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1.
Metabolism ; 101: 153999, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31672447

RESUMO

BACKGROUND: Adipose tissue plays a crucial role in diet- and obesity-related insulin resistance, with implications for several metabolic diseases. Identification of novel target genes and mechanisms that regulate adipocyte function could lead to improved treatment strategies. RND3 (RhoE/Rho8), a Rho-related GTP-binding protein that inhibits Rho kinase (ROCK) signaling, has been linked to diverse diseases such as apoptotic cardiomyopathy, heart failure, cancer and type 2 diabetes, in part by regulating cytoskeleton dynamics and insulin-mediated glucose uptake. RESULTS: We here investigated the expression of RND3 in adipose tissue in human obesity, and discovered a role for RND3 in regulating adipocyte metabolism. In cross-sectional and prospective studies, we observed 5-fold increased adipocyte levels of RND3 mRNA in obesity, reduced levels after surgery-induced weight loss, and positive correlations of RND3 mRNA with adipocyte size and surrogate measures of insulin resistance (HOMA2-IR and circulating triglyceride/high-density lipoprotein cholesterol (TAG/HDL-C) ratio). By screening for RND3-dependent gene expression following siRNA-mediated RND3 knockdown in differentiating human adipocytes, we found downregulation of inflammatory genes and upregulation of genes related to adipocyte ipolysis and insulin signaling. Treatment of adipocytes with tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), hypoxia or cAMP analogs increased RND3 mRNA levels 1.5-2-fold. Functional assays in primary human adipocytes confirmed that RND3 knockdown reduces cAMP- and isoproterenol-induced lipolysis, which were mimicked by treating cells with ROCK inhibitor. This effect could partly be explained by reduced protein expression of adipose triglyceride lipase (ATGL) and phosphorylated hormone-sensitive lipase (HSL). CONCLUSION: We here uncovered a novel differential expression of adipose RND3 in obesity and insulin resistance, which may at least partly depend on a causal effect of RND3 on adipocyte lipolysis.


Assuntos
Adipócitos/metabolismo , Lipólise/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Células Cultivadas , Estudos Transversais , Regulação da Expressão Gênica , Humanos , Resistência à Insulina , Obesidade/metabolismo , Estudos Prospectivos , RNA Mensageiro/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
2.
Inflammopharmacology ; 27(6): 1309-1318, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31168686

RESUMO

SB-706375 is a selective receptor antagonist of human urotensin-II (hU-II), which can block the aorta contraction induced by hU-II in rats. The effect of SB-706375 on myocardial ischaemia-reperfusion (I/R) injury is unclear. The major objective of this study was to investigate whether SB-706375 has a protective effect on myocardial I/R injury in rats and explore its possible mechanisms. Isolated hearts of Adult Sprague-Dawley were perfused in a Langendorff apparatus, and haemodynamic parameters, lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI), RhoA, and the protein expressions of U-II receptor (UTR), receptor-interacting protein 3 (RIP3), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) were assessed. We found that SB-706375 (1 × 10-6 and 1 × 10-5 mol/L) significantly inhibited the changes of haemodynamic parameters and reduced LDH and CK-MB activities and also cTnI level in the coronary effluents in the heart subjected to myocardial I/R injury. Further experiments studies showed that SB-706375 obviously prevented myocardial I/R increased RhoA activity and UTR, RIP3, ROCK1, and ROCK2 protein expressions. ROCK inhibition abolished the improving effect of SB-706375 on myocardial I/R-induced haemodynamic change in the isolated perfused rat heart. These findings suggested that SB-706375 provides cardio-protection against I/R injury in isolated rats by blocking UTR-RhoA/ROCK-RIP3 pathway.


Assuntos
Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Pirrolidinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Animais , Feminino , Masculino , Necrose , Ratos , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Receptores Acoplados a Proteínas-G/análise , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia
3.
Cells ; 8(5)2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075957

RESUMO

Rho guanosine triphosphatases (GTPases) are key regulators in a number of cellular functions, including actin cytoskeleton remodeling and vesicle traffic. Traditionally, Rho GTPases are studied because of their function in cell migration and cancer, while their roles in metabolism are less documented. However, emerging evidence implicates Rho GTPases as regulators of processes of crucial importance for maintaining metabolic homeostasis. Thus, the time is now ripe for reviewing Rho GTPases in the context of metabolic health. Rho GTPase-mediated key processes include the release of insulin from pancreatic ß cells, glucose uptake into skeletal muscle and adipose tissue, and muscle mass regulation. Through the current review, we cast light on the important roles of Rho GTPases in skeletal muscle, adipose tissue, and the pancreas and discuss the proposed mechanisms by which Rho GTPases act to regulate glucose metabolism in health and disease. We also describe challenges and goals for future research.


Assuntos
Glucose/metabolismo , Insulina/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Tecido Adiposo/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Homeostase , Humanos , Camundongos , Músculo Esquelético/metabolismo , Pâncreas/metabolismo , Ratos
4.
Biomed Pharmacother ; 112: 108651, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30784931

RESUMO

The protective role of alkaloids from Nelumbinis Plumula (AFNP) on the aorta during hypertension is not yet fully understood. We hypothesize that AFNP exerts protective effects against Ang II-induced hypertension by mediating RhoA/ROCK pathway and phenotypic switching during hypertension. In the present study, we evaluated the effect of AFNP on angiotensin II (Ang II)-induced actin cytoskeleton reorganization and aorta remodeling, as well as the involvement of RhoA/Rho-associated coiled kinase (ROCK) pathway in protecting against hypertension. We used rat aortic tissues to investigate the vasodilatation effect of AFNP on Ang II-induced constriction. AFNP was shown to significantly relax the endothelium-intact arteries induced by Ang II. We further investigated the vasodilation effect of AFNP in endothelium denuded arteries, which showed that the action of AFNP was endothelial independent. Male SHR rats were treated with saline or AFNP and morphological changes were examined following 8 weeks. AFNP treatment normalized the effects of hypertension in SHRs. HE staining showed that AFNP treatment improved the tunica media and wall thickness and ratio of MT/LD and MA/LA. Western blotting showed that AFNP treatment markedly decreased the Ang II-induced expression of collagen I and increased α-SMA in aorta. Furthermore, MTT assay showed that AFNP inhibited the proliferation of Ang II treated VSMCs in a concentration-dependent manner. AFNP treatment also ameliorated F-actin cytoskeleton remodeling in Ang II treated VSMCs, as visualized under fluorescence microscopy. Western blot analysis showed that RhoA transposition and ROCK activation and phosphorylation of MYPT1 was increased following Ang II treatment but were inhibited by AFNP treatment, showing that the cardio-protective effect of AFNP is likely mediated by the RhoA/ROCK signaling pathway. The anti-hypertension and aortic protection effects of AFNP are due to non-endothelial dependent inhibition of the VSMC cytoskeleton remodeling and regulation of RhoA/ROCK pathway.


Assuntos
Alcaloides/farmacologia , Aorta Torácica/efeitos dos fármacos , Nelumbo , Remodelação Vascular/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Alcaloides/isolamento & purificação , Animais , Aorta Torácica/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Sementes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Remodelação Vascular/fisiologia
5.
Development ; 146(5)2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30770391

RESUMO

Root hairs are protrusions from root epidermal cells with crucial roles in plant soil interactions. Although much is known about patterning, polarity and tip growth of root hairs, contributions of membrane trafficking to hair initiation remain poorly understood. Here, we demonstrate that the trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) contribute to activation and plasma membrane accumulation of Rho-of-plant (ROP) small GTPases during hair initiation, identifying YIP4a/b as central trafficking components in ROP-dependent root hair formation.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Genes de Plantas , Proteínas de Membrana/farmacologia , Raízes de Plantas/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/farmacologia , Membrana Celular/fisiologia , Genótipo , Proteínas de Membrana/genética , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Mutação , Fenótipo , Transporte Proteico , Sementes , Rede trans-Golgi/fisiologia
6.
Eur J Pharmacol ; 843: 27-33, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445018

RESUMO

Improper cervical function may lead premature or late-term birth. The RhoA/Rho-kinase (ROCK) signalling pathway takes part in cellular functions including smooth muscle contraction. No information is available about the cervical expression of the RhoA/ROCK system during pregnancy. Our aim was to detect the mRNA and protein expression of ROCK enzymes in rat cervices and to evaluate the effects of RhoA/ROCK inhibitors on cervical resistance. The mRNA and protein expressions of RhoA, ROCK I and II were measured in non-pregnant, pregnant and postpartum rat cervices and during parturition by Real-time qPCR and Western blot. The cervical resistance modifying effects of RhoA (simvastatin) and ROCK (fasudil, Y-27632) (10-6M) were investigated in tissue bath experiments. RhoA mRNA was increased on post-partum day 3, while the RhoA protein expression was decreased near and during parturition. ROCK I mRNA and protein expressions were fluctuating with a decrease in protein expression during parturition. ROCK II mRNA and protein expressions were sharply reduced during parturition. Simvastatin increased the cervical resistance on pregnancy days 20 and 22 while Y-27632 and fasudil reduced the resistance on pregnancy days 20. The decrease in RhoA/ROCK expression near parturition may take part in cervical ripening, especially in the final processes leading to delivery. ROCK inhibitors might be potential drug candidates to treat insufficient cervical ripening late-term pregnancies. The effect of simvastatin possibly due to its unique smooth muscle contracting activity in pregnant cervix. Compounds with simvastatin-like action might be new drug candidates for preterm cervical ripening.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Amidas/farmacologia , Maturidade Cervical/efeitos dos fármacos , Colo do Útero/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Piridinas/farmacologia , Sinvastatina/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Maturidade Cervical/fisiologia , Colo do Útero/fisiologia , Feminino , Técnicas In Vitro , Contração Muscular/fisiologia , Gravidez , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia
7.
Fungal Genet Biol ; 120: 30-41, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30205199

RESUMO

Aspergillus fumigatus is a major pathogen of invasive pulmonary aspergillosis. The small GTPase, Rho1, of A. fumigatus is reported to comprise a potential regulatory subunit of ß-1,3-glucan synthase and is indispensable for fungal viability; however, the role of AfRho1 on the growth, cell wall integrity, and pathogenesis of A. fumigatus is still poorly understood. We constructed A. fumigatus mutants with conditional- and overexpression of Rho1 and found that defects of AfRho1 expression led to the reduction of ß-1,3-glucan and glucosamine moieties on the cell wall, with down-regulated transcription of genes in the cell wall integrity signaling pathway and a decrease of calcofluor white (CFW)-stimulated mitogen-activated protein kinase (MpkA) phosphorylation and cytoplasmic leakage compared to those of the wild-type strain (WT). In addition, down-regulation of AfRho1 expression caused much higher sensitivity of A. fumigatus to H2O2 and alkaline pH compared to that of WT. Decrease of AfRho1 expression also attenuated the A. fumigatus pathogenicity in Galleria mellonella and inhibited conidial internalization into lung epithelial cells and inflammatory factor release. In contrast, overexpression of Rho1 did not alter A. fumigatus morphology, susceptibility to cell wall stresses, or pathogenicity relative to its parental strain. Taken together, our findings support AfRho1 as an essential regulator of the cell wall integrity, stress response, and pathogenesis of A. fumigatus.


Assuntos
Aspergillus fumigatus/enzimologia , Parede Celular/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Células A549 , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mariposas/microbiologia , Estresse Fisiológico , Virulência/genética
8.
Naunyn Schmiedebergs Arch Pharmacol ; 391(11): 1275-1283, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30073384

RESUMO

Rho/Rho-kinase (ROCK) signaling contributes to neuroinflammation, epileptogenesis, and seizures in convulsive-type epilepsies. However, this pathway has not been investigated in absence epilepsy. We investigated RhoA activity in genetic absence epilepsy rats from Strasburg (GAERS) and the effects of ROCK inhibitors Y-27632 and fasudil on spike-and-wave discharges (SWDs) of GAERS. ROCK level and activity were measured by Western blot analysis in the brain areas involved in absence seizures (i.e., cortex and thalamus) and hippocampus. Male GAERS were stereotaxically implanted with bilateral cortical electrodes for electroencephalogram (EEG) recordings and/or guide cannula into the right ventricle. ROCK inhibitors were administered by intraperitoneal injection (1-10 mg/kg for Y-27632 or fasudil) or intracerebroventricular injection (7-20 nmol/5 µl for Y-27632 or 10-100 nmol/5 µl for fasudil). EEG was recorded under freely moving conditions. Compared with Wistar rats, GAERS exhibited increased RhoA activity in the somatosensory cortex but not in the thalamus or hippocampus. The single systemic administration of Y-27632 and fasudil partially suppressed the duration and frequency of absence seizure, respectively. However, local brain administration caused a widespread suppressive effect on the total seizure duration, number of seizures, and the average individual seizure length. In summary, Rho/ROCK signaling may be involved in the pathophysiology of absence epilepsy. Furthermore, ROCK inhibitors can control the expression of absence seizure in GAERS, thus indicating that Y-27632 and fasudil have the potential to be used as novel anti-absence drugs.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Amidas/farmacologia , Encéfalo/efeitos dos fármacos , Epilepsia Tipo Ausência/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Encéfalo/fisiologia , Eletroencefalografia/efeitos dos fármacos , Masculino , Ratos Wistar , Proteínas rho de Ligação ao GTP/fisiologia
9.
Braz J Med Biol Res ; 51(10): e7423, 2018 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-30066727

RESUMO

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.


Assuntos
Movimento Celular/fisiologia , Escherichia coli Enteropatogênica/patogenicidade , Células Epiteliais/microbiologia , Sistemas de Secreção Tipo III/fisiologia , Fatores de Virulência/genética , Proteínas rho de Ligação ao GTP/fisiologia , Apoptose , Western Blotting , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Virulência/fisiologia
10.
PLoS Genet ; 14(8): e1007584, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30080872

RESUMO

The mechanisms that control tissue patterning and cell behavior are extensively studied separately, but much less is known about how these two processes are coordinated. Here we show that the Drosophila transcription factor Dysfusion (Dysf) directs leg epithelial folding and joint formation through the regulation of Rho1 activity. We found that Dysf-induced Rho1 activity promotes apical constriction specifically in folding epithelial cells. Here we show that downregulation of Rho1 or its downstream effectors cause defects in fold and joint formation. In addition, Rho1 and its effectors are sufficient to induce the formation of epithelial folds when misexpressed in a flat epithelium. Furthermore, as apoptotic cells can actively control tissue remodeling, we analyzed the role of cell death in the formation of tarsal folds and its relation to Rho1 activity. Surprisingly, we found no defects in this process when apoptosis is inhibited. Our results highlight the coordination between a patterning transcription factor and the cellular processes that cause the cell shape changes necessary to sculpt a flat epithelium into a three dimensional structure.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Morfogênese/genética , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Polaridade Celular , Forma Celular , Drosophila/genética , Proteínas de Drosophila/genética , Células Epiteliais/fisiologia , Epitélio/fisiologia , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Dobramento de Proteína , Proteínas rho de Ligação ao GTP/genética
11.
Blood ; 132(6): 565-576, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-29891535

RESUMO

Aging-associated remodeling of the immune system impairs its functional integrity and contributes to increased morbidity and mortality in the elderly. Aging of hematopoietic stem cells (HSCs), from which all cells of the adaptive immune system ultimately originate, might play a crucial role in the remodeling of the aged immune system. We recently reported that aging of HSCs is, in part, driven by elevated activity of the small RhoGTPase Cdc42 and that aged HSCs can be rejuvenated in vitro by inhibition of the elevated Cdc42 activity in aged HSCs with the pharmacological compound CASIN. To study the quality of immune systems stemming selectively from young or aged HSCs, we established a HSC transplantation model in T- and B-cell-deficient young RAG1-/- hosts. We report that both phenotypic and functional changes in the immune system on aging are primarily a consequence of changes in the function of HSCs on aging and, to a large extent, independent of the thymus, as young and aged HSCs reconstituted distinct T- and B-cell subsets in RAG1-/- hosts that mirrored young and aged immune systems. Importantly, aged HSCs treated with CASIN reestablished an immune system similar to that of young animals, and thus capable of mounting a strong immune response to vaccination. Our studies further imply that epigenetic signatures already imprinted in aged HSCs determine the transcriptional profile and function of HSC-derived T and B cells.


Assuntos
Envelhecimento/imunologia , Senescência Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Subpopulações de Linfócitos/imunologia , Animais , Feminino , Perfilação da Expressão Gênica , Genes RAG-1 , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doadores de Tecidos , Vacinação , Vacinas de DNA/imunologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/fisiologia
12.
PLoS Genet ; 14(5): e1007388, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29813053

RESUMO

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division.


Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/fisiologia , Citocinese/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Fuso Acromático/fisiologia , Benzenossulfonatos/química , Proteína Quinase CDC2/fisiologia , Núcleo Celular/fisiologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Proteínas Quinases/fisiologia , Schizosaccharomyces/ultraestrutura , Fuso Acromático/ultraestrutura , Telófase/fisiologia , Fatores de Tempo , Proteínas rho de Ligação ao GTP/fisiologia
13.
J Neurosci ; 38(20): 4666-4677, 2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29686046

RESUMO

Mitochondrial Ca2+ uptake is gated by the mitochondrial calcium uniplex, which is comprised of mitochondrial calcium uniporter (MCU), the Ca2+ pore-forming subunit of the complex, and its regulators. Ca2+ influx through MCU affects both mitochondrial function and movement in neurons, but its direct role in mitochondrial movement has not been explored. In this report, we show a link between MCU and Miro1, a membrane protein known to regulate mitochondrial movement. We find that MCU interacts with Miro1 through MCU's N-terminal domain, previously thought to be the mitochondrial targeting sequence. Our results show that the N-terminus of MCU has a transmembrane domain that traverses the outer mitochondrial membrane, which is dispensable for MCU localization into mitochondria. However, this domain is required for Miro1 interaction and is critical for Miro1 directed movement. Together, our findings reveal Miro1 as a new component of the MCU complex, and that MCU is an important regulator of mitochondrial transport.SIGNIFICANCE STATEMENT Mitochondrial calcium level is critical for mitochondrial metabolic activity and mitochondrial transport in neurons. While it has been established that calcium influx into mitochondria is modulated by mitochondrial calcium uniporter (MCU) complex, how MCU regulates mitochondrial movement still remains unclear. Here, we discover that the N-terminus of MCU plays a different role than previously thought; it is not required for mitochondrial targeting but is essential for interaction with Miro1, an outer mitochondrial membrane protein important for mitochondrial movement. Furthermore, we show that MCU-Miro1 interaction is required to maintain mitochondrial transport. Our data identify that Miro1 is a novel component of the mitochondrial calcium uniplex and demonstrate that coupling between MCU and Miro1 as a novel mechanism modulating both mitochondrial Ca2+ uptake and mitochondrial transport.


Assuntos
Canais de Cálcio/fisiologia , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Neurônios/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Axônios/metabolismo , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/fisiologia , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células Cultivadas , Feminino , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/fisiologia , Gravidez , Proteínas rho de Ligação ao GTP/genética
14.
PLoS One ; 13(3): e0194003, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29518139

RESUMO

Netrin receptors of the DCC/NEO/UNC-40/Frazzled family have well established roles in cell migration and axon guidance but can also regulate epithelial features such as adhesion, polarity and adherens junction (AJ) stability. Previously, we have shown that overexpression of Drosophila Frazzled (Fra) in the peripodial epithelium (PE) inhibits wing disc eversion and also generates cellular protrusions typical of motile cells. Here, we tested whether the molecular pathways by which Fra inhibits eversion are distinct from those driving motility. We show that in disc proper (DP) epithelial cells Fra, in addition to inducing F-Actin rich protrusions, can affect localization of AJ components and columnar cell shape. We then show that these phenotypes have different requirements for the three conserved Fra cytoplasmic P-motifs and for downstream genes. The formation of protrusions required the P3 motif of Fra, as well as integrins (mys and mew), the Rac pathway (Rac1, wave and, arpc3) and myosin regulatory light chain (Sqh). In contrast, apico-basal cell shape change, which was accompanied by increased myosin phosphorylation, was critically dependent upon the P1 motif and was promoted by RhoGef2 but inhibited by Rac1. Fra also caused a loss of AJ proteins (DE-Cad and Arm) from basolateral regions of epithelial cells. This phenotype required all 3 P-motifs, and was dependent upon the polarity factor par6. par6 was not required for protrusions or cell shape change, but was required to block eversion suggesting that control of AJ components may underlie the ability of Fra to promote epithelial stability. The results imply that multiple molecular pathways act downstream of Fra in epithelial cells.


Assuntos
Caderinas/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Receptores de Netrina/fisiologia , Junções Aderentes/metabolismo , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Ciclo Celular , Movimento Celular , Polaridade Celular , Forma Celular , Extensões da Superfície Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais/citologia , Integrinas/fisiologia , Larva , Miosinas/metabolismo , Receptores de Netrina/química , Receptores de Netrina/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transgenes , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia
15.
PLoS Pathog ; 14(1): e1006840, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29370294

RESUMO

Epichloë festucae is an endophytic fungus which systemically colonizes temperate grasses to establish symbiotic associations. Maintaining symptomless infection is a key requirement for endophytes, a feature that distinguishes them from pathogenic fungi. While pathogenic fungi extend their hyphae by tip growth, hyphae of E. festucae systemically colonize the intercellular space of expanding host leaves via a unique mechanism of hyphal intercalary growth. This study reports that two homologous Rho GTPases, Cdc42 and RacA, have distinctive roles in the regulation of E. festucae growth in planta. Here we highlight the vital role of Cdc42 for intercalary hyphal growth, as well as involvement of RacA in regulation of hyphal network formation, and demonstrate the consequences of mutations in these genes on plant tissue infection. Functions of Cdc42 and RacA are mediated via interactions with BemA and NoxR respectively, which are expected components of the ROS producing NOX complex. Symbiotic defects found in the racA mutant were rescued by introduction of a Cdc42 with key amino acids substitutions crucial for RacA function, highlighting the significance of the specific interactions of these GTPases with BemA and NoxR for their functional differentiation in symbiotic infection.


Assuntos
Epichloe/fisiologia , Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Simbiose , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Endófitos/metabolismo , Endófitos/fisiologia , Epichloe/metabolismo , Proteínas Fúngicas/fisiologia , Interações Hospedeiro-Patógeno/genética , Lolium/microbiologia , Organismos Geneticamente Modificados , Simbiose/genética , Simbiose/fisiologia , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/fisiologia
16.
Medicina (B Aires) ; 77(6): 497-504, 2017.
Artigo em Espanhol | MEDLINE | ID: mdl-29223942

RESUMO

Rho GTPases are molecular switches that control the different cellular processes. Deregulation of these proteins is associated to transformation and malignant progression in several cancer types. Given the evidence available of the role of Rho GTPases in cancer it is suggested that these proteins can serve as potential therapeutic targets. This review focuses on the strategies used to develop Rho GTPases modulators and their potential use in therapeutic settings.


Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Humanos , Neoplasias/enzimologia , Proteínas rho de Ligação ao GTP/fisiologia
17.
J Physiol Pharmacol ; 68(3): 439-451, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28820400

RESUMO

Activation of RhoA and Rho-associated kinases (ROCKs) is known to play a pivotal role in the regulation of smooth muscle contraction via phosphorylation of myosin-light chain and myosin phosphatase. There are few data on the RhoA and ROCKs expression levels in rat uteri. Therefore, our aim was to investigate the mRNA and protein concentration of RhoA and ROCKs in rat uterus during pregnancy, during parturition and post-partum using real time PCR and Western blot analysis. The other purpose was to evaluate the effects of the ROCK (Y-27632, fasudil and RKI 1441) and RhoA inhibitors (simvastatin) on uterine contractility in isolated organ bath experiments. The mRNA and protein levels of RhoA decreased on the 5th day of pregnancy to day 22, then a sharp increase was detected at term. The mRNA and protein concentration of ROCKs was down-regulated in the early stage of pregnancy, while it sharply increased during parturition. The RhoA-inhibitor simvastatin relaxed the uterus contractions, although its inhibitory effects were not followed by the alteration of RhoA. The strongest inhibitory effect of non-selective ROCK inhibitor fasudil was found on non-pregnant uterus, while it elicited milder relaxation on day 22, during parturition and postpartum day 1. The maximum relaxing effects of Y-27632 and RKI 1441 were altered in a proportional way with the target protein expressions. The RhoA/ROCK signalling pathway might be a potential target for the development of new tocolytic agents; however, high specificity to RhoA, ROCK I or ROCK II seems to be fundamental to the high efficacy of uterine relaxation.


Assuntos
Útero/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Animais , Feminino , Parto/fisiologia , Gravidez , Ratos Sprague-Dawley , Contração Uterina , Útero/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
18.
Mol Biol Cell ; 28(16): 2159-2169, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28615318

RESUMO

It has long been postulated, although never directly demonstrated, that mitochondria are strategically positioned in the cytoplasm to meet local requirements for energy production. Here we show that positioning of mitochondria in mouse embryonic fibroblasts (MEFs) determines the shape of intracellular energy gradients in living cells. Specifically, the ratio of ATP to ADP was highest at perinuclear areas of dense mitochondria and gradually decreased as more-peripheral sites were approached. Furthermore, the majority of mitochondria were positioned at the ventral surface of the cell, correlating with high ATP:ADP ratios close to the ventral membrane, which rapidly decreased toward the dorsal surface. We used cells deficient for the mitochondrial Rho-GTPase 1 (Miro1), an essential mediator of microtubule-based mitochondrial motility, to study how changes in mitochondrial positioning affect cytoplasmic energy distribution and cell migration, an energy-expensive process. The mitochondrial network in Miro1-/- MEFs was restricted to the perinuclear area, with few mitochondria present at the cell periphery. This change in mitochondrial distribution dramatically reduced the ratio of ATP to ADP at the cell cortex and disrupted events essential for cell movement, including actin dynamics, lamellipodia protrusion, and membrane ruffling. Cell adhesion status was also affected by changes in mitochondrial positioning; focal adhesion assembly and stability was decreased in Miro1-/- MEFs compared with Miro1+/+  MEFs. Consequently Miro1-/- MEFs migrated slower than control cells during both collective and single-cell migration. These data establish that Miro1-mediated mitochondrial positioning at the leading edge provides localized energy production that promotes cell migration by supporting membrane protrusion and focal adhesion stability.


Assuntos
Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Adesão Celular , Movimento Celular/fisiologia , Células Cultivadas , Citoplasma/metabolismo , Metabolismo Energético , Camundongos , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo
19.
Exp Eye Res ; 158: 23-32, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27593914

RESUMO

Glaucoma is a leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is considered to be a predominant risk factor for primary open angle glaucoma, the most prevalent form of glaucoma. Although the etiological mechanisms responsible for increased IOP are not completely clear, impairment in aqueous humor (AH) drainage through the conventional or trabecular pathway is recognized to be a primary cause in glaucoma patients. Importantly, lowering of IOP has been demonstrated to reduce progression of vision loss and is a mainstay of treatment for all types of glaucoma. Currently however, there are limited therapeutic options available for lowering IOP especially as it relates to enhancement of AH outflow through the trabecular pathway. Towards addressing this challenge, bench and bedside research conducted over the course of the last decade and a half has identified the significance of inhibiting Rho kinase for lowering IOP. Rho kinase is a downstream effector of Rho GTPase signaling that regulates actomyosin dynamics in numerous cell types. Studies from several laboratories have demonstrated that inhibition of Rho kinase lowers IOP via relaxation of the trabecular meshwork which enhances AH outflow. By contrast, activation of Rho GTPase/Rho kinase signaling in the trabecular outflow pathway increases IOP by altering the contractile, cell adhesive and permeability barrier characteristics of the trabecular meshwork and Schlemm's canal tissues, and by influencing extracellular matrix production and fibrotic activity. This article, written in honor of the late David Epstein, MD, summarizes findings from both basic and clinical studies that have been instrumental for recognition of the importance of the Rho/Rho kinase signaling pathway in regulation of AH outflow, and in the development of Rho kinase inhibitors as promising IOP- lowering agents for glaucoma treatment.


Assuntos
Glaucoma de Ângulo Aberto/enzimologia , Glaucoma de Ângulo Aberto/terapia , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Animais , Humor Aquoso/metabolismo , Humanos , Pressão Intraocular/fisiologia , Limbo da Córnea/metabolismo , Testes Imediatos , Malha Trabecular/metabolismo
20.
Int J Cancer ; 140(4): 747-755, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27616679

RESUMO

Melanoma is a highly lethal cutaneous tumor, killing affected patients through development of multiple poorly immunogenic metastases. Suboptimal activation of immune system by melanoma cells is often due to molecular modifications occurring during tumor progression that prevent efficient recognition of melanoma cells by immune effectors. Statins are HMG-CoA reductase inhibitors, which block the mevalonate synthesis pathway, used by millions of people as hypocholesterolemic agents in cardiovascular and cerebrovascular diseases. They are also known to inhibit Rho GTPase activation and Rho dependent signaling pathways. Rho GTPases are regarded as molecular switches that regulate a wide spectrum of cellular functions and their dysfunction has been characterized in various oncogenic process notably in melanoma progression. Moreover, these molecules can modulate the immune response. Since 10 years we have demonstrated that Statins and other Rho GTPases inhibitors are critical regulators of molecules involved in adaptive and innate anti-melanoma immune response. In this review we summarize our major observations demonstrating that these pharmacological agents stimulate melanoma immunogenicity and suggest a potential use of these molecules to promote anti-melanoma immune response.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antineoplásicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Melanoma/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Prenilação de Proteína/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Progressão da Doença , Ativação Enzimática/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Melanoma/imunologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Ácido Mevalônico/metabolismo , Camundongos , Terapia de Alvo Molecular , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/fisiologia
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