scholarly journals Aurora A activates D-TACC–Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

2005 ◽  
Vol 170 (7) ◽  
pp. 1039-1046 ◽  
Author(s):  
Teresa P. Barros ◽  
Kazuhisa Kinoshita ◽  
Anthony A. Hyman ◽  
Jordan W. Raff
Keyword(s):  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Mutant Form ◽  
Dependent Manner ◽  
Aurora A ◽  
Animal Cells

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A–dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC–Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.

scholarly journals p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

2007 ◽  
Vol 18 (10) ◽  
pp. 3741-3751 ◽  
Author(s):  
Kiyoko Ogawa-Goto ◽  
Keiko Tanaka ◽  
Tomonori Ueno ◽  
Keisuke Tanaka ◽  
Takeshi Kurata ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Specific Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

TheS. pombeaurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation

2001 ◽  
Vol 114 (24) ◽  
pp. 4371-4384 ◽  
Author(s):  
Janni Petersen ◽  
Jeannie Paris ◽  
Martin Willer ◽  
Michel Philippe ◽  
Iain M. Hagan
Keyword(s):  
Fission Yeast ◽  
Histone H3 ◽  
Central Zone ◽  
Chromosome Condensation ◽  
Yeast Cells ◽  
Aurora B ◽  
Dependent Manner ◽  
Aurora A ◽  
Spindle Formation

Metazoans contain three aurora-related kinases. Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis. Less is known about the function of aurora C. S. pombe contains a single aurora-related kinase, Ark1. Although Ark1 protein levels remained constant as cells progressed through the mitotic cell cycle, its distribution altered during mitosis and meiosis. Throughout G2 Ark1 was concentrated in one to three nuclear foci that were not associated with the spindle pole body/centromere complex. Following commitment to mitosis Ark1 associated with chromatin and was particularly concentrated at several sites including kinetochores/centromeres. Kinetochore/centromere association diminished during anaphase A, after which it was distributed along the spindle. The protein became restricted to a small central zone that transiently enlarged as the spindle extended. As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells. A number of studies have linked this modification with chromosome condensation. Ark1 immuno-precipitates phosphorylated serine 10 of histone H3 in vitro. This activity was highest in mitotic extracts. The absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1+ gene had been deleted (ark1.Δ1); the inability of these cells to resolve their chromosomes during anaphase and the co-localisation of this phospho-epitope with Ark1 early in mitosis, all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo. ark1.Δ1 cells also exhibited a reduction in kinetochore activity and a minor defect in spindle formation. Thus the enzyme activity, localisation and phenotype arising from our manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems.

scholarly journals Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP

2004 ◽  
Vol 15 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Shin-ichi Kawaguchi ◽  
Yixian Zheng
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Biochemical Studies ◽  
Ring Complex ◽  
Centrosome Protein

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

scholarly journals In Vitro Inhibitory Mechanism Effect of TRAIP on the Function of TRAF2 Revealed by Characterization of Interaction Domains

2018 ◽  
Vol 19 (8) ◽  
pp. 2457 ◽  
Author(s):  
Eijaz Bhat ◽  
Chang Kim ◽  
Sunghwan Kim ◽  
Hyun Park
Keyword(s):  
Coiled Coil ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Inhibitory Mechanism ◽  
Concentration Dependent Manner ◽  
Critical Function

TRAF-interacting protein (TRAIP), a negative regulator of TNF-induced-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, inhibits adaptor protein TRAF2 by direct interaction and is critical in apoptosis, cell proliferation, antiviral response, and embryonic development. Although the critical function of TRAIP in NF-κB signaling is well-known, the molecular inhibitory mechanism of TRAIP remains unclear. We found that the TRAIP coiled-coil domain altered its stoichiometry between dimer and trimer in a concentration-dependent manner. Additionally, the TRAIP RING domain induced even higher-ordered assembly, which was necessary for interacting with the TRAF-N domain of TRAF2 but not TRAF1. Characterization of the TRAF-N domains of TRAF1 and TRAF2, the tentative TRAIP-binding region of TRAFs, suggested the molecular basis of the inhibitory effect of TRAIP on TRAF2 in NF-κB signaling.

scholarly journals Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

2021 ◽  
Vol 22 (8) ◽  
pp. 4036
Author(s):  
Małgorzata Śliwinska ◽  
Katarzyna Robaszkiewicz ◽  
Piotr Wasąg ◽  
Joanna Moraczewska
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thin Filament ◽  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Troponin Complex ◽  
Increased Sensitivity

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca2+-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca2+, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca2+]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca2+]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca2+-dependent regulation of actin–myosin interactions.

The tyrosine phosphatase CD148 interacts with the p85 regulatory subunit of phosphoinositide 3-kinase

2008 ◽  
Vol 413 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Nobuo Tsuboi ◽  
Tadahiko Utsunomiya ◽  
Richard L. Roberts ◽  
Hideyuki Ito ◽  
Keiko Takahashi ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Growth Factor Receptor ◽  
Regulatory Subunit ◽  
Mutant Form ◽  
Dependent Manner ◽  
Screening Assay ◽  
Intact Cells ◽  
Pi3k Activity ◽  
Phosphoinositide 3 Kinase

CD148 is a transmembrane tyrosine phosphatase that has been implicated in the regulation of cell growth and transformation. However, the signalling mechanisms of CD148 are incompletely understood. To identify the specific intracellular molecules involved in CD148 signalling, we carried out a modified yeast two-hybrid screening assay. Using the substrate-trapping mutant form of CD148 (CD148 D/A) as bait, we recovered the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase). CD148 D/A, but not catalytically active CD148, interacted with p85 in a phosphorylation-dependent manner in vitro and in intact cells. Growth factor receptor and PI3K activity were also trapped by CD148 D/A via p85 from pervanadate-treated cell lysates. CD148 prominently and specifically dephosphorylated p85 in vitro. Co-expression of CD148 reduced p85 phosphorylation induced by active Src, and attenuated the increases in PI3K activity, yet CD148 did not alter the basal PI3K activity. Finally, CD148 knock-down by siRNA (short interfering RNA) increased PI3K activity on serum stimulation. Taken together, these results demonstrate that CD148 may interact with and dephosphorylate p85 when it is phosphorylated and modulate the magnitude of PI3K activity.

scholarly journals dCHD3, a Novel ATP-Dependent Chromatin Remodeler Associated with Sites of Active Transcription

2008 ◽  
Vol 28 (8) ◽  
pp. 2745-2757 ◽  
Author(s):  
Magdalena Murawska ◽  
Natascha Kunert ◽  
Joke van Vugt ◽  
Gernot Längst ◽  
Elisabeth Kremmer ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Protein ◽  
Polytene Chromosomes ◽  
Dependent Manner ◽  
Chromatin Remodelers ◽  
Active Transcription ◽  
Nucleosome Binding

ABSTRACT ATP-dependent chromatin remodelers of the CHD family play important roles during differentiation and development. Three CHD proteins, dMi-2, dChd1, and Kismet, have been described for Drosophila melanogaster. Here, we study dCHD3, a novel member of the CHD family. dCHD3 is related in sequence to dMi-2 but lacks several domains implicated in dMi-2 function. We demonstrate that dCHD3 is a nuclear protein and that expression is tightly regulated during fly development. Recombinant dCHD3 remodels mono- and polynucleosomes in an ATP-dependent manner in vitro. Its chromodomains are critical for nucleosome binding and remodeling. Unlike dMi-2, dCHD3 exists as a monomer. Nevertheless, both proteins colocalize with RNA polymerase II to actively transcribed regions on polytene chromosomes, suggesting that both remodelers participate in the process of transcription.

scholarly journals Kinetic analysis of transcellular passage of the cobalamin–transcobalamin complex in Caco-2 monolayers

2019 ◽  
Vol 30 (4) ◽  
pp. 467-477 ◽  
Author(s):  
Christian B. Juul ◽  
Sergey N. Fedosov ◽  
Ebba Nexo ◽  
Christian W. Heegaard
Keyword(s):  
Dissociation Constants ◽  
Cellular Transport ◽  
Dependent Manner ◽  
Animal Cells ◽  
Receptor Associated Protein ◽  
Ligand Free ◽  
Apparent Concentration ◽  
Ligand Interactions ◽  
Specific Receptors

We suggest a novel kinetic approach to quantifying receptor–ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [57Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC–Cbl ( Kd = 13 nM) and RAP ( Kd = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.

scholarly journals Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells

2021 ◽  
Vol 12 ◽  
pp. 204173142097832
Author(s):  
Yoann Torres ◽  
Maude Gluais ◽  
Nicolas Da Silva ◽  
Sylvie Rey ◽  
Agathe Grémare ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Species Differences ◽  
Human Fibroblasts ◽  
Growth Dynamics ◽  
Large Animal ◽  
Dependent Manner ◽  
Tissue Architecture ◽  
Animal Cells

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

scholarly journals A novel RAB11-containing adaptor complex anchoring myosin-5 to secretory vesicles

2021 ◽  
Author(s):  
Mario Pinar ◽  
Ana Alonso ◽  
Vivian de los Ríos ◽  
Ignacio Bravo-Plaza ◽  
Álvaro Gandara ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Analytical Ultracentrifugation ◽  
Coiled Coil ◽  
Shotgun Proteomics ◽  
Secretory Vesicles ◽  
Dependent Manner ◽  
Apical Dome ◽  
Basal Septum ◽  
Dependent Transport

AbstractHyphal fungi grow rapidly by apical extension, providing a notorious example of polarized growth. The continuous supply of secretory vesicles necessary to meet the demands of the extending tip and the long intracellular distances existing between the tip and the basal septum, often localized > 100 µm away from the former, impose the need of efficient networks of intracellular traffic involving exquisite cooperation between microtubule- and actin-mediated transport. In Aspergillus nidulans kinesin-1 conveys secretory vesicles to the hyphal tip, where they are transferred to myosin-5, which focuses them at the growing apex, thereby determining cell shape. This relay mechanism and the central role played by myosin-5 in hyphal morphogenesis suggested that the mechanisms anchoring secretory vesicles to this motor should involve specific adaptor(s) ensuring the robustness of actomyosin-dependent transport.Secretory vesicles are charged with RAB11, a regulatory GTPase that determines the Golgi to post-Golgi identity transition. By using a combination of shotgun proteomics, GST-RAB pull-down assays, in vitro reconstitution experiments, targeted reverse genetics and multidimensional fluorescence microscopy with endogenously tagged proteins we show that RAB11, the master regulator of fungal exocytosis, mediates myosin-5 engagement both by contacting the motor and by recruiting UDS1, a homologue of an as yet uncharacterized Schizosaccharomyces protein ‘upregulated during mitosis’, which we demonstrate to be a novel RAB11 effector. Analytical ultracentrifugation determined that UDS1 is an elongated dimer and negative-stain electron microscopy showed that, in agreement, UDS1 is rod-shaped. UDS1 does not contact myosin-5 directly, but rather recruits the coiled-coil HMSV, which bridges RAB11/UDS1 to myosin-5. An HMSV-scaffolded complex containing UDS1 and myosin-5 is present in cells, and a RAB11-UDS1-HMSV complex can be reconstituted in vitro in a RAB nucleotide state-dependent manner. In the absence of UDS1/HMSV the steady state levels of myosin-5 at the apical vesicle supply center diminish markedly, such that microtubule-dependent transport spreading vesicles across the apical dome predominates over apex-focused actin-mediated transport. As a consequence, RAB11 and chitin-synthase B (a cargo of the RAB11 pathway) are not focused at the apex, being distributed instead across the apical dome. Therefore, the RAB11 effector UDS1/HMSV cooperates with the GTPase to adapt secretory vesicles to myosin-5, which is required for the apical targeting of RAB11 cargoes and thus for the normal morphology of the hyphae.

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