scholarly journals Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

2002 ◽  
Vol 156 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Toshiro Ohta ◽  
Russell Essner ◽  
Jung-Hwa Ryu ◽  
Robert E. Palazzo ◽  
Yumi Uetake ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Amino Acid Residues ◽  
Protein Overexpression ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Centrosomal Protein ◽  
Wide Range ◽  
Spindle Microtubules

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

scholarly journals Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry

2018 ◽  
Vol 29 (26) ◽  
pp. 3105-3118 ◽  
Author(s):  
Felix Bärenz ◽  
Yvonne T. Kschonsak ◽  
Annalena Meyer ◽  
Aliakbar Jafarpour ◽  
Holger Lorenz ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Microtubule Stability ◽  
Coiled Coil Domain ◽  
Intrinsic Symmetry ◽  
Spindle Microtubules

Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

Cloning, expression and characterization of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase of adult Haemonchus contortus†

2010 ◽  
Vol 85 (4) ◽  
pp. 421-429 ◽  
Author(s):  
K. Han ◽  
L. Xu ◽  
R. Yan ◽  
X. Song ◽  
X. Li
Keyword(s):  
Enzymatic Activity ◽  
Haemonchus Contortus ◽  
Expressed Sequence Tag ◽  
Sequence Similarity ◽  
Full Length ◽  
Amino Acid Residues ◽  
Gapdh Gene ◽  
Wide Range ◽  
Rapid Amplification

AbstractGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) regulates a wide range of biological processes, including pathogen evasion. In the present research, the GAPDH gene of Haemonchus contortus (HcGAPDH) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, AW670737) to amplify the 3′ and 5′ ends of HcGAPDH. The full length of cDNA from this gene was obtained by overlapping the sequences of 3′ and 5′ extremities and amplification by reverse transcription polymerase chain reaction (RT-PCR). The biochemical activities of the recombinant protein HcGAPDH, which was expressed in prokaryotic cells and purified by affinity chromatography, were analysed by assays of enzymatic activity, thermal stability and pH. The results showed that the cloned full-length cDNA comprised 1303 bp and encoded a peptide with 341 amino acid residues which showed sequence similarity to several known GAPDHs. The biochemical assay showed that the protein encoded by the HcGAPDH exhibited enzymatic activity with NAD+ as a cofactor. HcGAPDH was stable between pH 5 and 9 and maintained activity at high temperatures of up to 75°C. The natural GAPDH of Haemonchus contortus detected by immunoblot assay was approximately 38 kDa in size, and the recombinant HcGAPDH was recognized strongly by serum from naturally infected goats.

scholarly journals High-resolution structures of a heterochiral coiled coil

2015 ◽  
Vol 112 (43) ◽  
pp. 13144-13149 ◽  
Author(s):  
David E. Mortenson ◽  
Jay D. Steinkruger ◽  
Dale F. Kreitler ◽  
Dominic V. Perroni ◽  
Gregory P. Sorenson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Information ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Polypeptide Chains ◽  
Packing Interactions

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein–protein interactions. Coiled–coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled–coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

scholarly journals A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface

IUCrJ ◽  
2020 ◽  
Vol 7 (3) ◽  
pp. 462-479 ◽  
Author(s):  
Danielle Karoline Silva do Vale Castro ◽  
Sabrina Matos de Oliveira da Silva ◽  
Humberto D'Muniz Pereira ◽  
Joci Neuby Alves Macedo ◽  
Diego Antonio Leonardo ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Coiled Coil ◽  
Amino Acid Residues ◽  
Binding Domains ◽  
Cellular Events ◽  
Gtp Binding ◽  
Wide Range ◽  
Specific Subgroup ◽  
Order Complexes ◽  
Polybasic Region

Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

scholarly journals Interchromosomal Crossover in Human Cells Is Associated with Long Gene Conversion Tracts

2007 ◽  
Vol 27 (15) ◽  
pp. 5261-5274 ◽  
Author(s):  
Efrem A. H. Neuwirth ◽  
Masamitsu Honma ◽  
Andrew J. Grosovsky
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Human Cells ◽  
Lymphoblastoid Cell Lines ◽  
Analysis Methodology ◽  
Wide Range ◽  
Specific Association ◽  
Gene Conversions ◽  
Conversion Tract

ABSTRACT Crossovers have rarely been observed in specific association with interchromosomal gene conversion in mammalian cells. In this investigation two isogenic human B-lymphoblastoid cell lines, TI-112 and TSCER2, were used to select for I-SceI-induced gene conversions that restored function at the selectable thymidine kinase locus. Additionally, a haplotype linkage analysis methodology enabled the rigorous detection of all crossover-associated convertants, whether or not they exhibited loss of heterozygosity. This methodology also permitted characterization of conversion tract length and structure. In TI-112, gene conversion tracts were required to be complex in tract structure and at least 7.0 kb in order to be selectable. The results demonstrated that 85% (39/46) of TI-112 convertants extended more than 11.2 kb and 48% also exhibited a crossover, suggesting a mechanistic link between long tracts and crossover. In contrast, continuous tracts as short as 98 bp are selectable in TSCER2, although selectable gene conversion tracts could include a wide range of lengths. Indeed, only 16% (14/95) of TSCER2 convertants were crossover associated, further suggesting a link between long tracts and crossover. Overall, these results demonstrate that gene conversion tracts can be long in human cells and that crossovers are observable when long tracts are recoverable.

Overexpression of normal and mutant Arp1alpha (centractin) differentially affects microtubule organization during mitosis and interphase

1999 ◽  
Vol 112 (20) ◽  
pp. 3507-3518 ◽  
Author(s):  
I.B. Clark ◽  
D.I. Meyer
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Dynactin Complex

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin's most abundant component, Arp1alpha was transiently overexpressed in mammalian cells. Arp1alpha overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1alpha in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1alpha that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1alpha proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1alpha, NuMA localization remained unaffected. Although expression of mutant Arp1alpha proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1alpha interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1alpha mutants. We conclude that the amino acids altered in the Arp1alpha mutant proteins participate in stabilizing interactions between overexpressed Arp1alpha and components of the endogenous dynactin complex as well as the NuMA protein.

scholarly journals Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

1995 ◽  
Vol 129 (4) ◽  
pp. 1049-1059 ◽  
Author(s):  
R Kuriyama ◽  
M Kofron ◽  
R Essner ◽  
T Kato ◽  
S Dragas-Granoic ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Amino Acid Residues ◽  
Central Stalk ◽  
Vitro Analysis ◽  
Kinesin Superfamily ◽  
In Vitro Analysis

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

scholarly journals Prophase Microtubule Arrays Undergo Flux-like Behavior in Mammalian Cells

2007 ◽  
Vol 18 (10) ◽  
pp. 3993-4002 ◽  
Author(s):  
Nick P. Ferenz ◽  
Patricia Wadsworth
Keyword(s):  
Mammalian Cells ◽  
Marked Reduction ◽  
Fluorescent Protein ◽  
Model Systems ◽  
Microtubule Organization ◽  
Spindle Microtubule ◽  
Nuclear Envelope Breakdown ◽  
Wide Range ◽  
Green Fluorescent ◽  
Poleward Flux

In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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