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1.
Nat Commun ; 9(1): 4600, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389913

RESUMO

Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.


Assuntos
Matriz Extracelular/metabolismo , Pneumopatias/metabolismo , Cadeias Pesadas de Miosina/deficiência , Miosina não Muscular Tipo IIB/deficiência , Sequência de Aminoácidos , Animais , Regulação para Baixo/genética , Enfisema/patologia , Etilnitrosoureia , Feminino , Pneumopatias/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Mutagênese/genética , Mutação de Sentido Incorreto/genética , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/química , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Organogênese , Fenótipo , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/metabolismo , Regulação para Cima/genética
2.
Glia ; 62(4): 580-91, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24470341

RESUMO

The oligodendrocyte (OL), the myelinating cell of the central nervous system, undergoes dramatic changes in the organization of its cytoskeleton as it differentiates from a precursor (oligodendrocyte precursor cells) to a myelin-forming cell. These changes include an increase in its branching cell processes, a phenomenon necessary for OL to myelinate multiple axon segments. We have previously shown that levels and activity of non-muscle myosin II (NMII), a regulator of cytoskeletal contractility, decrease as a function of differentiation and that inhibition of NMII increases branching and myelination of OL in coculture with neurons. We have also found that mixed glial cell cultures derived from NMIIB knockout mice display an increase in mature myelin basic protein-expressing OL compared with wild-type cultures. We have now extended our studies to investigate the role of NMIIB ablation on myelin repair following focal demyelination by lysolecithin. To this end, we generated an oligodendrocyte-specific inducible knockout model using a Plp-driven promoter in combination with a temporally activated CRE-ER fusion protein. Our data indicate that conditional ablation of NMII in adult mouse brain, expedites lesion resolution and remyelination by Plp+ oligodendrocyte-lineage cells when compared with that observed in control brains. Taken together, these data validate the function of NMII as that of a negative regulator of OL myelination in vivo and provide a novel target for promoting myelin repair in conditions such as multiple sclerosis.


Assuntos
Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/fisiopatologia , Regeneração Nervosa/fisiologia , Miosina não Muscular Tipo IIB/deficiência , Animais , Antígenos/metabolismo , Proteínas Relacionadas à Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Corpo Caloso/patologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/genética , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Lisofosfatidilcolinas , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/metabolismo , Miosina não Muscular Tipo IIB/genética , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/patologia , Proteoglicanas/metabolismo
3.
Dev Biol ; 369(2): 356-61, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22820068

RESUMO

Cytokinesis, the final stage of cell division, bisects the cytoplasm into two daughter cells. In mitotic cells, this process depends on the activity of non-muscle myosin II (NMII), a family of actin-binding motor-proteins that participate in the formation of the cleavage furrow. The relevance of NMII for meiotic cell division, however, is poorly understood. The NMII family consists of three members, NMIIA, NMIIB, and NMIIC, containing different myosin heavy chains (MYH9, MYH10, and MYH14, respectively). We find that a single non-muscle myosin II, NMIIB, is required for meiotic cytokinesis in male but not female mice. Specifically, NMIIB-deficient spermatocytes exhibit cytokinetic failure in meiosis I, resulting in bi-nucleated secondary spermatocytes. Additionally, cytokinetic failure at meiosis II gives rise to bi-nucleated or even tetra-nucleated spermatids. These multi-nucleated spermatids fail to undergo normal differentiation, leading to male infertility. In spite of the presence of multiple non-muscle myosin II isoforms, we demonstrate that a single member, NMIIB, plays an essential and non-redundant role in cytokinesis during meiotic cell divisions of the male germline.


Assuntos
Citocinese/fisiologia , Meiose/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Espermatogênese/fisiologia , Animais , Divisão Celular/genética , Divisão Celular/fisiologia , Citocinese/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/deficiência , Miosina não Muscular Tipo IIB/genética , Espermátides/metabolismo , Espermátides/ultraestrutura , Espermatócitos/metabolismo , Espermatócitos/ultraestrutura , Espermatogênese/genética , Espermatogônias/metabolismo , Espermatogônias/ultraestrutura , Testículo/citologia , Testículo/metabolismo
4.
Dev Dyn ; 237(12): 3577-90, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18697221

RESUMO

Flectin, a protein previously described to be expressed in a left-dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC-II) protein class. During looping, both NMHC-IIA and NMHC-IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC-IIB, rather than NMHC-IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full-length human NMHC-IIA and -IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC-IIB antibody does not. Abnormally looping hearts of the Nodal(Delta 600) homozygous mouse embryos show decreased NMHC-IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC-II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping.


Assuntos
Coração/embriologia , Miocárdio/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Humanos , Camundongos , Camundongos Knockout , Proteína Nodal/genética , Proteína Nodal/metabolismo , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/deficiência , Miosina não Muscular Tipo IIB/genética , Ligação Proteica , Proteômica , RNA Mensageiro/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Mol Biol Cell ; 18(3): 1009-17, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17202408

RESUMO

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.


Assuntos
Miosina não Muscular Tipo IIB/química , Miosina não Muscular Tipo IIB/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Forma Celular , Citoesqueleto/metabolismo , Fibroblastos/citologia , Genes Dominantes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Subfragmentos de Miosina/química , Miosina não Muscular Tipo IIB/deficiência , Fenótipo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade
6.
J Physiol ; 577(Pt 3): 1033-42, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16973711

RESUMO

The functional significance of smooth muscle-specific h1-calponin up-regulation in the smooth muscle contractility of SM-B null mice was studied by generating double knockout mice lacking both h1-calponin and SM-B myosin. The double knockout mice appear healthy, reproduce well and do not show any smooth muscle pathology. Loss of h1-calponin in the SM-B null mice bladder resulted in increased maximal shortening velocity (V(max)) and steady-state force generation. The force dilatation pressure, which was decreased in the SM-B null mesenteric vessels, was restored to wild-type levels in the double knockout vessels. In contrast, the half-time to maximal constriction was significantly increased in the double knockout vessels similar to that of SM-B null mice and indicating decreased shortening velocity in the double knockout vessels. Biochemical analyses showed that there is a significant reduction in smooth muscle alpha-actin levels, whereas h-caldesmon levels are increased in the double knockout bladder and mesenteric vessels, suggesting that these changes may also partly contribute to the altered contractile function. Taken together, our studies suggest that up-regulation of h1-calponin in the SM-B null mice may be necessary to maintain a reduced level of cross-bridge cycling over time in the absence of SM-B myosin and play an important role in regulating the smooth muscle contraction.


Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Proteínas dos Microfilamentos/deficiência , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Músculo Liso/fisiologia , Miosina não Muscular Tipo IIB/deficiência , Bexiga Urinária/fisiologia , Vasoconstrição/fisiologia , Actinas/antagonistas & inibidores , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Feminino , Técnicas In Vitro , Masculino , Artérias Mesentéricas/anatomia & histologia , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Knockout , Músculo Liso/anatomia & histologia , Músculo Liso Vascular/anatomia & histologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Leves de Miosina/metabolismo , Isoformas de Proteínas/metabolismo , Bexiga Urinária/anatomia & histologia , Bexiga Urinária/metabolismo , Calponinas
7.
Mol Immunol ; 39(13): 783-9, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12617993

RESUMO

Methods for cell type specific targeted intracellular delivery of proteins in vivo remain limited. A murine monoclonal anti-dsDNA antibody, mAb 3E10, was selectively transported into skeletal muscle cells in vivo. The antibody bound a 200 kDa protein only found in lysates of skeletal muscle by Western blotting. The 200 kDa protein was purified from muscle lysate by antibody affinity chromatography and identified as the skeletal muscle specific heavy chain of myosin IIb by electrospray mass spectrometry. Antibody binding specificity for myosin IIb was demonstrated in Western blots by binding myosin in skeletal muscle lysates from mice null for myosin IId but not in mice null for myosin IIb. Myosin IIb is implicated in the specific targeting of mAb 3E10 to skeletal muscle.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Músculo Esquelético/imunologia , Miosina não Muscular Tipo IIB/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/administração & dosagem , Anticorpos Antinucleares/metabolismo , Anticorpos Monoclonais/farmacocinética , Especificidade de Anticorpos , Feminino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Miosina não Muscular Tipo IIB/deficiência , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Ratos , Distribuição Tecidual
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