RESUMO
Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.
Assuntos
Lipoilação , Proteínas de Membrana/antagonistas & inibidores , Proteínas Motores Moleculares/química , Cadeias Leves de Miosina/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Toxoplasma/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Interações Hospedeiro-Parasita/fisiologia , Humanos , Estágios do Ciclo de Vida , Masculino , Proteínas de Membrana/genética , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Movimento , Mutação , Cadeias Leves de Miosina/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genéticaRESUMO
PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed. RESULTS: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors. CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR.
Assuntos
Carcinoma Pulmonar de Lewis/radioterapia , Cadeias Leves de Miosina/efeitos da radiação , Microambiente Tumoral/efeitos da radiação , Animais , Azepinas/administração & dosagem , Vasos Sanguíneos/patologia , Vasos Sanguíneos/efeitos da radiação , Linfócitos T CD8-Positivos/citologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Histonas/metabolismo , Histonas/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismo , Naftalenos/administração & dosagem , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos da radiação , Radioterapia/métodos , Dosagem Radioterapêutica , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiaçãoRESUMO
PURPOSE: The aim of this study was to evaluate the neuroprotective effect of tanshinone IIA (TSA) on focal cerebral ischemia in rats and to investigate whether it was associated with Nogo-A/NgR1/RhoA/Rho-associated protein kinase 2 (ROCKII)/myosin light chain (MLC) signaling. METHODS: In this study, focal cerebral ischemia animal model was used. Neurological deficit scores and infarction volume were investigated to evaluate the neuroprotection of TSA. Hematoxylin-eosin staining, Nissl staining, and immunofluorescence staining were conducted to detect ischemic changes in brain tissue and changes in neurofilament protein 200 (NF200) and growth-associated protein-43 (GAP-43) expression, respectively. Western blotting and qRT-PCR analyses were used to detect the expression levels of NF200, GAP-43 and Nogo-A/NgR1/RhoA/ROCKII/MLC pathway-related signaling molecules. RESULTS: TSA treatment can improve the survival rate of rats, reduce the neurological score and infarct volume, and reduce neuron damage. In addition, TSA also increased axon length and enhanced expression of NF200 and GAP-43. Importantly, TSA significantly attenuated the expression of Nogo-A, NgR1, RhoA, ROCKII, and p-MLC, and thus inhibiting the activation of this signaling pathway. CONCLUSION: TSA promoted axonal regeneration by inhibiting the Nogo-A/NgR1/RhoA/ROCKII/MLC signaling pathway, thereby exerting neuroprotective effects in cerebral ischemia rats, which provided support for the clinical application of TSA in stroke treatment.
Assuntos
Abietanos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Axônios/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Abietanos/química , Abietanos/isolamento & purificação , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Axônios/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Humanos , Estrutura Molecular , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismo , Proteínas Nogo/antagonistas & inibidores , Proteínas Nogo/metabolismo , Receptor Nogo 1/antagonistas & inibidores , Receptor Nogo 1/metabolismo , Ratos , Ratos Sprague-Dawley , Salvia miltiorrhiza/química , Transdução de Sinais/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rhoRESUMO
PURPOSE: Our previous studies have indicated that non-muscle myosin heavy chain IIA (NMMHC IIA) is involved in H2O2-induced neuronal apoptosis, which is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. However, the neuroprotective effect of NMMHC IIA inhibition with an adeno-associated virus (AAV) vector after transient middle cerebral artery occlusion (MCAO) and its role in caspases-3/ROCK1/MLC pathway remain blurred. METHODS: Green fluorescent protein (GFP) and a small hairpin RNA targeting Myh9 (encoding NMMHC IIA) were cloned and packaged into the AAV9 vector. AAV-shMyh9 or control vector were injected into C57BL/6J mice four weeks prior to 60 min MCAO. Twenty-four hours after reperfusion, functional and histological analyses of the mice were performed. RESULTS: In this study, AAV-shMyh9 was used to down-regulate NMMHC IIA expression in mice. We found that down-regulation of NMMHC IIA could improve neurological scores and histological injury in ischemic mice. Ischemic attack also activated neuronal apoptosis, and this effect was partially attenuated when NMMHC IIA was inhibited by AAV-shMyh9. In addition, AAV-shMyh9 significantly reduced cerebral ischemic/reperfusion (I/R)-induced NMMHC IIA-actin interaction, caspase-3 cleavage, Rho-associated kinase1 (ROCK1) activation and myosin light-chains (MLC) phosphorylation. CONCLUSION: Consequently, we showed that AAV-shMyh9 inhibits I/R-induced neuronal apoptosis linked with caspase-3/ROCK1/MLC/NMMHC IIA-actin cascade, which has also been confirmed to be a positive feedback loop. These findings put some insights into the neuroprotective effect of AAV-shMyh9 associated with the regulation of NMMHC IIA-related pathway under ischemic attack and provide a therapeutic strategy for ischemic stroke.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Caspase 3/metabolismo , Cadeias Leves de Miosina/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Leves de Miosina/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Miosina não Muscular Tipo IIA , Fosforilação/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Quinases Associadas a rho/metabolismoRESUMO
The numbers of patients with inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), have been increasing over time, worldwide; however, the pathogenesis of IBD is multifactorial and has not been fully understood. Myosin light chain 9 and 12a and 12b (Myl9/12) are known as ligands of the CD69 molecule. They create "Myl9 nets" that are often detected in inflamed site, which play a crucial role in regulating the recruitment and retention of CD69-expressing effector cells in inflamed tissues. We demonstrated the strong expression of Myl9/12 in the inflamed gut of IBD patients and mice with DSS-induced colitis. The administration of anti-Myl9/12 Ab to mice with DSS-induced colitis ameliorated the inflammation and prolonged their survival. The plasma Myl9 levels in the patients with active UC and CD were significantly higher than those in patients with disease remission, and may depict the disease severity of IBD patients, especially those with UC. Thus, our results indicate that Myl9/12 are involved in the pathogenesis of IBD, and are likely to be a new therapeutic target for patients suffering from IBD.
Assuntos
Suscetibilidade a Doenças , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Cadeias Leves de Miosina/genética , Adulto , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores , Estudos de Casos e Controles , Diagnóstico Diferencial , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismoRESUMO
BACKGROUND: Restrictive cardiomyopathy is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy directed at the underlying cause. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model. METHODS: A short hairpin RNA (M7.8L) was selected from a pool for specificity and efficacy. Two groups of myosin regulatory light chain N47K transgenic mice were injected with M7.8L packaged in adeno-associated virus 9 at 3 days of age and 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine maximal oxygen uptake and left ventricular mass. At the end of treatment, heart, lung, liver, and kidney tissue was harvested to determine viral tropism and for transcriptomic and proteomic analysis. Cardiomyocytes were isolated for single-cell studies. RESULTS: A one-time injection of AAV9-M7.8L RNAi in 3-day-old humanized regulatory light chain mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed at 16 weeks postinjection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight, and attenuated a pathological increase in left ventricular mass. Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of contraction, relaxation, and calcium kinetics. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II and the transcription activator Brg1 were reduced, suggesting recovery toward a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts, and endogenous microRNAs were preserved, suggesting that the RNAi pathway was not saturated. CONCLUSIONS: Our results show the feasibility, efficacy, and safety of RNAi therapeutics directed towards human restrictive cardiomyopathy. This is a promising step toward targeted therapy for a prevalent human disease.
Assuntos
Cardiomiopatia Restritiva/patologia , Cadeias Leves de Miosina/metabolismo , Interferência de RNA , Alelos , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatia Restritiva/prevenção & controle , DNA Helicases/genética , DNA Helicases/metabolismo , Modelos Animais de Doenças , Redes Reguladoras de Genes , Vetores Genéticos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Contração Muscular , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/genética , RNA Interferente Pequeno/metabolismoRESUMO
Piperine, an alkaloid from black pepper, was found to inhibit the super-relaxed state (SRX) of myosin in fast-twitch skeletal muscle fibers. In this work we report that the piperine molecule binds heavy meromyosin (HMM), whereas it does not interact with the regulatory light chain (RLC)-free subfragment-1 (S1) or with control proteins from the same muscle molecular machinery, G-actin and tropomyosin. To further narrow down the location of piperine binding, we studied interactions between piperine and a fragment of skeletal myosin consisting of the full-length RLC and a fragment of the heavy chain (HCF). The sequence of HCF was designed to bind RLC and to dimerize via formation of a stable coiled coil, thus producing a well-folded isolated fragment of the myosin neck. Both chains were co-expressed in Escherichia coli, the RLC/HCF complex was purified and tested for stability, composition and binding to piperine. RLC and HCF chains formed a stable heterotetrameric complex (RLC/HCF)2 which was found to bind piperine. The piperine molecule was also found to bind isolated RLC. Piperine binding to RLC in (RLC/HCF)2 altered the compactness of the complex, suggesting that the mechanism of SRX inhibition by piperine is based on changing conformation of the myosin.
Assuntos
Alcaloides/metabolismo , Alcaloides/farmacologia , Benzodioxóis/metabolismo , Benzodioxóis/farmacologia , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Sequência de Aminoácidos , Animais , Camundongos , Modelos Moleculares , Mutação , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Cadeias Leves de Miosina/química , Ligação Proteica , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacosRESUMO
The coordination of individual cell behaviors is a crucial step in the assembly and morphogenesis of tissues. Xenopus mesendoderm cells migrate collectively along a fibronectin (FN) substrate at gastrulation, but how the adhesive and mechanical forces required for these movements are generated and transmitted is unclear. Traction force microscopy (TFM) was used to establish that traction stresses are limited primarily to leading edge cells in mesendoderm explants, and that these forces are balanced by intercellular stresses in follower rows. This is further reflected in the morphology of these cells, with broad lamellipodial protrusions, mature focal adhesions and a gradient of activated Rac1 evident at the leading edge, while small protrusions, rapid turnover of immature focal adhesions and lack of a Rac1 activity gradient characterize cells in following rows. Depletion of keratin (krt8) with antisense morpholinos results in high traction stresses in follower row cells, misdirected protrusions and the formation of actin stress fibers anchored in streak-like focal adhesions. We propose that maintenance of mechanical integrity in the mesendoderm by keratin intermediate filaments is required to balance stresses within the tissue to regulate collective cell movements.
Assuntos
Gastrulação/fisiologia , Queratinas/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus/embriologia , Xenopus/fisiologia , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Miosinas Cardíacas/antagonistas & inibidores , Miosinas Cardíacas/metabolismo , Movimento Celular/fisiologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/fisiologia , Adesões Focais/fisiologia , Técnicas de Silenciamento de Genes , Filamentos Intermediários/fisiologia , Queratina-8/antagonistas & inibidores , Queratina-8/genética , Queratina-8/fisiologia , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/fisiologia , Modelos Biológicos , Morfogênese/fisiologia , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismo , Transdução de Sinais , Estresse Mecânico , Xenopus/genética , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C ß2 (PKCß2) at Ser660. Inhibiting (protein kinase C ß) PKCß blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCß2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection.
Assuntos
Ácido Butírico/farmacologia , Miosinas Cardíacas/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Proteína Quinase C beta/metabolismo , Junções Íntimas/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Western Blotting , Células CACO-2 , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Miosinas Cardíacas/antagonistas & inibidores , Humanos , Imunoprecipitação , Cadeias Leves de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
The role of myosin light chain II (MLCII) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a fourpoint bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runtrelated transcription factor2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLCII to total MLCII was increased significantly (P<0.05). Silencing MLCII by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML7, reduced the CUCSassociated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLCII.
Assuntos
Miosinas Cardíacas/genética , Condrócitos/citologia , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Côndilo Mandibular/crescimento & desenvolvimento , Cadeias Leves de Miosina/genética , Osteogênese/genética , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Animais , Azepinas/administração & dosagem , Miosinas Cardíacas/antagonistas & inibidores , Miosinas Cardíacas/biossíntese , Diferenciação Celular/genética , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Côndilo Mandibular/citologia , Côndilo Mandibular/metabolismo , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/biossíntese , Naftalenos/administração & dosagem , Fosforilação , Pressão , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RatosRESUMO
Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Miosinas Cardíacas/antagonistas & inibidores , Cadeias Leves de Miosina/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Antígenos CD/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Barreira Hematoencefálica/metabolismo , Caderinas/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Glucose/metabolismo , Ocludina/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Ratos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
During oocyte meiosis, the bipolar spindle forms in the central cytoplasm and then migrates to the cortex. Subsequently, the oocyte extrudes the polar body through two successive asymmetric divisions, which are regulated primarily by actin filaments. Myosin light chain2 (MLC2) phosphorylation plays pivotal roles in smooth muscle contraction, stress fiber formation, cell motility and cytokinesis. However, whether MLC2 phosphorylation participates in the oocyte polarization and asymmetric division has not been clarified. The present study investigated the expression and functions of MLC2 during mouse oocyte meiosis. Our result showed that p-MLC2 was localized in the oocyte cortex, with a thickened cap above the chromosomes. Meanwhile, p-MLC2 was also localized in the poles of spindle. Disruption of MLC2 activity by MLC2 knock down (KD) caused the failure of polar body extrusion. Immunofluorescent staining showed that a large proportion of oocytes arrested in telophase stage and failed to undergo cytokinesis after culturing for 12 hours. In the meantime, actin filament staining at oocyte membrane and cytoplasm were reduced in MLC2 KD oocytes. Finally, we found that the phosphorylation of MLC2 protein levels was decreased after disruption of RhoA activity. Above all, our data indicated that the RhoA-mediated MLC2 regulates the actin organization for cytokinesis during mouse oocyte maturation.
Assuntos
Actinas/metabolismo , Citocinese , Meiose , Cadeias Leves de Miosina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Citocinese/efeitos dos fármacos , Camundongos , Microscopia Confocal , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/genética , Oócitos/citologia , Oócitos/metabolismo , Oogênese , Compostos Orgânicos/farmacologia , Fosforilação/efeitos dos fármacos , Corpos Polares , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , TelófaseRESUMO
To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mielopoese/imunologia , RNA Mensageiro/metabolismo , Ribonuclease III/imunologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Células Dendríticas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mielopoese/genética , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonuclease III/deficiência , Ribonuclease III/genéticaRESUMO
Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge.
Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Adesões Focais/fisiologia , Proteínas dos Microfilamentos/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Pseudópodes/patologia , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Apoptose , Western Blotting , Proliferação de Células , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Microscopia de Fluorescência , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Pseudópodes/metabolismo , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
Pluripotent human embryonic stem cells (hESCs) have the capability of differentiating into different lineages based on specific environmental cues. We had previously shown that hESCs can be primed to differentiate into either neurons or glial cells, depending on the arrangement, geometry and size of their substrate topography. In particular, anisotropically patterned substrates like gratings were found to favour the differentiation of hESCs into neurons rather than glial cells. In this study, our aim is to elucidate the underlying mechanisms of topography-induced differentiation of hESCs towards neuronal lineages. We show that high actomyosin contractility induced by a nano-grating topography is crucial for neuronal maturation. Treatment of cells with the myosin II inhibitor (blebbistatin) and myosin light chain kinase inhibitor (ML-7) greatly reduces the expression level of microtubule-associated protein 2 (MAP2). On the other hand, our qPCR array results showed that PAX5, BRN3A and NEUROD1 were highly expressed in hESCs grown on nano-grating substrates as compared to unpatterned substrates, suggesting the possible involvement of these genes in topography-mediated neuronal differentiation of hESCs. Interestingly, YAP was localized to the cytoplasm of differentiating hESCs. Taken together, our study has provided new insights in understanding the mechanotransduction of topographical cues during neuronal differentiation of hESCs.
Assuntos
Actomiosina/metabolismo , Células-Tronco Embrionárias/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Azepinas/química , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Citoesqueleto/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Cadeias Leves de Miosina/antagonistas & inibidores , Miosina Tipo II/antagonistas & inibidores , Naftalenos/química , Neuroglia/metabolismo , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Estresse Mecânico , Regulação para Cima , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Porcine induced pluripotent stem cells (piPSCs) had been reported during the past 5years, but there were few reports on how the cell signaling works in piPSCs. In order to clarify the signaling work that dominated the characteristic difference of two types of piPSCs which were derived from Oct4, Sox2, Klf4 and c-Myc (termed 4F piPSCs) and Oct4, Sox2, Klf4, c-Myc, Tbx3 and Nr5α2 (termed 6F piPSCs) respectively, we performed this study. 4F piPSCs and 6F piPSCs were cultured in medium with or without the ROCK inhibitor Y27632 after dissociating into single cells, the efficiency of a single cell colony and the number of AP positive colonies were assessed. The total RhoA and GTP-bind RhoA were detected in 4F piPSCs and 6F piPSCs before and after digestion into single cells. To explore the relationship between RHO-ROCK-MLC signaling pathway and the two factors Tbx3 and Nr5α2, the 4F piPSCs were infected with lenti-virus Tbx3 and Nr5α2 (termed 4F+TND). Results showed that the viability of cells could be enhanced by Y27632 and the RHO-ROCK-MLC signaling pathway was activated after dissociation into single cells in 4F piPSCs but not in 6F piPSCs. And, the 4F+TND piPSCs could be passaged and keep in high viability after dissociation into single cells, though the morphology of colonies did not change. These results indicated that the Tbx3 and Nr5α2 can improve the viability of piPSCs after dissociation into single cells by inhibiting the RHO-ROCK-MLC signaling pathway. And this provides useful information for establishing porcine pluripotent cells in future study.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas com Domínio T/fisiologia , Amidas/farmacologia , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos ICR , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismo , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Suínos , Proteínas com Domínio T/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2(kd/kd)) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2(+/+)) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2(kd/kd) mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2(kd/kd) neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2(kd/kd) neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Membrana/genética , Neurogênese/fisiologia , Neurônios/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Miosinas Cardíacas/antagonistas & inibidores , Miosinas Cardíacas/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/patologia , Fosforilação , Transporte Proteico , Células Piramidais/fisiologia , Piridinas/farmacologia , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by "click" chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor.
Assuntos
Antiparasitários/farmacologia , Compostos de Benzilideno/farmacologia , Movimento Celular/efeitos dos fármacos , Cadeias Leves de Miosina/antagonistas & inibidores , Parasitos/fisiologia , Piperidonas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Toxoplasma/fisiologia , Sequência de Aminoácidos , Animais , Antiparasitários/química , Compostos de Benzilideno/química , Técnicas de Introdução de Genes , Humanos , Masculino , Dados de Sequência Molecular , Peso Molecular , Mutação , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Parasitos/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Peptídeos/química , Piperidonas/química , Proteínas Recombinantes/química , Células Sf9 , Bibliotecas de Moléculas Pequenas/química , Toxoplasma/efeitos dos fármacosRESUMO
The iron-regulated metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), is up-regulated by cellular iron depletion mediated by iron chelators and can inhibit cancer cell migration. However, the mechanism of how NDRG1 achieves this effect remains unclear. In this study, we implemented established and newly constructed NDRG1 overexpression and knockdown models using the DU145, HT29, and HCT116 cancer cell lines to investigate the molecular basis by which NDRG1 exerts its inhibitory effect on cell migration. Using these models, we demonstrated that NDRG1 overexpression inhibits cell migration by preventing actin-filament polymerization, stress fiber assembly and formation. In contrast, NDRG1 knockdown had the opposite effect. Moreover, we identified that NDRG1 inhibited an important regulatory pathway mediated by the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2) pathway that modulates stress fiber assembly. The phosphorylation of MLC2 is a key process in inducing stress fiber contraction, and this was shown to be markedly decreased or increased by NDRG1 overexpression or knockdown, respectively. The mechanism involved in the inhibition of MLC2 phosphorylation by NDRG1 was mediated by a significant (P < 0.001) decrease in ROCK1 expression that is a key kinase involved in MLC2 phosphorylation. Considering that NDRG1 is up-regulated after cellular iron depletion, novel thiosemicarbazone iron chelators (e.g., di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) were demonstrated to inhibit ROCK1/pMLC2-modulated actin-filament polymerization, stress fiber assembly, and formation via a mechanism involving NDRG1. These results highlight the role of the ROCK1/pMLC2 pathway in the NDRG1-mediated antimetastatic signaling network and the therapeutic potential of iron chelators at inhibiting metastasis.
Assuntos
Miosinas Cardíacas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quelantes de Ferro/farmacologia , Cadeias Leves de Miosina/metabolismo , Fibras de Estresse/metabolismo , Quinases Associadas a rho/metabolismo , Actinas/genética , Actinas/metabolismo , Miosinas Cardíacas/antagonistas & inibidores , Miosinas Cardíacas/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Silenciamento de Genes , Células HCT116 , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Terapia de Alvo Molecular , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/genética , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiossemicarbazonas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaRESUMO
Vascular endothelial cadherin (VE-cad) tyrosine (Tyr) phosphorylation has been implicated in the disruption of adherens junctions (AJs) induced by inflammatory reactions. The impacts of statins on integrity of AJs and VE-cad Tyr phosphorylation have not been explored. The effects of atorvastatin on IL-1ß and monocyte-induced VE-cad Tyr phosphorylation in human umbilical vein endothelial cells (ECs) were studied. In ECs treated with interleukin (IL)-1ß for 30 min, VE-cad Tyr phosphorylation, dissociation of the VE-cad/ß-catenin complex and transendothelial migration (TEM) of monocytes were increased. These processes were mediated by activation of HRas and RhoA that leads to phosphorylation of myosin light chain (MLC). Atorvastatin inhibited IL-1ß-induced Tyr phosphorylation of VE-cad by inhibiting RhoA and by dephosphorylating MLC. The attenuating effect of atorvastatin on VE-cad Tyr phosphorylation was reversed when RhoA was activated or MLC phosphatase was inhibited. Furthermore, inhibiting farnesyl transferase or geranylgeranyl transferase reproduced the inhibitory effects of atorvastatin on VE-cad Tyr phosphorylation. In addition, atorvastatin inhibited monocyte-induced VE-cad Tyr phosphorylation in ECs and attenuated IL-1ß-induced TEM of monocytes. Our study introduces a novel pleiotropic effect of atorvastatin and suggests that statins protect the integrity of AJs in ECs by inhibiting RhoA-mediated Tyr phosphorylation of VE-cad.