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1.
Mucosal Immunol ; 11(2): 415-426, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28832026

RESUMO

Barrier dysfunction has been implicated in the pathophysiology of eosinophilic esophagitis (EoE). Transforming growth factor-ß1 (TGF-ß1), a potent pleiotropic molecule, is increased in EoE; however, no study has evaluated its influence on esophageal epithelial barrier. We hypothesized that TGF-ß1 regulates barrier dysfunction in EoE. We aimed to determine the role of TGF-ß1 in the epithelial barrier in models of EoE. To examine the impact of TGF-ß1 on esophageal barrier, immortalized human esophageal epithelial (EPC2-hTERT) cells were exposed to TGF-ß1 during the three-dimensional air-liquid interface (3D-ALI) model in vitro. TGF-ß1 exposure diminished EPC2-hTERT barrier function as measured by transepithelial electrical resistance (TEER) and 3 kDa Fluorescein isothiocyanate dextran paracellular flux (FITC Flux), and hematoxylin and eosin (H&E) assessment revealed prominent cellular separation. In analysis of epithelial barrier molecules, TGF-ß1 led to the specific reduction in expression of the tight-junction molecule, claudin-7 (CLDN7), and this was prevented by TGF-ß-receptor I inhibitor. Short hairpin ribonucleic acid (shRNA)-mediated CLDN7 knockdown diminished epithelial barrier function, whereas CLDN7 overexpression resulted in protection from TGF-ß1-mediated barrier dysfunction. In pediatric EoE biopsies CLDN7 expression was decreased and altered localization was observed with immunofluorescence analysis, and the TGF-ß1 downstream transcription factor, phosphorylated SMAD2/3 (pSMAD2/3), was increased. Our data suggest that TGF-ß1 participates in esophageal epithelial barrier dysfunction through CLDN7 dysregulation.


Assuntos
Claudinas/metabolismo , Esofagite Eosinofílica/imunologia , Eosinófilos/imunologia , Células Epiteliais/fisiologia , Esôfago/patologia , Junções Íntimas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Biópsia , Técnicas de Cultura de Células , Células Cultivadas , Criança , Claudinas/genética , Regulação para Baixo , Impedância Elétrica , Células Epiteliais/patologia , Humanos , RNA Interferente Pequeno/genética
2.
Methods Cell Biol ; 131: 177-97, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26794514

RESUMO

We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture.


Assuntos
MicroRNAs/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Plasmídeos/genética , Células de Purkinje/citologia , Transfecção/métodos , Animais , Sinalização do Cálcio/genética , Técnicas de Cultura de Células , Células Cultivadas , Imunofluorescência , Técnicas de Silenciamento de Genes , Perda Auditiva Neurossensorial/genética , Camundongos , Piebaldismo/genética , Transtornos da Pigmentação/genética , Regiões Promotoras Genéticas/genética
3.
Anal Biochem ; 308(2): 314-9, 2002 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12419345

RESUMO

Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [14C]N-acetylaspartate (NAA) from L-[14C]Asp; (b) use of [14C]NAA as substrate in the assay; and (c) separation and quantitation of the product L-[14C]Asp using a TLC system. This method can detect as low as 10pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.


Assuntos
Amidoidrolases/metabolismo , Ácido Aspártico/análogos & derivados , Oligodendroglia/enzimologia , Animais , Ácido Aspártico/síntese química , Encéfalo/enzimologia , Células Cultivadas , Cromatografia em Camada Fina , Glutamatos/síntese química , Radiometria , Ratos , Espectrofotometria
4.
J Cell Biol ; 155(6): 893-8, 2001 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-11733546

RESUMO

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia-axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553-562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Neurônios/metabolismo , Animais , Axônios/química , Axônios/metabolismo , Células COS , Técnicas de Cocultura , Gânglios Espinais/citologia , Proteínas Associadas aos Microtúbulos/análise , Glicoproteína Associada a Mielina/análise , Glicoproteína Associada a Mielina/genética , Neuroglia/metabolismo , Neurônios/química , Neurônios/ultraestrutura , Fosforilação , Ligação Proteica/fisiologia , Ratos , Transfecção
5.
J Cell Biol ; 155(5): 703-4, 2001 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-11724811

RESUMO

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.


Assuntos
Miosina Tipo I/classificação , Terminologia como Assunto , Animais , Humanos , Miosina Tipo I/genética
6.
Mol Biol Cell ; 12(9): 2742-55, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11553713

RESUMO

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages.


Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Fagossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo , Tamanho Celular , Células Cultivadas , Galinhas , Citosol/metabolismo , Deleção de Genes , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Mutantes , Proteínas dos Microfilamentos/isolamento & purificação , Microscopia de Fluorescência , Microesferas , Peso Molecular , Movimento (Física) , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/isolamento & purificação , Miosina Tipo V/química , Miosina Tipo V/isolamento & purificação , Fagossomos/química , Fenótipo , Ligação Proteica
7.
J Cell Biol ; 153(7): 1479-97, 2001 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-11425877

RESUMO

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.


Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto , Proteínas dos Microfilamentos/metabolismo , Miosinas/metabolismo , Domínios de Homologia de src/fisiologia , Fatores de Despolimerização de Actina , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Animais , Sítios de Ligação/fisiologia , Caenorhabditis elegans , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Estruturas da Membrana Celular/fisiologia , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Clonagem Molecular , Destrina , Dictyostelium , Drosophila , Endocitose/fisiologia , Humanos , Substâncias Macromoleculares , Camundongos , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos
8.
J Cell Biol ; 152(4): 835-42, 2001 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-11266473

RESUMO

Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti-CD3-coated beads. However, rapid anti-CD3-induced granule secretion was drastically defective in both CD8(+) and CD4(+) T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor-stimulated interferon-gamma secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.


Assuntos
Exocitose , Imunoconjugados , Células Matadoras Naturais/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4 , Citotoxicidade Imunológica , Interferon gama/metabolismo , Camundongos , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/citologia , Timo/citologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP
9.
J Cell Sci ; 114(Pt 6): 1091-100, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11228153

RESUMO

The peripheral accumulation of melanosomes characteristic of wild-type mouse melanocytes is driven by a cooperative process involving long-range, bidirectional, microtubule-dependent movements coupled to capture and local movement in the actin-rich periphery by myosin Va, the product of the dilute locus. Genetic evidence suggests that Rab27a, the product of the ashen locus, functions with myosin Va in this process. Here we show that ashen melanocytes, like dilute melanocytes, exhibit normal dendritic morphology and melanosome biogenesis, an abnormal accumulation of end-stage melanosomes in the cell center, and rapid, bidirectional, microtubule-dependent melanosome movements between the cell center and the periphery. This phenotype suggests that ashen melanocytes, like dilute melanocytes, are defective in peripheral melanosome capture. Consistent with this, introduction into ashen melanocytes of cDNAs encoding wild-type and GTP-bound versions of Rab27a restores the peripheral accumulation of melanosomes in a microtubule-dependent manner. Conversely, introduction into wild-type melanocytes of the GDP-bound version of Rab27a generates an ashen/dilute phenotype. Rab27a colocalizes with end-stage melanosomes in wild-type cells, and is most concentrated in melanosome-rich dendritic tips, where it also colocalizes with myosin Va. Finally, neither endogenous myosin Va nor an expressed, GFP-tagged, myosin Va tail domain fusion protein colocalize with melanosomes in ashen melanocytes, in contrast to that seen previously in wild-type cells. These results argue that Rab27a serves to enable the myosinVa-dependent capture of melanosomes delivered to the periphery by bidirectional, microtubule-dependent transport, and that it does so by recruiting the myosin to the melanosome surface. We suggest that Rab27a, in its GTP-bound and melanosome-associated form, predominates in the periphery, and that it is this form that recruits the myosin, enabling capture. These results argue that Rab27a serves as a myosin Va 'receptor', and add to the growing evidence that Rab GTPases regulate vesicle motors as well as SNARE pairing.


Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Cadeias Pesadas de Miosina , Miosina Tipo V , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Citoesqueleto , Dendritos/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Melanócitos/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Organelas , Fenótipo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP
10.
J Neurochem ; 76(6): 1925-34, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11259511

RESUMO

Treatment of cultured rat oligodendroglial progenitors with either platelet-derived growth factor (PDGF) or fibroblast growth factor-2 (FGF-2) activated extracellular signal regulated kinase 2 (ERK2). Activation was transient in response to PDGF, whereas it was greater and more prolonged in response to FGF-2. ERK2 activation by PDGF was preceded by a very rapid, robust and transient tyrosine phosphorylation of the PDGF receptor. Although there was consistently more activation of ERK2 in response to FGF-2 than to PDGF, immunostaining of FGF receptors 1 (FGFR1) and 2 (FGFR2) and their tyrosine phosphorylation in progenitors was very weak, and both receptors were up-regulated during differentiation to oligodendrocytes. Tyrosine phosphorylation of the FGF receptors was maximal from 15 to 60 min of treatment and was sustained for many hours. Binding of radioiodinated FGF-2 to FGFR1 was predominant in progenitors, whereas binding to FGFR2 was predominant in oligodendrocytes. ERK2 activation by PDGF was more sensitive to inhibition of tyrosine kinases, whereas ERK2 activation by FGF-2 was relatively more sensitive to inhibitors of protein kinase C. These differences in signal transduction pathways probably contribute to the different cellular responses of oligodendroglial lineage cells to PDGF and FGF-2, respectively.


Assuntos
Encéfalo/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Oligodendroglia/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Diferenciação Celular , Células Cultivadas , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Fosforilação , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
11.
Appl Opt ; 40(24): 4243-53, 2001 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-18360462

RESUMO

The performance of adaptive systems that consist of microscale on-chip elements [microelectromechanical mirror (mu-mirror) arrays and a VLSI stochastic gradient descent microelectronic control system] is analyzed. The mu-mirror arrays with 5 x 5 and 6 x 6 actuators were driven with a control system composed of two mixed-mode VLSI chips implementing model-free beam-quality metric optimization by the stochastic parallel perturbative gradient descent technique. The adaptation rate achieved was near 6000 iterations/s. A secondary (learning) feedback loop was used to control system parameters during the adaptation process, further increasing the adaptation rate.

12.
Pigment Cell Res ; 13(4): 241-7, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10952391

RESUMO

Molecular motors drive most if not all organelle movements in Eukaryotic cells. These proteins are thought to bind to the organelle surface and, through the action of their mechanochemical domains, to translocate the organelle along a cytoskeletal track. In the case of the myosin family of molecular motors, the cytoskeletal track is filamentous actin. Microtubules serve as the cytoskeletal track for the kinesins and dyneins. While a considerable amount is known about the motors and tracks responsible for the bi-directional movement of pigment granules in fish and frog melanophores, relatively little is known about how melanosomes in mammalian melanocytes are transported out the cells dendritic arbor, accumulated at the ends of these dendrites, and transferred to keratinocytes. In this short review, we focus on the use of video microscopy to address these questions in mouse melanocytes, and we describe how an analysis of melanosome dynamics within wild type and dilute melanocytes shaped our thinking regarding the role of an unconventional myosin in melanosome transport and distribution.


Assuntos
Proteínas de Filamentos Intermediários/fisiologia , Melanócitos/ultraestrutura , Melanossomas/fisiologia , Proteínas Motores Moleculares/fisiologia , Cadeias Pesadas de Miosina , Miosina Tipo V , Animais , Transporte Biológico , Células Cultivadas , Melanócitos/citologia , Melanossomas/ultraestrutura , Camundongos , Microscopia de Vídeo , Movimento
13.
Nature ; 405(6788): 804-7, 2000 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10866203

RESUMO

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.


Assuntos
Actinas/metabolismo , Miosinas/metabolismo , Actinas/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Miosinas/ultraestrutura , Ligação Proteica
14.
Curr Opin Cell Biol ; 12(1): 42-51, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10679359

RESUMO

To date, fourteen classes of unconventional myosins have been identified. Recent reports have implicated a number of these myosins in organelle transport, and in the formation, maintenance and/or dynamics of actin-rich structures involved in a variety of cellular processes including endocytosis, cell migration, and sensory transduction. Characterizations of organelle dynamics in pigment cells and neurons have further defined the contributions made by unconventional myosins and microtubule motors to the transport and distribution of organelles. Several studies have provided evidence of complexes through which cooperative organelle transport may be coordinated. Finally, the myosin superfamily has been shown to contain at least one processive motor and one backwards motor.


Assuntos
Miosinas/metabolismo , Actinas/metabolismo , Animais , Transporte Biológico , Dineínas/metabolismo , Endocitose , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Neoplasias , Organelas/metabolismo , Transdução de Sinais
15.
J Biol Chem ; 275(6): 4329-35, 2000 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-10660602

RESUMO

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.


Assuntos
Difosfato de Adenosina/farmacologia , Proteínas de Ligação a Calmodulina/química , Miosina Tipo V , Proteínas do Tecido Nervoso/química , Actinas/química , Animais , Encéfalo/metabolismo , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Calmodulina/farmacologia , Proteínas de Ligação a Calmodulina/genética , Ativação Enzimática , Fluorescência , Cinética , Camundongos , Microscopia Eletrônica , Subfragmentos de Miosina/química , Subfragmentos de Miosina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Concentração Osmolar , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Pirenos , Proteínas Recombinantes/química
16.
Biochemistry ; 38(22): 7243-52, 1999 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10353836

RESUMO

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.


Assuntos
Acanthamoeba/química , Citoesqueleto de Actina/química , Subfragmentos de Miosina/química , Acanthamoeba/genética , Acanthamoeba/ultraestrutura , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Animais , Birrefringência , Magnésio/química , Microscopia Eletrônica , Dados de Sequência Molecular , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/ultraestrutura , Mutação Puntual , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Ultracentrifugação
17.
Proc Natl Acad Sci U S A ; 95(26): 15200-5, 1998 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-9860946

RESUMO

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.


Assuntos
Acanthamoeba/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Miosinas/química , Miosinas/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , Spodoptera , Transfecção
18.
Proc Natl Acad Sci U S A ; 95(26): 15206-11, 1998 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-9860947

RESUMO

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)-AMIA, AMIB, and AMIC-are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin's actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-A resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31 degrees on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998) J. Cell Biol. 141, 155-162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.


Assuntos
Acanthamoeba/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Miosinas/química , Miosinas/metabolismo , Estrutura Secundária de Proteína , Actinas/ultraestrutura , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Miosinas/ultraestrutura , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Serina , Spodoptera , Transfecção
19.
Cell Motil Cytoskeleton ; 40(3): 286-303, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9678671

RESUMO

Class V unconventional myosins are two-headed, nonfilamentous, actin-based mechanoenzymes that appear to be expressed ubiquitously. Mice possess at least two myosin V heavy chain genes (dilute and myr6) whose approximately 190 kDa protein products are referred to as myosin Va and Vb, respectively. Using antibodies that are specific for the Va isoform and immunofluorescence microscopy, we show here that myosin Va localizes to the microtubule organizing center (MTOC) in interphase cells, and to the mitotic asters, spindle, and midbody of dividing cells. These associations, which in the case of mitotic cells are characterized by the concentration of myosin Va in the immediate vicinity of the microtubules, were observed in a variety of cell types, including primary and immortal mouse melanocytes and fibroblasts, Hela cells, and Cos cells. Importantly, these associations were not observed in melanocytes and fibroblasts cultured from dilute null mice, indicating that the staining of these microtubule-rich domains was due to the presence of myosin Va, as opposed to another protein(s) containing a shared epitope(s) with myosin Va. When cells were extracted with detergent prior to fixation, myosin Va remained associated with each of these microtubule-rich domains, suggesting that these associations are not due to the possible presence of membranes at these sites. This fact, and our observation that these microtubule-rich domains contain little if any F-actin (based on phalloidin staining), suggest that myosin Va may bind to microtubules either directly or through a microtubule-associated protein. Finally, we found that dilute null fibroblasts in primary culture are twice as likely to be binucleate as wild type fibroblasts of the same genetic background (35% vs. 17%). Together, these results indicate that myosin Va associates with microtubule-rich domains in both interphase and dividing cells, and plays a role in the efficiency of cell division in culture.


Assuntos
Interfase/fisiologia , Melanócitos/química , Microtúbulos/química , Miosinas/análise , Células 3T3 , Animais , Células COS , Divisão Celular/fisiologia , Núcleo Celular , Centrossomo/química , Células HeLa , Humanos , Melanócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Mitose/fisiologia , Cadeias Pesadas de Miosina/análise , Células Tumorais Cultivadas
20.
J Muscle Res Cell Motil ; 18(5): 517-27, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9350005

RESUMO

Mice with mutations at the dilute locus, which encodes the heavy chain of a type V unconventional myosin, exhibit a reduction in coat colour intensity. This defect is thought to be caused by the absence in dilute melanocytes of the extensive dendritic arbor through which these cells normally deliver pigment-laden melanosomes to keratinocytes. The data on which this conclusion has been based can also be explained, however, by a defect in the outward transport of melanosomes within melanocytes of normal shape. To resolve this question, we compared the shape and pigment distribution within melanocytes present in primary cultures prepared from the epidermis of C57BL/6J pups that were either wild type (D/D) at dilute or homozygous for the dilute null allele d120J. These same comparisons were also performed on melanocytes in situ, where antibodies to the membrane tyrosine kinase receptor cKIT were used to visualize melanocyte cell shape independent of pigment distribution. Wild type melanocytes were found to be dendritic and to have melanosomes distributed throughout their dendrites both in vitro and in situ. Mutant melanocytes were also found to be dendritic in both cases, but their melanosomes were highly concentrated in the cell body and largely excluded from dendrites. We conclude, therefore, that the predominant defect in dilute melanocytes is in melanosome distribution, not cell shape. These results argue that the myosin V isoform encoded by the dilute locus functions in dendritic extensions to move melanosomes from their site of formation within the cell body to their site of intercellular transfer at dendritic tips. This conclusion is consistent with our recent demonstration by immunolocalization that the dilute myosin V isoform associates with melanosomes in mouse melanocytes.


Assuntos
Células Epidérmicas , Epiderme/fisiologia , Melanócitos/citologia , Melanócitos/fisiologia , Miosinas/fisiologia , Animais , Animais Recém-Nascidos , Tamanho Celular , Células Cultivadas , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pigmentos Biológicos/análise
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