Role of a novel coiled-coil domain-containing protein CCDC69 in regulating central spindle assembly

The formation of the central spindle (or the spindle midzone) is essential for cytokinesis in animal cells. In this study, we report that coiled-coil domain-containing protein 69 (CCDC69) is implicated in controlling the assembly of central spindles and the recruitment of midzone components. Exogenous expression of CCDC69 in HeLa cells interfered with microtubule polymerization and disrupted the formation of bipolar mitotic spindles. Endogenous CCDC69 proteins were localized to the central spindle during anaphase. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and disrupted the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and polo-like kinase 1 (Plk1) at the central spindle. Aurora B kinase was found to bind to CCDC69 and this binding depended on the coiled-coil domains at the C-terminus of CCDC69. Further, disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Our results indicate that CCDC69 acts as a scaffold to regulate the recruitment of midzone components and the assembly of central spindles.     

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.     

BubR1 is a spindle assembly checkpoint protein regulating meiotic cell cycle progression of mouse oocyte

BubR1 (Bub1-related kinase or MAD3/Bub1b) is an essential component of the spindle assembly checkpoint (SAC) and plays an important role in kinetochore localization of other spindle checkpoint proteins in mitosis. But its roles in mammalian oocyte meiosis are unclear. In the present study, we examined the expression, localization and function of BubR1 during mouse oocyte meiotic maturation. The expression level of BubR1 increased progressively from germinal vesicle to metaphase II stages. Immunofluorescent analysis showed that BubR1 localized to kinetochores from the germinal vesicle breakdown to the prometaphase I stages, co-localizing with polo-like kinase 1, while it disappeared from the kinetochores at the metaphase I stage. Spindle disruption by nocodazole treatment caused relocation of BubR1 to kinetochores at metaphase I, anaphase I and metaphase II stages; spindle microtubules were disrupted by low temperature treatment in the BubR1-depleted oocytes in meiosis I, suggesting that BubR1 monitors kinetochore-microtubule (K-MT) attachments. Over-expression of exogenous BubR1 arrested oocyte meiosis maturation at the M I stage or earlier; in contrast, dominant-negative BubR1 and BubR1 depletion accelerated meiotic progression. In the BubR1-depleted oocytes, higher percentage of chromosome misalignment was observed and more oocytes overrode the M I stage arrest induced by low concentration of nocodazole. Our data suggest that BubR1 is a spindle assembly checkpoint protein regulating meiotic progression of oocytes.     

Xenopus marginal coil (Xmc), a novel FGF inducible cytosolic coiled-coil protein regulating gastrulation movements

Gastrulation in vertebrates is a highly dynamic process driven by convergent extension movements of internal mesodermal cells, under the regulatory activity of the Spemann-Mangold or gastrula organizer. In a large-scale screen for genes expressed in the organizer, we have isolated a novel gene, termed Xmc, an acronym for Xenopus marginal coil. Xmc encodes a protein containing two widely spaced evolutionarily non-conserved coiled coils. Xmc protein is found in vesicular aggregates in the cytoplasm and associated with the inner plasma membrane. We show that Xmc is expressed in a dynamic fashion around the blastoporal circumference, in mesodermal cells undergoing morphogenetic movements, in a pattern similar to FGF target genes. Likewise, Xmc expression can be induced by ectopic XeFGF signaling and the early mesodermal expression is dependent on FGF receptor-mediated signaling. Morpholino-mediated translational 'knock-down' of Xmc results in embryos that display a reduced elongation of the antero-posterior axis and in a pronounced inhibition of morphogenetic movements in embryos and dorsal marginal zone explants. Xmc loss-of-function does not interfere with mesoderm induction or maintenance per se. Our results suggest that Xmc is a novel FGF target gene that is required for morphogenetic movements during gastrulation in Xenopus.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.     

Characterization of KIBRA,a novel WW domain-containing protein

In a yeast two hybrid screen with the human isoform of Dendrin (KIAA0749), a putative modulator of the postsynaptic cytoskeleton, we isolated a cDNA coding for a novel protein, KIBRA, possessing two amino-terminal WW domains, an internal C2-like domain and a carboxy-terminal glutamic acid-rich stretch. Northern blot analysis revealed that the expression of KIBRA mRNA was predominately found in kidney and brain. In vitro interaction studies revealed that the first KIBRA WW domain binds specifically to PPxY motifs. Transient transfection of monkey kidney cells with constructs encoding Myc-tagged KIBRA displayed a cytoplasmic localization and a perinuclear enrichment of the protein.     

Pilt is a coiled-coil domain-containing protein that localizes at the trans-Golgi complex and regulates its structure

Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure.

Structured summary of protein interactions

GM130 and Piltcolocalize by fluorescence microscopy (View interaction)Arf6(Q67L)physically interacts with Pilt by two hybrid (View Interaction: 1, 2)Piltphysically interacts with Arf6(Q67L) by pull down (View interaction)     

Arabidopsis At5g39790 encodes a chloroplast-localized,carbohydrate-binding,coiled-coil domain-containing putative scaffold protein

Background  

Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain.     

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana . The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.     

Regulation of coiled-coil assembly in tropomyosins

Tropomyosins (TMs) are a family of actin filament-binding proteins. They consist of nearly 100% alpha-helix and assemble into parallel coiled-coil dimers. In vertebrates, TMs are encoded by four genes that give rise to at least 17 distinct isoforms through the use of alternative RNA splicing and alternative promoters. We have studied various aspects of the coiled-coil interactions among muscle and nonmuscle isoforms by the use of transfection of epitope-tagged constructs, followed by immunoprecipitation, SDS-PAGE, and Western blot analyses. For coiled-coil interactions between high-molecular-weight isoforms (284 amino acids), the information for homo- versus heterodimerization is contained in large part within the alternatively spliced exons of nonmuscle and muscle (skeletal and smooth) isoforms. Furthermore, sequences located in alternatively spliced exons encoding amino acids 39-80 (exons 2a/2b), amino acids 189-213 (exons 6a/6b), and amino acids 258-284 (exons 9a/9d) are critical for the selective formation of homo- versus heterodimers. Among low-molecular-weight isoforms (248 amino acids), TM-4 and TM-5 can form either homodimers or heterodimers. The trigger sequence (amino acids 190-202) is required for homodimerization of TM-4, but not heterodimerization of TM-4 with TM-5. How the dimeric state of TMs might play a role in their cellular localization and function is discussed.     

WAC, a novel WW domain-containing adapter with a coiled-coil region, is colocalized with splicing factor SC35

WW domains mediate protein-protein interactions in many intracellular processes. In pre-mRNA splicing, WW domains participate in cross-intron bridging. These WW domains are characterized by a central aromatic block of three tyrosine residues. We identified a novel protein containing the same type of WW domain. The gene encoding the protein, named WAC, is located in human chromosome 10p11.2-10p12.1. A Drosophila melanogaster WAC homolog (CG8949) was identified as a Rosetta stone protein. Domain fusion analysis of the Rosetta stone protein linked WAC to splicing factor SNRP70. WAC existed mainly in a tyrosine-phosphorylated form. Immunofluorescence analysis colocalized WAC with SC35, the marker for pre-mRNA splicing machinery. Our analysis suggests that WAC represents a novel member of WW-domain-containing proteins for RNA processing.     

中枢血管紧张素对心血管活动调节作用

血管紧张素(Ang)广泛存在于中枢神经系统和外周组织中,对心血管活动和交感神经活动起重要调节作用。本文介绍了孤束核(NTS)、延髓头端腹侧区(RVLM)、延髓尾端腹侧区(CVLM)和室旁核(PVN)内Ang对心血管活动的影响,Ang对动脉压力感受性反射(ABR)和心交感传入反射(CSAR)的调节作用,肾素-血管紧张素系统的基因敲除研究,以及Ang与高血压和慢性心力衰竭的关系。     

Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.     

ICA69 is a novel Rab2 effector regulating ER-Golgi trafficking in insulinoma cells

Islet cell autoantigen of 69kDa (ICA69) is a small GTPase-binding protein of unknown function. ICA69 is enriched in the Golgi complex and its N-terminal half contains a BAR domain, a module that can bind/bend membranes and interacts with phospholipids. Here we show that in insulinoma INS-1 cells ICA69 binds to the small GTPase Rab2, which regulates the transport of COPI vesicles between the endoplasmic reticulum and the Golgi complex. Rab2 binds to ICA69 in a GTP-dependent fashion and recruits it to membranes. Over-expression of either Rab2 or ICA69 in INS-1 cells results in a phenotype characterized by: (i) impaired anterograde transport of the secretory granule protein precursors pro-ICA512 and chromogranin A; (ii) reduction of stimulated insulin secretion. Taken together, these data identify ICA69 as a novel Rab2 effector and point to its role in regulating the early transport of insulin secretory granule proteins.     

Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod-Zw10-Zwilch complex (RZZ) from aligned kinetochores (Howell, B.J., B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, and E.D. Salmon. 2001. J. Cell Biol. 155:1159-1172; Wojcik, E., R. Basto, M. Serr, F. Scaerou, R. Karess, and T. Hays. 2001. Nat. Cell Biol. 3:1001-1007). Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores.     

The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.     

Regulation of polysome assembly on the endoplasmic reticulum by a coiled-coil protein, p180

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.     

Developmental regulation of central spindle assembly and cytokinesis during vertebrate embryogenesis

Mitosis and cytokinesis not only ensure the proper segregation of genetic information but also contribute importantly to morphogenesis in embryos. Cytokinesis is controlled by the central spindle, a microtubule-based structure containing numerous microtubule motors and microtubule-binding proteins, including PRC1. We show here that central spindle assembly and function differ dramatically between two related populations of epithelial cells in developing vertebrate embryos examined in vivo. Compared to epidermal cells, early neural epithelial cells undergo exaggerated anaphase chromosome separation, rapid furrowing, and a marked reduction of microtubule density in the spindle midzone. Cytokinesis in normal early neural epithelial cells thus resembles that in cultured vertebrate cells experimentally depleted of PRC1. We find that PRC1 mRNA and protein expression is surprisingly dynamic in early vertebrate embryos and that neural-plate cells contain less PRC1 than do epidermal cells. Expression of excess PRC1 ameliorates both the exaggerated anaphase and reduced midzone microtubule density observed in early neural epithelial cells. These PRC1-mediated modifications to the cytokinetic mechanism may be related to the specialization of the midbody in neural cells. These data suggest that PRC1 is a dose-dependent regulator of the central spindle in vertebrate embryos and demonstrate unexpected plasticity to fundamental mechanisms of cell division.