDK substrates and have isolated a target, CP110, which is phosphorylated by CDKs in vitro and in vivo. Human CP110 localizes to centrosomes. Its expression is strongly induced at the G1-to-S phase transition, coincident with the initiation of centrosome duplication. RNAi-mediated depletion of CP110 indicates that this protein plays an essential role in centrosome duplication. Long-term disruption of CP110 phosphorylation leads to unscheduled centrosome separation and overt polyploidy. Our data suggest that CP110 is a physiological centrosomal CDK target that promotes centrosome duplication, and its deregulation may contribute to genomic instability.     

Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains

The maternally expressed C. elegans gene spd-5 encodes a centrosomal protein with multiple coiled-coil domains. During mitosis in mutants with reduced levels of SPD-5, microtubules assemble but radiate from condensed chromosomes without forming a spindle, and mitosis fails. SPD-5 is required for the centrosomal localization of gamma-tubulin, XMAP-215, and Aurora A kinase family members, but SPD-5 accumulates at centrosomes in mutants lacking these proteins. Furthermore, SPD-5 interacts genetically with a dynein heavy chain. We propose that SPD-5, along with dynein, is required for centrosome maturation and mitotic spindle assembly.     

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.     

Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin

A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays a regulatory role in the myosin assembly. In the presence of calcium, Mts1 binds at the C-terminal end of the myosin heavy chain close to the site of phosphorylation by protein kinase CK2 (Ser1944). In the present study, we have shown that interaction of Mts1 with the human platelet myosin or C-terminal fragment of the myosin heavy chain inhibits phosphorylation of the myosin heavy chain by protein kinase CK2 in vitro. Mts1 might also bind directly the beta subunit of protein kinase CK2, thereby modifying the enzyme activity. Our results indicate that myosin oligomers were disassembled in the presence of Mts1. The short C-terminal fragment of the myosin heavy chain was totally soluble in the presence of an equimolar amount of Mts1 at low ionic conditions (50 mM NaCl). Depolymerization was found to be calcium-dependent and could be blocked by EGTA. Our data suggest that Mts1 can increase myosin solubility and therefore suppress its assembly.     

Selective removal of the carboxyl-terminal tail end of the Dictyostelium myosin II heavy chain by chymotrypsin

Dictyostelium myosin II is a conventional myosin consisting of two heavy chains of 243,000 Da and two pairs of light chains of 16,000 and 18,000 Da. In this paper, we show that the heavy chain of myosin II can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular weights of 195,000 and 38,000 Da as estimated from sodium dodecyl sulfate-polyacrylamide gels. Chymotryptic cleavage at this site occurs most readily in the absence of salt and is greatly inhibited as the salt concentration is increased from 0 to 60 mM. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 of the myosin II heavy chain. If the fragment extends to the carboxyl terminus of the myosin II heavy chain, it would have a molecular weight of 33,700. Upon digestion of myosin II which has been phosphorylated with a high molecular weight Dictyostelium myosin heavy chain kinase (C?té, G.P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072), all of the phosphate is recovered on the 33,700-Da tail-end fragment. Chymotrypsin-cleaved myosin II is shown to be capable of forming filaments at salt concentrations between 20 and 100 mM as judged by its ability to be sedimented by centrifugation. Only the large fragment of myosin II is found in the pellet; the 33,700-dalton fragment remains soluble. Chymotrypsin-cleaved myosin II can bind to actin and displays a high Ca2+-activated ATPase activity but has very low actin-activated ATPase activity.     

Dynamic recruitment of Nek2 kinase to the centrosome involves microtubules, PCM-1, and localized proteasomal degradation

Centrosomes undergo dramatic changes in composition and activity during cell cycle progression. Yet mechanisms involved in recruiting centrosomal proteins are poorly understood. Nek2 is a cell cycle-regulated protein kinase required for regulation of centrosome structure at the G2/M transition. Here, we have addressed the processes involved in trafficking of Nek2 to the centrosome of human adult cells. We find that Nek2 exists in small, highly dynamic cytoplasmic particles that move to and from the centrosome. Many of these particles align along microtubules and a motif was identified in the Nek2 C-terminal noncatalytic domain that allows both microtubule binding and centrosome localization. FRAP experiments reveal that 70% of centrosomal Nek2 is rapidly turned over (t(1/2) approximately 3 s). Microtubules facilitate Nek2 trafficking to the centrosome but only over long distances. Cytoplasmic Nek2 particles colocalize in part with PCM-1 containing centriolar satellites and depletion of PCM-1 interferes with centrosomal recruitment of Nek2 and its substrate C-Nap1. Finally, we show that proteasomal degradation is necessary to allow rapid recruitment of new Nek2 molecules to the centrosome. Together, these data highlight multiple processes involved in regulating the abundance of Nek2 kinase at the centrosome including microtubule binding, the centriolar satellite component PCM-1, and localized protein degradation.