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1.
JCI Insight ; 5(21)2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33001861

RESUMO

Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type-specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl- cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress-mediated progressive renal tubulointerstitial disease. These observations establish cell type-specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin.


Assuntos
Estresse do Retículo Endoplasmático , Epitélio/patologia , Nefropatias/patologia , Túbulos Renais/patologia , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Animais , Epitélio/metabolismo , Feminino , Nefropatias/etiologia , Nefropatias/metabolismo , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Resposta a Proteínas não Dobradas , Uromodulina/genética , Uromodulina/metabolismo
2.
J Cell Sci ; 130(16): 2696-2706, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28687623

RESUMO

Nonmuscle myosin IIB (NMIIB; heavy chain encoded by MYH10) is essential for cardiac myocyte cytokinesis. The role of NMIIB in other cardiac cells is not known. Here, we show that NMIIB is required in epicardial formation and functions to support myocardial proliferation and coronary vessel development. Ablation of NMIIB in epicardial cells results in disruption of epicardial integrity with a loss of E-cadherin at cell-cell junctions and a focal detachment of epicardial cells from the myocardium. NMIIB-knockout and blebbistatin-treated epicardial explants demonstrate impaired mesenchymal cell maturation during epicardial epithelial-mesenchymal transition. This is manifested by an impaired invasion of collagen gels by the epicardium-derived mesenchymal cells and the reorganization of the cytoskeletal structure. Although there is a marked decrease in the expression of mesenchymal genes, there is no change in Snail (also known as Snai1) or E-cadherin expression. Studies from epicardium-specific NMIIB-knockout mice confirm the importance of NMIIB for epicardial integrity and epicardial functions in promoting cardiac myocyte proliferation and coronary vessel formation during heart development. Our findings provide a novel mechanism linking epicardial formation and epicardial function to the activity of the cytoplasmic motor protein NMIIB.


Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Pericárdio/citologia , Pericárdio/embriologia , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Coração/embriologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/genética , Organogênese/genética
3.
Mol Biol Cell ; 28(14): 1924-1936, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28592632

RESUMO

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration.


Assuntos
Movimento Celular/fisiologia , Miosina não Muscular Tipo IIB/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Actinas/fisiologia , Actomiosina/fisiologia , Antígeno CD146/metabolismo , Antígeno CD146/fisiologia , Polaridade Celular/fisiologia , Miosinas , Miosina não Muscular Tipo IIB/fisiologia , Proteínas Wnt , Proteína Wnt-5a
4.
Cell Biol Int ; 41(6): 586-598, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28328100

RESUMO

At the early stage of atherosclerosis, neointima is formed due to the migration of vascular smooth muscle cells (VSMCs) from the media to the intima. VSMCs are surrounded by highly adhesive 3D matrices. They take specific strategies to cross various 3D matrices in the media, including heterogeneous collagen and mechanically strong basement membrane. Migration of VSMCs is potentially caused by biomechanical mechanism. Most in vitro studies focus on cell migration on 2D substrates in response to biochemical factors. How the cells move through 3D matrices under the action of mechanosensing machineries remains unexplored. In this review, we propose that several interesting tension-dependent machineries act as "tractor"-posterior myosin II accumulation, and "wrecker"-anterior podosome maintaining, to power VSMCs ahead. VSMCs embedded in 3D matrices may accumulate a minor myosin II isoform, myosin IIB, at the cell rear. Anisotropic myosin IIB distribution creates cell rear, polarizes cell body, pushes the nucleus and reshapes the cell body, and cooperates with a uniformly distributed myosin IIA to propel the cell forward. On the other hand, matrix digestion by podosome further promote the migration when the matrix becomes denser. Actomyosin tension activates Src to induce podosome in soft 3D matrices and retain the podosome integrity to steadily digest the matrix.


Assuntos
Polaridade Celular/fisiologia , Mecanotransdução Celular/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Aterosclerose/fisiopatologia , Linhagem Celular , Movimento Celular/fisiologia , Células Cultivadas , Colágeno , Humanos , Miócitos de Músculo Liso , Neointima/fisiopatologia , Miosina não Muscular Tipo IIB/metabolismo , Miosina não Muscular Tipo IIB/fisiologia
5.
Mol Biol Cell ; 28(2): 240-251, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27881665

RESUMO

The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells.


Assuntos
Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIA/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Imagem Molecular , Contração Muscular/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/metabolismo , Miosina não Muscular Tipo IIB/fisiologia , Imagem Óptica , Fosforilação , Domínios Proteicos , Quinases Associadas a rho/metabolismo
6.
Oncotarget ; 7(30): 47586-47592, 2016 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-27285763

RESUMO

Despite many advances in the treatment of breast cancer, it remains one of the leading causes of death among women. One hurdle for effective therapy is the treatment of the highly invasive and tumorigenic subpopulation of tumors called cancer stem cells (CSCs). CSCs, when stimulated with EGF, migrate through a physiological 3D collagen matrix at a higher velocity than non-stem cancer cells (non-SCCs). This increased invasion is due, in part, by an enhanced nuclear translocation ability of CSCs. We observed no difference between CSC and non-SCC in cellular migration rates on a 2D surface. Furthermore, during transwell migration using large diameter transwell pores, both CSC and non-SCC populations migrated with similar efficiency. However, when challenged with more restrictive transwells, CSCs were dramatically more capable of transwell migration. These results implicate nuclear translocation as a major rate limiting factor for CSC dissemination. We further show that non-muscle myosin IIB is critical for this enhanced nuclear translocation and the ability for cancer stem cells to efficiently migrate through restrictive 3D environments. These studies suggest that cytoskeletal elements upregulated in CSCs, such as myosin IIB, may be valuable targets for intervention in cancer stem cell dispersal from tumors.


Assuntos
Núcleo Celular/metabolismo , Células-Tronco Neoplásicas/patologia , Miosina não Muscular Tipo IIB/fisiologia , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Movimento Celular , Humanos , Invasividade Neoplásica
7.
J Biol Chem ; 290(42): 25395-410, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26330558

RESUMO

N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity.


Assuntos
Actinas/fisiologia , Plasticidade Neuronal , Miosina não Muscular Tipo IIB/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Ativação Enzimática , Hipocampo/fisiologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Proteína Quinase C/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley
8.
J Cell Biol ; 210(4): 583-94, 2015 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-26261182

RESUMO

Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing.


Assuntos
Movimento Celular , Núcleo Celular/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo
9.
J Cell Biol ; 209(1): 11-2, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25869662

RESUMO

A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell's rear. This motif represents a new paradigm for what determines directional cell migration.


Assuntos
Polaridade Celular , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Animais , Humanos
10.
J Cell Biol ; 209(1): 23-32, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25869664

RESUMO

In this study, we show that the role of nonmuscle myosin II (NMII)-B in front-back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935-1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B-like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front-back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.


Assuntos
Polaridade Celular , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Actomiosina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Adesão Celular , Movimento Celular , Cricetinae , Cricetulus , Células HEK293 , Humanos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Miosina não Muscular Tipo IIB/química , Estabilidade Proteica , Estrutura Terciária de Proteína
11.
Dev Biol ; 397(1): 103-15, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25446029

RESUMO

During brain morphogenesis, the neuroepithelium must fold in specific regions to delineate functional units, and give rise to conserved embryonic brain shape. Individual cell shape changes are the basis for the morphogenetic events that occur during whole tissue shaping. We used the zebrafish to study the molecular mechanisms that regulate the first fold in the vertebrate brain, the highly conserved midbrain-hindbrain boundary (MHB). Since the contractile state of the neuroepithelium is tightly regulated by non-muscle myosin II (NMII) activity, we tested the role of NMIIA and NMIIB in regulating cell shape changes that occur during MHB morphogenesis. Using morpholino knockdown, we show that NMIIA and NMIIB are both required for normal MHB tissue angle. Quantification of cell shapes revealed that NMIIA is required for the shortening of cells specifically at the MHB constriction (MHBC), while NMIIB is required for the proper width of cells throughout the MHB region. NMIIA and NMIIB knockdown also correlated with abnormal distribution of actin within the cells of the MHBC. Thus, NMIIA and NMIIB perform distinct functions in regulating cell shape during MHB morphogenesis.


Assuntos
Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Miosina não Muscular Tipo IIA/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Actinas/fisiologia , Animais , Encéfalo/fisiologia , Forma Celular , Perfilação da Expressão Gênica , Morfogênese , Contração Muscular , Cadeias Pesadas de Miosina/fisiologia
12.
J Am Soc Nephrol ; 26(5): 1081-91, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25168025

RESUMO

The kidney develops from reciprocal interactions between the metanephric mesenchyme and ureteric bud. The mesenchyme transforms into epithelia and forms complicated nephron structures, whereas the ureteric bud extends its pre-existing epithelial ducts. Although the roles are well established for extracellular stimuli, such as Wnt and Notch, it is unclear how the intracellular cytoskeleton regulates these morphogenetic processes. Myh9 and Myh10 encode nonmuscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases and focal segmental glomerulosclerosis in adults. Here, we analyzed the roles of Myh9 and Myh10 in the developing kidney. Ureteric bud-specific depletion of Myh9 resulted in no apparent phenotypes, whereas mesenchyme-specific Myh9 deletion caused proximal tubule dilations and renal failure. Mesenchyme-specific Myh9/Myh10 mutant mice died shortly after birth and showed a severe defect in nephron formation. The nascent mutant nephrons failed to form a continuous lumen, which likely resulted from impaired apical constriction of the elongating tubules. In addition, nephron progenitors lacking Myh9/Myh10 or the possible interactor Kif26b were less condensed at midgestation and reduced at birth. Taken together, nonmuscle myosin II regulates the morphogenesis of immature nephrons derived from the metanephric mesenchyme and the maintenance of nephron progenitors. Our data also suggest that Myh9 deletion in mice results in failure to maintain renal tubules but not in glomerulosclerosis.


Assuntos
Morfogênese , Cadeias Pesadas de Miosina/fisiologia , Néfrons/embriologia , Miosina não Muscular Tipo IIA/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Animais , Animais Recém-Nascidos , Mesoderma/fisiologia , Camundongos Endogâmicos C57BL , Néfrons/metabolismo , Isoformas de Proteínas/metabolismo
13.
Cell Mol Life Sci ; 70(1): 1-21, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22565821

RESUMO

Members of the nonmuscle myosin-2 (NM-2) family of actin-based molecular motors catalyze the conversion of chemical energy into directed movement and force thereby acting as central regulatory components of the eukaryotic cytoskeleton. By cyclically interacting with adenosine triphosphate and F-actin, NM-2 isoforms promote cytoskeletal force generation in established cellular processes like cell migration, shape changes, adhesion dynamics, endo- and exo-cytosis, and cytokinesis. Novel functions of the NM-2 family members in autophagy and viral infection are emerging, making NM-2 isoforms regulators of nearly all cellular processes that require the spatiotemporal organization of cytoskeletal scaffolding. Here, we assess current views about the role of NM-2 isoforms in these activities including the tight regulation of NM-2 assembly and activation through phosphorylation and how NM-2-mediated changes in cytoskeletal dynamics and mechanics affect cell physiological functions in health and disease.


Assuntos
Modelos Biológicos , Miosina não Muscular Tipo IIA/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Animais , Transporte Biológico , Adesão Celular , Movimento Celular , Citocinese , Endocitose , Exocitose , Humanos , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Transdução de Sinais , Internalização do Vírus
14.
Dev Neurobiol ; 73(5): 333-53, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22949126

RESUMO

In response to a wound, astrocytes in culture extend microtubule-rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. ß1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in ß1 integrin null astrocytes. Expression of a ß1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric ß1 subunit that can be activated by an antibody assemble centrosomes suggesting that ß1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non-muscle myosin II (NMII), mimics the response of ß1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin-mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity.


Assuntos
Astrócitos/ultraestrutura , Centrossomo/ultraestrutura , Integrina beta1/fisiologia , Cicatrização/fisiologia , Animais , Adesão Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Polaridade Celular , Células Cultivadas/fisiologia , Embrião de Galinha , DNA Complementar/genética , Matriz Extracelular/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Integrina beta1/biossíntese , Integrina beta1/genética , Camundongos , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Miosina não Muscular Tipo IIB/fisiologia , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/fisiologia , Retina/citologia , Retina/embriologia , Suspensões
15.
Front Biosci (Landmark Ed) ; 17(2): 545-55, 2012 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-22201759

RESUMO

Nonmuscle myosin II-B (NM II-B) plays an important role in cardiac development and function. Genetic ablation of NM II-B in mice results in both cellular and structural defects involving cardiac myocytes. These abnormalities include a ventricular septal defect, double outlet of the right ventricle, myocyte hypertrophy and premature onset of myocyte binucleation due to abnormalities in cytokinesis. The mice die by embryonic day (E) 14.5 due to defects in heart development. Conditional ablation of NM II-B in cardiac myocytes after E11.5 allows study of NM II-B function in adult myocytes. BaMHC/BaMHC mice are born with enlarged cardiac myocytes, some of which are multinucleated. Between 6-10 months of age they develop a cardiomyopathy. Many of these mice develop a marked widening of the intercalated discs. The loss of NM II-B from the intercalated discs primarily affects the adhesion junctions rather than the gap junctions and desmosomes. Interestingly, the loss of NM II-B results in a decrease in the actin binding protein mXin which also has been shown to cause disruption of the intercalated disc in addition to cardiac arrhythmias (Gustafson-Wagner et al. Am J Physiol Heart Circ Physiol. 2007, 293:H2680-92). Finally we review the evidence showing that ablation of NM II-C (which also localizes to the intercalated disc) in mouse hearts deficient in NM II-B expression results in destabilization of N-cadherin and beta-catenin in the intercalated disc.


Assuntos
Coração Fetal/embriologia , Coração Fetal/fisiologia , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/fisiologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Modelos Moleculares , Miocárdio/metabolismo , Miosina não Muscular Tipo IIB/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia
16.
J Neuroimmunol ; 237(1-2): 101-5, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21737147

RESUMO

The molecular mechanisms that underlie the axonal damage that accompanies CNS inflammation are largely unknown. Here, we investigate the effects of immune cells on neuronal viability and axonal growth and show that conditioned media from myeloid lineage cells inhibit neurite outgrowth without causing apoptosis. Treatment with monocyte conditioned medium enhances myosin light chain phosphorylation in neurons and the neurite outgrowth inhibitory effect of myeloid lineage cells can be attenuated with the myosin II inhibitor blebbistatin. Our results suggest that in the context of CNS inflammation myeloid cells may limit axonal repair in the CNS via a myosin II-dependent mechanism.


Assuntos
Diferenciação Celular/imunologia , Inibidores do Crescimento/metabolismo , Células Mieloides/metabolismo , Neuritos/imunologia , Miosina não Muscular Tipo IIB/fisiologia , Adulto , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/patologia , Linhagem da Célula/imunologia , Células Cultivadas , Inibidores do Crescimento/fisiologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Microglia/metabolismo , Microglia/patologia , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/imunologia , Células Mieloides/patologia , Neuritos/patologia , Ratos , Ratos Sprague-Dawley
17.
Mol Cell Biol ; 31(10): 2162-70, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21402784

RESUMO

Genome-wide association studies linked single-nucleotide polymorphisms (SNPs) at the MYH9 locus to chronic kidney disease among African-Americans, particularly glomerular diseases such as HIV nephropathy and idiopathic focal and segmental glomerulosclerosis (FSGS). However, these MYH9 SNPs are intronic, and despite extensive sequencing, a causal variant remains elusive. To investigate the role of MYH9 in kidney disease, we selectively deleted Myh9 from mouse podocytes and found that mutant C57BL/6 mice did not develop renal insufficiency or proteinuria compared to control littermates, even when the mice were aged for 9 months. To explain the surprisingly normal phenotype, we considered genetic redundancy with the paralog Myh10 in podocytes, but we found that Myh10 was not expressed in podocytes in Myh9-deficient or control mice. We tested whether Myh9 podocyte deletion predisposed mice to glomerulopathy in response to injury by doxorubicin hydrochloride (Adriamycin), and we found that Myh9 podocyte-deleted mice developed proteinuria and glomerulosclerosis, while control mice were resistant. In summary, Myh9 podocyte deletion in C57BL/6 mice results in susceptibility to experimental doxorubicin hydrochloride glomerulopathy. We review evidence that MYH9 dysfunction in humans results in similar susceptibility and place our data, the first examination of Myh9 kidney disease in experimental animals, in the context of recent findings in human kidney disease, including the role of APOL1.


Assuntos
Nefropatias/genética , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/fisiologia , Podócitos , Animais , Apolipoproteína L1 , Apolipoproteínas/genética , Linhagem Celular , Doxorrubicina/administração & dosagem , Predisposição Genética para Doença , Glomerulosclerose Segmentar e Focal/genética , Humanos , Nefropatias/induzido quimicamente , Lipoproteínas HDL/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/fisiologia , Proteinúria/genética , Deleção de Sequência
18.
Nucleic Acids Res ; 39(12): 5098-108, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21398640

RESUMO

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.


Assuntos
Actinas/fisiologia , DNA Mitocondrial/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina não Muscular Tipo IIA/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Actinas/análise , Actinas/antagonistas & inibidores , Animais , Células Cultivadas , DNA Mitocondrial/química , DNA Mitocondrial/isolamento & purificação , Inativação Gênica , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/isolamento & purificação , Cadeias Pesadas de Miosina/antagonistas & inibidores , Miosina não Muscular Tipo IIA/análise , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Ratos
19.
J Neurophysiol ; 105(5): 1966-76, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21325687

RESUMO

The synaptic vesicle population in a nerve terminal is traditionally divided into subpopulations according to physiological criteria; the readily releasable pool (RRP), the recycling pool, and the reserve pool. It is recognized that the RRP subserves synaptic transmission evoked by low-frequency neural activity and that the recycling and reserve populations are called on to supply vesicles as neural activity increases. Here we investigated the contribution of nonmuscle myosin II (NMMII) to synaptic transmission with emphasis on the role a motor protein could play in the supply of vesicles. We used Drosophila genetics to manipulate NMMII and assessed synaptic transmission at the larval neuromuscular junction. We observed a positive correlation between synaptic strength at low-frequency stimulation and NMMII expression: reducing NMMII reduced the evoked response, while increasing NMMII increased the evoked response. Further, we found that NMMII contributed to the spontaneous release of vesicles differentially from evoked release, suggesting differential contribution to these two release mechanisms. By measuring synaptic responses under conditions of differing external calcium concentration in saline, we found that NMMII is important for normal synaptic transmission under high-frequency stimulation. This research identifies diverse functions for NMMII in synaptic transmission and suggests that this motor protein is an active contributor to the physiology of synaptic vesicle recruitment.


Assuntos
Proteínas de Drosophila/fisiologia , Junção Neuromuscular/fisiologia , Plasticidade Neuronal/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Potenciais Sinápticos/fisiologia , Vesículas Sinápticas/fisiologia
20.
J Hepatol ; 54(1): 132-41, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20932596

RESUMO

BACKGROUND & AIMS: Upon liver injury, hepatic stellate cells (HSCs) undergo dramatic morphological and functional changes including migration and contraction. In the present study, we investigated the role of myosin II isoforms in the development of the contractile phenotype of mouse HSCs, which are considered therapeutic targets to decrease portal hypertension and fibrosis. METHODS: We characterized the expression of myosin IIA and IIB in primary mouse HSCs and addressed their function by gene knock-down using isoform-specific siRNAs. RESULTS: We found that myosin IIA and IIB are differentially expressed and localized and have clearly different functions in HSCs. Myosin IIA is mainly located in the subcortical area of quiescent HSCs and at α-SMA-containing stress fibres after activation, while myosin IIB is located in the cytoplasm and at the edge of protrusions of quiescent HSCs, at stress fibres of activated cells, and at the leading edge of lamellipodia. Knock-down of myosin IIA in HSCs influences cell size and shape, results in the disruption of stress fibres and in a decrease of focal adhesions, and inhibits contractility and intra-cellular Ca(2+) release but increases cell migration. Myosin IIB contributes to the extension of lamellipodia and cell spreading but has no direct role in stress fibres and focal adhesion formation, contraction, or intra-cellular Ca(2+) signalling. CONCLUSIONS: In mouse HSCs, myosin IIA and IIB clearly fulfil distinct roles. Our results provide an insight into the contractile machinery of HSCs, that could be important in the search for new molecules to treat portal hypertension.


Assuntos
Células Estreladas do Fígado/fisiologia , Miosina não Muscular Tipo IIA/fisiologia , Miosina não Muscular Tipo IIB/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/fisiologia , Retículo Endoplasmático/fisiologia , Endotelina-1/farmacologia , Adesões Focais/fisiologia , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Motores Moleculares/antagonistas & inibidores , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/fisiologia , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Miosina não Muscular Tipo IIB/genética , Pseudópodes/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Fibras de Estresse/fisiologia , Vinculina/metabolismo
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