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1.
Nat Commun ; 11(1): 4471, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901010

RESUMO

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.


Assuntos
DNA Mitocondrial/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Transporte Biológico Ativo , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Cinesina/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteínas rho de Ligação ao GTP/metabolismo
2.
Nat Commun ; 11(1): 4477, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901019

RESUMO

Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Here, using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), we show that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. We demonstrate that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease.


Assuntos
Drosophila melanogaster/embriologia , Células Germinativas Embrionárias/fisiologia , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Padronização Corporal/fisiologia , Caderinas/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Células Germinativas Embrionárias/citologia , Microscopia de Fluorescência por Excitação Multifotônica , Miosina Tipo II/metabolismo , Transdução de Sinais , Análise de Célula Única , Proteínas rho de Ligação ao GTP/metabolismo
3.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651375

RESUMO

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Calorimetria , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica , Fosforilação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
4.
Nat Cell Biol ; 22(7): 791-802, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483386

RESUMO

Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1-Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1-Rok-myosin II pulsatility, little is known about how F-actin organization influences this process. Here, we show that the medioapical cortex consists of two entangled F-actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1-dependent manner. The other forms a persistent and homogeneous network independent of Rho1. We identify the formin Frl (also known as Fmnl) as a critical nucleator of the persistent network, since modulating its level in mutants or by overexpression decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue-scale morphogenetic defects.


Assuntos
Citoesqueleto de Actina/fisiologia , Drosophila melanogaster/metabolismo , Células Epiteliais/patologia , Forminas/fisiologia , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Polaridade Celular , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Morfogênese , Miosina Tipo II/genética , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética
5.
Am J Physiol Heart Circ Physiol ; 319(2): H377-H391, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32559140

RESUMO

Pulmonary arterial hypertension (PAH) is a fatal progressive disease characterized by an increased blood pressure in the pulmonary arteries. RhoA/Rho-kinase (RhoA/ROCK) signaling activation is often associated with PAH. The purpose of this study is to investigate the role and mechanisms of long noncoding RNA (lncRNA) smooth muscle-induced lncRNA (SMILR) to activate the RhoA/ROCK pathway in PAH. SMILR, microRNA-141 (miR-141), and RhoA were identified by qRT-PCR in PAH patients' serum. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), wound-healing assay, cell counting kit-8 (CCK-8) assay, and flow cytometry were performed to determine cell viability, migration, proliferation, and cell cycle in human pulmonary arterial smooth muscle cells (hPASMCs) and primary PASMCs from PAH patients. We also performed bioinformatical prediction, luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) to assess the interaction among SMILR, miR-141, and RhoA. The RhoA/ROCK pathway and proliferation-related proteins were measured by Western blotting. Finally, we introduced the small hairpin (sh)SMILR to monocrotaline-induced PAH rat model and used the hemodynamic measurement, qRT-PCR, and immunohistochemistry to examine the therapeutic effects of shSMILR. SMILR and RhoA expression were upregulated, while miR-141 expression was downregulated in PAH patients. SMILR directly interacted with miR-141 and negatively regulated its expression. Knockdown of SMILR suppressed PASMC proliferation and migration induced by hypoxia. Furthermore, overexpression of miR-141 could inhibit the RhoA/ROCK pathway by binding to RhoA, thereby repressing cell proliferation-related signals. Knockdown of SMILR significantly inhibited the Rho/ROCK activation and vascular remodeling in monocrotaline-induced rats. Knockdown of SMILR effectively elevated miR-141 expression and in turn inhibited the RhoA/ROCK pathway to regulate vascular remodeling and reduce blood pressure in PAH.NEW & NOTEWORTHY Smooth muscle enriched long noncoding RNA (SMILR), as a long noncoding RNA (lncRNA), was increased in pulmonary arterial hypertension (PAH) patients and in vitro and in vivo models. SMILR activated RhoA/ROCK signaling by targeting miR-141 to disinhibit its downstream target RhoA. SMILR knockdown or miR-141 overexpression inhibited hypoxia-induced cell proliferation and migration via repressing RhoA/ROCK signaling in pulmonary arterial smooth muscle cells (PASMCs), which was confirmed in vivo experiments that knockdown of SMILR inhibited vascular remodeling and alleviated PAH in rats. SMILR may be a promising and novel therapeutic target for the treatment and drug development of PAH.


Assuntos
MicroRNAs/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Hipertensão Arterial Pulmonar/enzimologia , RNA Longo não Codificante/metabolismo , Remodelação Vascular , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , RNA Longo não Codificante/genética , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética
6.
PLoS One ; 15(4): e0232025, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353019

RESUMO

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.


Assuntos
Proteínas dos Microfilamentos/genética , Placentação/genética , Proteínas rho de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Desenvolvimento Embrionário , Feminino , Forminas/genética , Forminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Placenta/embriologia , Gravidez , Via de Sinalização Wnt , Proteínas rho de Ligação ao GTP/metabolismo
7.
Gene ; 753: 144809, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470503

RESUMO

Small GTPases function as molecular switches to active or inactive signaling cascades via binding or hydrolyzing GTP. A type of plant specific small GTPases, the ROPs are known to be involved in plant growth, development and immunity. We determined whether ROPs are conserved in Solanaceous species and whether they are involved in plant growth, development and resistance against Phytophthora capsisi. In genome-wide screening, a total of 66 ROPs in six Solanaceous species (SolROPs) were identified, including 16 ROPs in Solanum tuberosum L. (potato), 9 in Solanum lycopersicum L. (tomato), 5 in Solanum melongena L. (eggplant), 9 in Capsicum annuum L. (pepper), 13 in Nicotiana benthamiana Domin and 14 in Nicotiana tabacum L. (tobacco). Phylogenetic analysis revealed that 11 AtROPs and 66 SolROPs fall into five distinct clades (I-V) and hence a novel and systematic gene nomenclature was proposed. In addition, a comprehensive expression analysis was performed by making use of an online database. This revealed that ROP genes are differentially expressed during plant growth and development. Moreover, gene expression of SlROP-II.1 in S. lycopersicum could be significantly induced by P. capsici. Subsequently, SlROP-II.1 and its homologues in N. benthamiana and C. annuum (NbROP-II.1 and CaROP-II.1) were selected for functional analysis using virus-induced gene silencing. Infection assays with P. capsici on silenced plants revealed that SlROP-II.1, NbROP-II.1 and CaROP-II.1 play a role in P. capsici resistance, suggesting conserved function of ROP-II clade across different Solanaceous species. In addition, NbROP-II.1 is also involved in regulating plant growth and development. This study signified the diversity of Solanaceous ROPs and their potential roles in plant growth, development and immunity.


Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de Plantas/genética , Solanaceae/enzimologia , Solanaceae/genética , Proteínas rho de Ligação ao GTP/genética , Capsicum/enzimologia , Capsicum/genética , Genoma de Planta , Estudo de Associação Genômica Ampla/métodos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Filogenia , Doenças das Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais , Tabaco/enzimologia , Tabaco/genética , Proteínas rho de Ligação ao GTP/metabolismo
8.
PLoS One ; 15(4): e0231739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352989

RESUMO

OBJECTIVES: We previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvß3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation. METHODS AND RESULTS: Using intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition. CONCLUSION: The FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.


Assuntos
Permeabilidade Capilar/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Hemorragia/patologia , Microvasos/patologia , Quinases da Família src/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Fibrinogênio/toxicidade , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Hemorragia/induzido quimicamente , Humanos , Microscopia Intravital , Pulmão/irrigação sanguínea , Masculino , Mesentério/irrigação sanguínea , Mesentério/diagnóstico por imagem , Microvasos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/genética
9.
Phytomedicine ; 69: 153193, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32120245

RESUMO

BACKGROUND: Although mechanical barriers and modern surgical techniques have been developed to prevent postoperative adhesion formation, high incidence of adhesions still represents an important challenge in abdominal surgery. So far, there has been no available therapeutic drug in clinical practice. PURPOSE: In this study, we explored the efficacy of sodium aescinate (AESS) treatment against postoperative peritoneal adhesions, the potential molecular mechanism was also investigated. STUDY DESIGN AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into 6 groups for the study: the blank, vehicle, positive control and three AESS administration groups (0.5, 1 and 2 mg/kg/d, intravenous administration for 7 days). Adhesions were induced by discretely ligating peritoneal sidewall. An IL-1ß-induced HMrSV5 cell model was also performed to explore possible functional mechanism. RESULTS: The results indicated that the incidence and severity of peritoneal adhesions were significantly lower in the AESS-treated groups than that in the vehicle and positive control group. AESS-treated groups showed that the secretion, activity, and expression of tPA in rat peritoneum were notably increased. The FIB levels in rat plasma were decreased. The immunohistochemical staining analysis demonstrated that collagen I and α-SMA deposition were significantly attenuated in AESS-treated peritoneal tissues. Besides, we found that AESS treatment reduced the protein levels of p-MYPT1. To further explore the mechanisms of AESS, both activator and inhibitors of RhoA/ROCK pathway were employed in this study. It was found that AESS-induced up-regulation of tPA was reversed by activator of ROCK, but the effects of ROCK inhibitors were consistent with AESS. CONCLUSION: Taken together, the findings of in vivo and in vitro experiments proved that AESS could significantly suppress postoperative peritoneal adhesion formation through inhibiting the RhoA/ROCK signaling pathway. Our researches provide important pharmacological basis for AESS development as a potential therapeutic agent on peritoneal adhesions.


Assuntos
Doenças Peritoneais/tratamento farmacológico , Complicações Pós-Operatórias/tratamento farmacológico , Saponinas/farmacologia , Triterpenos/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Fibrinogênio/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Masculino , Doenças Peritoneais/patologia , Doenças Peritoneais/prevenção & controle , Peritônio/citologia , Peritônio/cirurgia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/prevenção & controle , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Aderências Teciduais
10.
Adv Exp Med Biol ; 1223: 99-127, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32030687

RESUMO

The Rho-ROCK signaling network has a range of specialized functions of key biological importance, including control of essential developmental processes such as morphogenesis and physiological processes including homeostasis, immunity, and wound healing. Deregulation of Rho-ROCK signaling actively contributes to multiple pathological conditions, and plays a major role in cancer development and progression. This dynamic network is critical in modulating the intricate communication between tumor cells, surrounding diverse stromal cells and the matrix, shaping the ever-changing microenvironment of aggressive tumors. In this chapter, we overview the complex regulation of the Rho-ROCK signaling axis, its role in health and disease, and analyze progress made with key approaches targeting the Rho-ROCK pathway for therapeutic benefit. Finally, we conclude by outlining likely future trends and key questions in the field of Rho-ROCK research, in particular surrounding Rho-ROCK signaling within the tumor microenvironment.


Assuntos
Neoplasias/metabolismo , Transdução de Sinais , Microambiente Tumoral , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Humanos , Neoplasias/enzimologia
11.
Biochemistry (Mosc) ; 85(1): 54-67, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079517

RESUMO

KLF2 is a member of the Krüppel-like transcription factor family of proteins containing highly conserved DNA-binding zinc finger domains. KLF2 participates in the differentiation and regulation of the functional activity of monocytes, T lymphocytes, adipocytes, and vascular endothelial cells. The activity of KLF2 is controlled by several regulatory systems, including the MEKK2,3/MEK5/ERK5/MEF2 MAP kinase cascade, Rho family G-proteins, histone acetyltransferases CBP and p300, and histone deacetylases HDAC4 and HDAC5. Activation of KLF2 in endothelial cells induces eNOS expression and provides vasodilatory effect. Many KLF2-dependent genes participate in the suppression of blood coagulation and aggregation of T cells and macrophages with the vascular endothelium, thereby preventing atherosclerosis progression. KLF2 can have a dual effect on the gene transcription. Thus, it induces expression of multiple genes, but suppresses transcription of NF-κB-dependent genes. Transcription factors KLF2 and NF-κB are reciprocal antagonists. KLF2 inhibits induction of NF-κB-dependent genes, whereas NF-κB downregulates KLF2 expression. KLF2-mediated inhibition of NF-κB signaling leads to the suppression of cell response to the pro-inflammatory cytokines IL-1ß and TNFα and results in the attenuation of inflammatory processes.


Assuntos
Células Endoteliais da Veia Umbilical Humana/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Fatores de Transcrição Kruppel-Like/fisiologia , Leucócitos/imunologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Leucócitos/citologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
12.
Nat Commun ; 11(1): 1050, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103012

RESUMO

Organisms respond to tissue damage through the upregulation of protective responses which restore tissue structure and metabolic function. Mitochondria are key sources of intracellular oxidative metabolic signals that maintain cellular homeostasis. Here we report that tissue and cellular wounding triggers rapid and reversible mitochondrial fragmentation. Elevated mitochondrial fragmentation either in fzo-1 fusion-defective mutants or after acute drug treatment accelerates actin-based wound closure. Wounding triggered mitochondrial fragmentation is independent of the GTPase DRP-1 but acts via the mitochondrial Rho GTPase MIRO-1 and cytosolic Ca2+. The fragmented mitochondria and accelerated wound closure of fzo-1 mutants are dependent on MIRO-1 function. Genetic and transcriptomic analyzes show that enhanced mitochondrial fragmentation accelerates wound closure via the upregulation of mtROS and Cytochrome P450. Our results reveal how mitochondrial dynamics respond to cellular and tissue injury and promote tissue repair.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas Mitocondriais/metabolismo , Cicatrização/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , GTP Fosfo-Hidrolases/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Oxirredução , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas rho de Ligação ao GTP/genética
13.
Biochim Biophys Acta Rev Cancer ; 1873(2): 188351, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32007596

RESUMO

Cancer is a multifaceted global disease. Transformation of a normal to a malignant cell takes several steps, including somatic mutations, epigenetic alterations, metabolic reprogramming and loss of cell growth control. Recently, the mevalonate pathway has emerged as a crucial regulator of tumor biology and a potential therapeutic target. This pathway controls cholesterol production and posttranslational modifications of Rho-GTPases, both of which are linked to several key steps of tumor progression. Inhibitors of the mevalonate pathway induce pleiotropic antitumor-effects in several human malignancies, identifying the pathway as an attractive candidate for novel therapies. In this review, we will provide an overview about the role and regulation of the mevalonate pathway in certain aspects of cancer initiation and progression and its potential for therapeutic intervention in oncology.


Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/metabolismo , Colesterol/biossíntese , Ácido Mevalônico/metabolismo , Neoplasias/metabolismo , Antineoplásicos/uso terapêutico , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Ensaios Clínicos como Assunto , Progressão da Doença , Geraniltranstransferase/antagonistas & inibidores , Geraniltranstransferase/metabolismo , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Resultado do Tratamento , Proteínas rho de Ligação ao GTP/metabolismo
14.
Dev Cell ; 52(4): 446-460.e5, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32032546

RESUMO

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Endotélio Vascular/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mecanotransdução Celular , Fatores de Transcrição/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endotélio Vascular/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Hemodinâmica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas rho de Ligação ao GTP/metabolismo
15.
Neoplasma ; 67(3): 614-622, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32009420

RESUMO

Human immediate early response 2 (IER2) has been implicated in tumor cell motility and metastasis; however, the underlying mechanisms in hepatocellular carcinoma (HCC) metastasis remain to be clarified. In this study, we demonstrate that dysregulation of IER2 was shown in HCC clinical samples, and IER2 expression resulted in the promotion of cell migration and invasion in vitro, and HCC tumor growth and pulmonary metastasis in vivo. Moreover, we showed that IER2 expression altered assembly of the actin cytoskeleton rearrangement. Furthermore, MAPK and PI3K/Akt signaling pathways induced by IER2 were confirmed to be probably involved in regulating the activity of Rho GTPases, such as RhoA, Rac1 and Cdc42. Collectively, our results indicated a significant role of IER2 in the HCC cell motility and metastasis through MAPK and PI3K/Akt signaling pathways to regulate the activity of Rho GTPases, thereby modulating actin cytoskeleton rearrangement, unveiling a novel mechanism of cell motility regulation induced by IER2.


Assuntos
Carcinoma Hepatocelular/patologia , Proteínas Imediatamente Precoces/genética , Neoplasias Hepáticas/patologia , Transativadores/genética , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias Hepáticas/genética , Fosfatidilinositol 3-Quinases , Transdução de Sinais
16.
J Vasc Res ; 57(3): 126-135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106116

RESUMO

Uterine artery myogenic tone (MT) develops during pregnancy in hemochorial placentates such as rats and humans. The physiological reason for its appearance is not clear, and we reasoned that it may be a late pregnancy (LP) event in preparation for controlling hemorrhage during parturition. We also hypothesized that gestational increases in RhoA-induced vascular smooth muscle (VSM) calcium sensitivity are contributory and occur under the tonic influence of nitric oxide (NO). Second-order pre-placental radial arteries from early-pregnant (day 12, n = 5), mid-pregnant (day 16, n = 5) and LP (day 20, n = 20) rats were used in combination with arteriography, VSM calcium measurements, pharmacological RHO/Rho-associated protein kinase (ROCK) and nitric oxide synthase (NOS) inhibition, and Western blotting. A subgroup of LP animals (LP + LN; n = 5) treated with L-NAME from gestational days 10 to 20 were used to determine the effects of NOS inhibition on MT and RhoA expression. MT was evident throughout pregnancy, but its expression in pressurized vessels was masked by endothelial NO-induced vasodilation during early gestation. RhoA protein expression was upregulated in LP and attenuated by in vivo NOS inhibition (as was MT). In vitro RHO/ROCK inhibition decreased MT in a concentration-dependent manner without reducing VSM calcium. In summary, pressure-dependent uterine artery tone increases with gestational age due to a combination of RhoA-mediated increases in VSM calcium sensitivity and a loss of endothelial NO influence.


Assuntos
Sinalização do Cálcio , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Vasoconstrição , Animais , Feminino , Idade Gestacional , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Gravidez , Ratos Sprague-Dawley , Artéria Uterina/metabolismo , Vasodilatação , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
17.
Cell Mol Life Sci ; 77(5): 859-874, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31960115

RESUMO

Phosphatidylethanolamine-binding protein 1 (PEBP1), a small 21 kDa protein, is implicated in several key processes of the living cell. The deregulation of PEBP1, especially its downregulation, leads to major diseases such as cancer and Alzheimer's disease. PEBP1 was found to interact with numerous proteins, especially kinases and GTPases, generally inhibiting their activity. To understand the basic functionality of this amazing small protein, we have considered several known processes that it modulates and we have discussed the role of each molecular target in these processes. Here, we propose that cortical actin organization, associated with membrane changes, is involved in the majority of the processes modulated by PEBP1. Furthermore, based on recent data, we summarize some key PEBP1-interacting proteins, and we report their respective functions and focus on their relationships with actin organization. We suggest that, depending on the cell status and environment, PEBP1 is an organizer of the actin-membrane composite material.


Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Sequência de Aminoácidos , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
18.
J Neurosci ; 40(8): 1778-1787, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31953371

RESUMO

Increase in blood-brain barrier (BBB) permeability is a crucial step in neuroinflammatory processes. We previously showed that N Methyl D Aspartate Receptor (NMDARs), expressed on cerebral endothelial cells forming the BBB, regulate immune cell infiltration across this barrier in the mouse. Here, we describe the mechanism responsible for the action of NMDARs on BBB permeabilization. We report that mouse CNS endothelial NMDARs display the regulatory GluN3A subunit. This composition confers to NMDARs' unconventional properties: these receptors do not induce Ca2+ influx but rather show nonionotropic properties. In inflammatory conditions, costimulation of human brain endothelial cells by NMDA agonists (NMDA or glycine) and the serine protease tissue plasminogen activator, previously shown to potentiate NMDAR activity, induces metabotropic signaling via the Rho/ROCK pathway. This pathway leads to an increase in permeability via phosphorylation of myosin light chain and subsequent shrinkage of human brain endothelial cells. Together, these data draw a link between NMDARs and the cytoskeleton in brain endothelial cells that regulates BBB permeability in inflammatory conditions.SIGNIFICANCE STATEMENT The authors describe how NMDARs expressed on endothelial cells regulate blood-brain barrier function via myosin light chain phosphorylation and increase in permeability. They report that these non-neuronal NMDARs display distinct structural, functional, and pharmacological features than their neuronal counterparts.


Assuntos
Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Miosinas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Células Endoteliais/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Masculino , Camundongos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Permeabilidade , Fosforilação/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tecidual/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
19.
Development ; 147(3)2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31932350

RESUMO

Nascent myotubes undergo a dramatic morphological transformation during myogenesis, in which the myotubes elongate over several cell diameters and are directed to the correct muscle attachment sites. Although this process of myotube guidance is essential to pattern the musculoskeletal system, the mechanisms that control myotube guidance remain poorly understood. Using transcriptomics, we found that components of the Fibroblast Growth Factor (FGF) signaling pathway were enriched in nascent myotubes in Drosophila embryos. Null mutations in the FGF receptor heartless (htl), or its ligands, caused significant myotube guidance defects. The FGF ligand Pyramus is expressed broadly in the ectoderm, and ectopic Pyramus expression disrupted muscle patterning. Mechanistically, Htl regulates the activity of Rho/Rac GTPases in nascent myotubes and effects changes in the actin cytoskeleton. FGF signals are thus essential regulators of myotube guidance that act through cytoskeletal regulatory proteins to pattern the musculoskeletal system.


Assuntos
Padronização Corporal/genética , Drosophila/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ectoderma/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/genética , Ligantes , Masculino , Desenvolvimento Musculoesquelético/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
20.
Mol Biol Cell ; 31(3): 143-148, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999511

RESUMO

While the organization of inanimate systems such as gases or liquids is predominantly thermodynamically driven-a mixture of two gases will tend to mix until they reach equilibrium-biological systems frequently exhibit organization that is far from a well-mixed equilibrium. The anisotropies displayed by cells are evident in some of the dynamic processes that constitute life including cell development, movement, and division. These anisotropies operate at different length-scales, from the meso- to the nanoscale, and are proposed to reflect self-organization, a characteristic of living systems that is becoming accessible to reconstitution from purified components, and thus a more thorough understanding. Here, some examples of self-organization underlying cellular anisotropies at the cellular level are reviewed, with an emphasis on Rho-family GTPases operating at the plasma membrane. Given the technical challenges of studying these dynamic proteins, some of the successful approaches that are being employed to study their self-organization will also be considered.


Assuntos
Anisotropia , Fenômenos Fisiológicos Celulares/fisiologia , Células/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Termodinâmica , Proteínas rho de Ligação ao GTP/metabolismo
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