RESUMO
Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic process involving focal adhesion proteins assembled at the integrin cytoplasmic tails and the inner leaflet of the plasma membrane. The weak multivalent protein interactions within the complex and with the plasma membrane suggest that liquid-liquid phase separation could play a role in the nascent adhesion assembly. Here, we report that solid-supported lipid membranes supplemented with phosphoinositides induce the phase separation of minimal integrin adhesion condensates composed of integrin ß1 tails, kindlin, talin, paxillin, and FAK at physiological ionic strengths and protein concentrations. We show that the presence of phosphoinositides is key to enriching kindlin and talin on the lipid membrane, which is necessary to further induce the phase separation of paxillin and FAK at the membrane. Our data demonstrate that lipid membrane surfaces set the local solvent conditions for steering the membrane-localized phase separation even in a regime where no condensate formation of proteins occurs in bulk solution.
Assuntos
Integrinas , Talina , Animais , Integrinas/metabolismo , Paxilina/metabolismo , Talina/metabolismo , Membrana Celular/metabolismo , Integrina beta1/metabolismo , Fosfatidilinositóis , Adesão Celular/fisiologiaRESUMO
BACKGROUND: Gastric cancer (GC) is one of the most common cancers with the majority of patients recognized in advanced stages. The efficacy of using docosahexaenoic acid (DHA) as a supplementary agent has been suggested in treatment along with chemotherapeutics including docetaxel. However, the molecular signatures of such beneficial effects are not well-understood. OBJECTIVE(S): We aimed to evaluate the effects of DHA and docetaxel on the expression level of metastasis-related genes, including MMP-2 and talin-2, and their controlling miRNAs, miR-106b and miR-194, in metastatic GC cell line, MKN45. METHOD(S): GC cell line, MKN45, was cultured, and determination of IC50 of DHA was done by MTT test. Cells were treated with docetaxel, DHA, and their combination for 24 h, and then total RNA was extracted and cDNA synthesis was done using standard protocols. The expression level of target genes, MMP-2 and talin-2, and miR-106b and miR-194 were determined by using quantitative real-time PCR. RESULTS: The expression level of MMP-2 was decreased significantly in all treated cells. However, talin-2 showed significant downregulation only after treatment with docetaxel. In contrary to increased expression after treatment with docetaxel, DHA led to a significant under-expression of miR-106b. The similar effect was seen for miR-194. CONCLUSION(S): Combination of docetaxel and DHA led to the significant downregulation of MMP-2. Also, DHA lowered the docetaxel-mediated upregulation of miR-106b oncomiR. In conclusion, supplementation of docetaxel therapy with DHA in GC patients would be considered as a beneficial approach in cancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Docetaxel/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Gástricas/patologia , Talina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/genética , MicroRNAs/genética , Neoplasias Gástricas/genética , Talina/genética , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. inhibits platelet aggregation, suggesting its significant anticoagulant and antithrombotic effects. However, the mechanisms have not been fully elucidated. HYPOTHESIS: We hypothesized that curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbß3 signaling pathway. STUDY DESIGN: We performed in vitro assays to evaluate the effect of curdione on thrombin-induced expression levels of the AMPK signaling molecule and integrin αIIbß3 signaling pathway components. METHODS: Platelet proteins were extracted from washed human platelets, and the effects of curdione on thrombin-induced platelet aggregation were evaluated. The expression levels of the AMPK signaling molecule and integrin αIIbß3 signaling pathway-related proteins were examined using western blot and RT-PCR. The binding of vinculin and talin were studied using immunoprecipitation, double immunofluorescence staining and microscale thermophoresis. RESULTS: Platelet aggregation analysis showed that 0.02â¯U/ml thrombin significantly induces platelet aggregation. Western blot and RT-PCR analysis revealed that AMPK inhibits the vinculin/talin-mediated integrin αIIbß3 signaling pathway, and curdione downregulates the thrombin-induced expression of phosphorylated AMPK (P-AMPK) and P-integrin at both the protein and mRNA levels and downregulates vinculin and talin at the protein level. Furthermore, microscale thermophoresis experiments showed that curdione inhibits the binding of vinculin and talin. The results from the immunoprecipitation and double immunofluorescence staining were consistent with the results of the microscale thermophoresis experiments. CONCLUSION: Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbß3 signaling pathway, which suggests its therapeutic potential in ethnomedicinal applications as an anti-platelet and anti-thrombotic compound to prevent thrombotic diseases.
Assuntos
Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Sesquiterpenos de Germacrano/farmacologia , Trombina/efeitos adversos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Curcuma/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Talina/metabolismo , Vinculina/metabolismoRESUMO
Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.
Assuntos
Antioxidantes/metabolismo , Catequina/análogos & derivados , Receptores ErbB/metabolismo , Integrina beta3/metabolismo , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Transdução de Sinais , Substituição de Aminoácidos , Animais , Antioxidantes/química , Antioxidantes/uso terapêutico , Células CHO , Catequina/química , Catequina/metabolismo , Catequina/uso terapêutico , Cricetulus , Suplementos Nutricionais , Dimerização , Receptores ErbB/agonistas , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Integrina alfa2/química , Integrina alfa2/genética , Integrina alfa2/metabolismo , Integrina beta3/química , Integrina beta3/genética , Ligantes , Bicamadas Lipídicas/química , Mutação , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/agonistas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Talina/antagonistas & inibidores , Talina/química , Talina/metabolismoRESUMO
Rhizoma Curcumae, the dry rhizomes derived from Curcuma aromatica Salisb., are a classical Chinese medicinal herb used to activate blood circulation, remove blood stasis and alleviate pain. Our previous study proved that curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. can inhibit platelet activation suggesting its significant anticoagulant and antithrombotic effects. However, the underlying mechanism of curdione mediated anti-platelet effect has not been fully elucidated. Platelet proteins extracted from washed human platelets, including normal group (treated with normal saline), thrombin group and curdione group were digested and analysed by nano ESI-LC-MS/MS. UniProt database and SIEVE software were employed to identify and reveal the differentially expressed proteins. Furthermore, possible mechanisms involved were explored by Ingenuity Pathway Analysis (IPA) Software and validated by western blot experiments. Twenty-two differentially expressed proteins between the normal and thrombin group were identified. Compared with the thrombin group, the curdione treatment was significantly down-regulated only 2 proteins (Talin1 and ß1-tubulin). Bioinformatics analysis showed that Talin1 and ß1-tubulin could be involved in the integrin signal pathway. The results of western blot analysis were consistent with that of the proteomics data. Vinculin, identified in IPA database was involved in the formation of cell cytoskeletal. The down-regulation of ß1-tubulin facilitated the decrease in vinculin/Talin1. Curdione regulated the expression of vinculin and Talin1 by ß1-tubulin affecting the integrin signalling pathway and eventually inhibiting platelet activation. The ß1-tubulin may be a potential target of curdione, which attenuates thrombin-induced human platelet activation.
Assuntos
Curcuma/química , Ativação Plaquetária/efeitos dos fármacos , Sesquiterpenos de Germacrano/farmacologia , Trombina/farmacologia , Tubulina (Proteína)/metabolismo , Plaquetas/efeitos dos fármacos , Regulação para Baixo , Humanos , Óleos Voláteis , Proteoma , Rizoma/química , Transdução de Sinais/efeitos dos fármacos , Talina/metabolismo , Vinculina/metabolismoRESUMO
Mu-opioid receptor regulates microRNA-190 (miR-190) in an agonist-dependent manner; fentanyl, but not morphine, decreases the miR-190 level in rat primary hippocampal neuron cultures and in mouse hippocampi. In this study, the correlation between the cellular content of miR-190 and the mRNA level of its host gene, talin2, suggested that fentanyl decreases the miR-190 level by inhibiting the transcription of talin2. Fentanyl-induced beta-arrestin2-mediated ERK phosphorylation led to the phosphorylation of Yin Yang 1 (YY1). In addition, YY1 phosphorylation impaired the association of YY1 with the -208 to -200 region on the Talin2 promoter, and this association was essential for YY1 to stimulate the transcription of talin2. Thus, fentanyl decreased the transcription of talin2 and subsequently the cellular level of miR-190 by inducing YY1 phosphorylation. In contrast, because morphine induces ERK phosphorylation via the protein kinase C pathway, morphine did not induce YY1 phosphorylation and had no effect on the transcription of talin2 and the cellular content of miR-190. This study therefore delineates a signaling pathway that mediates the effects of fentanyl on miR-190 expression.
Assuntos
Fentanila/farmacologia , MicroRNAs/genética , Receptores Opioides mu/agonistas , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Morfina/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Opioides mu/metabolismo , Talina/genética , Talina/metabolismoRESUMO
T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Comunicação Celular/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Ativação Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/fisiologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Comunicação Celular/genética , Linhagem Celular Tumoral , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/biossíntese , Antígeno-1 Associado à Função Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Talina/metabolismo , Regulação para Cima/imunologiaRESUMO
The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.
Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Epiderme Vegetal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Microtúbulos/metabolismo , Cebolas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Talina/genética , Talina/metabolismo , Nicotiana/metabolismoRESUMO
BACKGROUND: Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. RESULTS: Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. CONCLUSIONS: Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Núcleo Celular/metabolismo , Cebolas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Actinas/química , Actinas/genética , Animais , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microinjeções , Microscopia de Fluorescência , Cebolas/citologia , Faloidina/química , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química , Talina/genética , Talina/metabolismoRESUMO
We have isolated an integrin-beta and -alpha subunit from Podocoryne carnea (Cnidaria, Hydrozoa) and studied their expression in the life-cycle and during cell migration, in vitro transdifferentiation and regeneration. Comparison of the integrin expression pattern with a Podocoryne talin homologue by RT-PCR demonstrates that all three genes are maternal messages and continuously expressed in the life-cycle, in medusa development and in all medusae tissues. In situ hybridisation experiments confirm co-expression of both integrin subunits in the different life-stages. Integrin expression was furthermore studied in isolated striated muscle induced to transdifferentiate to new cell types, or grafted on ECM where the muscle adheres and migrates. Integrin expression was maintained continuously throughout both processes. These results suggest that in Podocoryne carnea processes such as cell migration and differentiation are not controlled by up- or downregulation of alternative integrin subunits, but by a single integrin heterodimer which activates different downstream signalling cascades.
Assuntos
Integrinas/genética , Cifozoários/genética , Talina/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cátions/metabolismo , Diferenciação Celular , Sequência Conservada , Cisteína/genética , DNA Complementar/química , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Integrinas/metabolismo , Larva/metabolismo , Dados de Sequência Molecular , Desenvolvimento Muscular , Músculos/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Talina/metabolismo , Distribuição TecidualRESUMO
Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.
Assuntos
Calpaína/metabolismo , Talina/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Biotina/metabolismo , Plaquetas/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Integrina beta3 , Integrinas/metabolismo , Cinética , Dados de Sequência Molecular , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.