RESUMEN
OBJECTIVES: Inappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress-induced senescence of nucleus pulposus (NP) cells. MATERIAL AND METHODS: Nucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co-immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF-A. In addition, the expression levels of p-RhoA/RhoA, ROCK1/2 and p-MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues. RESULTS: Compression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress-induced fibrotic phenotype mediated by MRTF-A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress-induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin. CONCLUSIONS: We for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.
Asunto(s)
Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Estrés Mecánico , Actinas/metabolismo , Actomiosina/metabolismo , Células Cultivadas , Senescencia Celular , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Metaloproteinasa 3 de la Matriz/metabolismo , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Núcleo Pulposo/citología , Núcleo Pulposo/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transactivadores/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.
Asunto(s)
Estimulantes del Sistema Nervioso Central/farmacología , Condicionamiento Clásico/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Consolidación de la Memoria/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , Metanfetamina/análogos & derivados , Metanfetamina/farmacología , Nicotina/farmacología , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Fármacos del Sistema Nervioso Periférico/farmacología , Animales , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Despite the use of adjuvant therapies, the cumulative proportion of live births remains at ~40%. Accumulating data show that low pregnancy rates, even in the presence of high fertility rates, are due to implantation failure. The present study aimed to identify and construct a profile of proteins that react with preimplantation factor (PIF) and to provide an understanding into the molecular mechanisms by which PIF promotes trophoblast invasion. Cytoplasmic proteins were immunoprecipitated with biotinlabeled synthetic PIF or intralipid and scrambled PIF (PIFscr). The protein profiles were analyzed using isobaric tags for relative and absolute quantification coupled with mass spectrometry. Immunoprecipitation and western blot analyses were used to assess the interactions between PIF and myosin heavy chain 10 (MYH10) and heat shock protein family D1. Small interfering RNAbased silencing was performed to examine the function of MYH10. In the results of the present study, 21 proteins were identified with interactions with PIF. The immunoprecipitation and western blot analyses revealed an interaction between PIF and MYH10. Silencing of the expression of MYH10 in HEC1B cells significantly attenuated cell migration and invasion capacities. These data support the conclusion that MYH10mediated cell migration and invasion act in conjunction with PIF to promote the trophoblast invasion procedure.
Asunto(s)
Implantación del Embrión/efectos de los fármacos , Péptidos/farmacología , Proteoma/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Células HEK293 , Humanos , Espectrometría de Masas , Cadenas Pesadas de Miosina/antagonistas & inhibidores , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Proteoma/efectos de los fármacos , Proteómica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 µM for SmM, 3.58 µM for NM2a, 2.30 µM for NM2b, and 1.57 µM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.
Asunto(s)
Proteínas Aviares/antagonistas & inhibidores , Proteínas Aviares/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/química , Miosinas del Músculo Liso/antagonistas & inhibidores , Miosinas del Músculo Liso/química , Sustitución de Aminoácidos , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos , Humanos , Ratones , Mutación Missense , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismoRESUMEN
Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB.
Asunto(s)
Adiponectina/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adiponectina/antagonistas & inhibidores , Animales , Transporte Biológico/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Ratones , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Relación Estructura-Actividad , Tiazolidinas/farmacologíaRESUMEN
Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories.
Asunto(s)
Estimulantes del Sistema Nervioso Central/farmacología , Espinas Dendríticas/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Memoria/efectos de los fármacos , Metanfetamina/farmacología , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Animales , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Femenino , Ratas , Recompensa , AutoadministraciónRESUMEN
Memories associated with drug use increase vulnerability to relapse in substance use disorder (SUD), and there are no pharmacotherapies for the prevention of relapse. Previously, we reported a promising finding that storage of memories associated with methamphetamine (METH), but not memories for fear or food reward, is vulnerable to disruption by actin depolymerization in the basolateral amygdala complex (BLC). However, actin is not a viable therapeutic target because of its numerous functions throughout the body. Here we report the discovery of a viable therapeutic target, nonmuscle myosin IIB (NMIIB), a molecular motor that supports memory by directly driving synaptic actin polymerization. A single intra-BLC treatment with Blebbistatin (Blebb), a small-molecule inhibitor of class II myosin isoforms, including NMIIB, produced a long-lasting disruption of context-induced drug seeking (at least 30 days). Further, postconsolidation genetic knockdown of Myh10, the heavy chain of the most highly expressed NMII in the BLC, was sufficient to produce METH-associated memory loss. Blebb was found to be highly brain penetrant. A single systemic injection of the compound selectively disrupted the storage of METH-associated memory and reversed the accompanying increase in BLC spine density. This effect was specific to METH-associated memory, as it had no effect on an auditory fear memory. The effect was also independent of retrieval, as METH-associated memory was disrupted 24 h after a single systemic injection of Blebb delivered in the home cage. Together, these results argue for the further development of small-molecule inhibitors of NMII as potential therapeutics for the prevention of SUD relapse triggered by drug associations.
Asunto(s)
Trastornos Relacionados con Anfetaminas/tratamiento farmacológico , Trastornos Relacionados con Anfetaminas/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Fármacos del Sistema Nervioso Central/administración & dosificación , Metanfetamina/administración & dosificación , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Trastornos Relacionados con Anfetaminas/patología , Amígdala del Cerebelo/metabolismo , Animales , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Comportamiento de Búsqueda de Drogas/fisiología , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Miedo/efectos de los fármacos , Miedo/fisiología , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Ratas , Prevención Secundaria , Autoadministración , Conducta Espacial/efectos de los fármacos , Conducta Espacial/fisiologíaRESUMEN
miR-200a has been implicated in the pathogenesis of meningiomas, one of the most common central nervous system tumors in humans. To identify how miR-200a contributes to meningioma pathogenesis at the molecular level, we used a comparative protein profiling approach using Gel-nanoLC-MS/MS and identified approximately 130 dysregulated proteins in miR-200a-overexpressing meningioma cells. Following the bioinformatic analysis to identify potential genes targeted by miR-200a, we focused on the non-muscle heavy chain IIb (NMHCIIb), and showed that miR-200a directly targeted NMHCIIb. Considering the key roles of NMHCIIb in cell division and cell migration, we aimed to identify whether miR-200a regulated these processes through NMHCIIb. We found that NMHCIIb overexpression partially rescued miR-200a-mediated inhibition of cell migration, as well as cell growth in vitro and in vivo. Moreover, siRNA-mediated silencing of NMHCIIb expression resulted in a similar migration phenotype in these cells and inhibited meningioma tumor growth in mice. Taken together, these results suggest that NMHCIIb might serve as a novel therapeutic target in meningiomas.
Asunto(s)
Neoplasias Meníngeas/patología , Meningioma/patología , MicroARNs/genética , Cadenas Pesadas de Miosina/genética , Miosina Tipo IIB no Muscular/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Meníngeas/genética , Meningioma/genética , Ratones , Ratones Desnudos , Cadenas Pesadas de Miosina/antagonistas & inhibidores , Cadenas Pesadas de Miosina/biosíntesis , Trasplante de Neoplasias , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño , Trasplante HeterólogoRESUMEN
The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²âº/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Citoesqueleto/fisiología , Hormonas de Invertebrados/metabolismo , Ovario/metabolismo , Palaemonidae/fisiología , Pigmentos Biológicos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Animales , Transporte Biológico/efectos de los fármacos , Brasil , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/fisiología , Extensiones de la Superficie Celular/ultraestructura , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/ultraestructura , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Dineínas/antagonistas & inhibidores , Dineínas/metabolismo , Femenino , Toxinas Marinas/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Miosinas/antagonistas & inhibidores , Miosinas/metabolismo , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/metabolismo , Oligopéptidos/metabolismo , Ovario/efectos de los fármacos , Ovario/ultraestructura , Palaemonidae/efectos de los fármacos , Palaemonidae/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Ácido Pirrolidona Carboxílico/análogos & derivados , Ácido Pirrolidona Carboxílico/metabolismo , Ríos , Moduladores de Tubulina/farmacologíaRESUMEN
Contraction of 3D collagen matrices by fibroblasts frequently is used as an in vitro model of wound closure. Different iterations of the model - all conventionally referred to as "contraction" - involve different morphological patterns. During floating matrix contraction, cells initially are round without stress fibers and subsequently undergo spreading. During stressed matrix contraction, cells initially are spread with stress fibers and subsequently undergo shortening. In the current studies, we used siRNA silencing of myosin IIA (MyoIIA) and myosin IIB (MyoIIB) to test the roles of myosin II isoforms in fibroblast interactions with 3D collagen matrices and collagen matrix contraction. We found that MyoIIA but not MyoIIB was required for cellular global inward contractile force, formation of actin stress fibers, and morphogenic cell clustering. Stressed matrix contraction required MyoIIA but not MyoIIB. Either MyoIIA or MyoIIB was sufficient for floating matrix contraction (FMC) stimulated by platelet-derived growth factor. Neither MyoIIA or MyoIIB was necessary for FMC stimulated by serum. Our findings suggest that myosin II-dependent motor mechanisms for collagen translocation during extracellular matrix remodeling differ depending on cell tension and growth factor stimulation.
Asunto(s)
Fibroblastos/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Fenómenos Biomecánicos , Adhesión Celular/fisiología , Agregación Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , ARN Interferente Pequeño/genética , Cicatrización de Heridas/fisiologíaRESUMEN
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.
Asunto(s)
Astrocitos/ultraestructura , Centrosoma/ultraestructura , Integrina beta1/fisiología , Cicatrización de Heridas/fisiología , Animales , Adhesión Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Polaridad Celular , Células Cultivadas/fisiología , Embrión de Pollo , ADN Complementario/genética , Matriz Extracelular/fisiología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Integrina beta1/biosíntesis , Integrina beta1/genética , Ratones , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/fisiología , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/fisiología , Retina/citología , Retina/embriología , SuspensionesRESUMEN
Recent evidence suggests that organization of the extracellular matrix (ECM) into aligned fibrils or fibril-like ECM topographies promotes rapid migration in fibroblasts. However, the mechanisms of cell migration that are altered by these changes in micro-environmental topography remain unknown. Here, using 1D fibrillar migration as a model system for oriented fibrillar 3D matrices, we find that fibroblast leading-edge dynamics are enhanced by 1D fibrillar micropatterns and demonstrate a dependence on the spatial positioning of cell adhesions. Although 1D, 2D and 3D matrix adhesions have similar assembly kinetics, both 1D and 3D adhesions are stabilized for prolonged periods, whereas both paxillin and vinculin show slower turnover rates in 1D adhesions. Moreover, actin in 1D adhesions undergoes slower retrograde flow than the actin that is present in 2D lamellipodia. These data suggest an increase in mechanical coupling between adhesions and protrusive machinery. Experimental reduction of contractility resulted in the loss of 1D adhesion structure and stability, with scattered small and unstable adhesions, and an uncoupling of adhesion protein-integrin stability. Genetic ablation of myosin IIA (MIIA) or myosin IIB (MIIB) isoforms revealed that MIIA is required for efficient migration in restricted environments as well as adhesion maturation, whereas MIIB helps to stabilize adhesions beneath the cell body. These data suggest that restricted cell environments, such as 1D patterns, require cellular contraction through MIIA to enhance adhesion stability and coupling to integrins behind the leading edge. This increase in mechanical coupling allows for greater leading-edge protrusion and rapid cell migration.
Asunto(s)
Microambiente Celular/fisiología , Fibroblastos/fisiología , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Seudópodos/fisiología , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Matriz Extracelular/metabolismo , Técnicas de Inactivación de Genes , Integrinas/metabolismo , Ratones , Células 3T3 NIH , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/genética , Paxillin/metabolismo , Plásmidos , Transfección , Vinculina/metabolismoRESUMEN
Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.
Asunto(s)
Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyesis , Miosina Tipo IIB no Muscular/metabolismo , Amidas/farmacología , Aminoquinolinas/farmacología , Células Cultivadas , Citocinesis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Eritroblastos/efectos de los fármacos , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Eritropoyesis/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Miosinas/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Actinas/fisiología , ADN Mitocondrial/metabolismo , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Actinas/análisis , Actinas/antagonistas & inhibidores , Animales , Células Cultivadas , ADN Mitocondrial/química , ADN Mitocondrial/aislamiento & purificación , Silenciador del Gen , Humanos , Ratones , Mitocondrias/química , Mitocondrias/ultraestructura , Proteínas Mitocondriales/aislamiento & purificación , Cadenas Pesadas de Miosina/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/análisis , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , RatasRESUMEN
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.
Asunto(s)
Células Estrelladas Hepáticas/fisiología , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Movimiento Celular/fisiología , Retículo Endoplásmico/fisiología , Endotelina-1/farmacología , Adhesiones Focales/fisiología , Técnicas de Silenciamiento del Gen , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Proteínas Motoras Moleculares/antagonistas & inhibidores , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/fisiología , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Seudópodos/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Fibras de Estrés/fisiología , Vinculina/metabolismoRESUMEN
Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.
Asunto(s)
Actinas/metabolismo , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Miosina Tipo IIB no Muscular/metabolismo , Sinapsis/fisiología , Animales , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Modelos Neurológicos , Cadenas Ligeras de Miosina/metabolismo , Miosinas/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/efectos de los fármacos , Factores de TiempoRESUMEN
Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe-linear-fail stress-strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and (14)C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Tendones/embriología , Tendones/fisiología , Actinas/antagonistas & inhibidores , Animales , Fenómenos Biomecánicos , Recuento de Células , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Embrión de Pollo , Citocalasina B/farmacología , Módulo de Elasticidad , Matriz Extracelular/ultraestructura , Colágenos Fibrilares/ultraestructura , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Metatarso/fisiología , Miosinas/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Octoxinol/farmacología , Procolágeno/metabolismo , Tendones/citología , Tendones/efectos de los fármacos , Tendones/metabolismo , Tendones/ultraestructura , Resistencia a la Tracción , Ingeniería de TejidosRESUMEN
The function and mechanism of macropinocytosis in cells outside of the immune system remain poorly understood. We used a neuroblastoma cell line, Neuro-2a, to study macropinocytosis in neuronal cells. We found that phorbol 12-myristate 13-acetate (PMA) and insulin-like growth factor 1 (IGF-1) induced two distinct types of macropinocytosis in the Neuro-2a cells. IGF-1-induced macropinocytosis occurs mostly around the cell bodies and requires phosphoinositide 3-kinase (PI3K), while PMA-induced macropinocytosis occurs predominantly in the neurites and is independent of PI3K activity. Both types of macropinocytosis were inhibited by a specific inhibitor of nonmuscle myosin II, blebbistatin. siRNA knockdown of nonmuscle myosin II isoforms, -IIA and -IIB, resulted in opposite effects on macropinocytosis induced by PMA or IGF. Myosin IIA knockdown significantly increased, whereas myosin IIB knockdown significantly decreased, macropinocytosis with correlating changes in membrane ruffle formation.
Asunto(s)
Miosina Tipo IIB no Muscular/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Pinocitosis/efectos de los fármacos , Ácidos Polimetacrílicos/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
PURPOSE: Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis. METHODS: Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. RESULTS: Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. CONCLUSIONS: Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.
Asunto(s)
Fagocitosis/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Miosinas del Músculo Esquelético/metabolismo , Actinas/metabolismo , Animales , Bovinos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/metabolismo , ARN Interferente Pequeño/fisiología , Ratas , Ratas Long-Evans , Ratas Mutantes , Ratas Sprague-Dawley , Epitelio Pigmentado de la Retina/efectos de los fármacos , Miosinas del Músculo Esquelético/antagonistas & inhibidores , Transfección , Tirosina Quinasa c-MerRESUMEN
We have used isoform-specific RNA interference knockdowns to investigate the roles of myosin IIA (MIIA) and MIIB in the component processes that drive cell migration. Both isoforms reside outside of protrusions and act at a distance to regulate cell protrusion, signaling, and maturation of nascent adhesions. MIIA also controls the dynamics and size of adhesions in central regions of the cell and contributes to retraction and adhesion disassembly at the rear. In contrast, MIIB establishes front-back polarity and centrosome, Golgi, and nuclear orientation. Using ATPase- and contraction-deficient mutants of both MIIA and MIIB, we show a role for MIIB-dependent actin cross-linking in establishing front-back polarity. From these studies, MII emerges as a master regulator and integrator of cell migration. It mediates each of the major component processes that drive migration, e.g., polarization, protrusion, adhesion assembly and turnover, polarity, signaling, and tail retraction, and it integrates spatially separated processes.