RESUMEN
Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.
Asunto(s)
Estrés Mecánico , Zixina/química , Citoesqueleto de Actina/efectos de los fármacos , Amidas/farmacología , Animales , Bovinos , Adhesión Celular , Citocalasina D/farmacología , Células Endoteliales , Recuperación de Fluorescencia tras Fotoblanqueo , Adhesiones Focales , Proteínas Fluorescentes Verdes , Microscopía Intravital , Cinética , Rayos Láser , Ratones , Ratones Endogámicos BALB C , Piridinas/farmacología , Proteínas Recombinantes de Fusión/química , Vinculina/química , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Traditionally, the screening of metabolites in microbial matrices is performed by monocultures. Nonetheless, the absence of biotic and abiotic interactions generally observed in nature still limit the chemical diversity and leads to "poorer" chemical profiles. Nowadays, several methods have been developed to determine the conditions under which cryptic genes are activated, in an attempt to induce these silenced biosynthetic pathways. Among those, the one strain, many compounds (OSMAC) strategy has been applied to enhance metabolic production by a systematic variation of growth parameters. The complexity of the chemical profiles from OSMAC experiments has required increasingly robust and accurate techniques. In this sense, deconvolution-based 1 HNMR quantification have emerged as a promising methodology to decrease complexity and provide a comprehensive perspective for metabolomics studies. Our present work shows an integrated strategy for the increased production and rapid quantification of compounds from microbial sources. Specifically, an OSMAC design of experiments (DoE) was used to optimize the microbial production of bioactive fusaric acid, cytochalasin D and 3-nitropropionic acid, and Global Spectral Deconvolution (GSD)-based 1 HNMR quantification was carried out for their measurement. The results showed that OSMAC increased the production of the metabolites by up to 33% and that GSD was able to extract accurate NMR integrals even in heavily coalescence spectral regions. Moreover, GSD-1 HNMR quantification was reproducible for all species and exhibited validated results that were more selective and accurate than comparative methods. Overall, this strategy up-regulated important metabolites using a reduced number of experiments and provided fast analyte monitor directly in raw extracts.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citocalasina D/metabolismo , Ácido Fusárico/biosíntesis , Metabolómica/métodos , Nitrocompuestos/metabolismo , Propionatos/metabolismo , Ascomicetos/aislamiento & purificación , Ascomicetos/metabolismo , Citocalasina D/análisis , Ácido Fusárico/análisis , Nitrocompuestos/análisis , Propionatos/análisis , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Pixuna virus (PIXV) is an enzootic member of the Venezuelan Equine Encephalitis Virus complex and belongs to the New World cluster of alphaviruses. Herein we explore the role of the cellular cytoskeleton during PIXV replication. We first identified that PIXV undergoes an eclipse phase consisting of 4 h followed by 20 h of an exponential phase in Vero cells. The infected cells showed morphological changes due to structural modifications in actin microfilaments (MFs) and microtubules (MTs). Cytoskeleton-binding agents, that alter the architecture and dynamics of MFs and MTs, were used to study the role of cytoskeleton on PIXV replication. The virus production was significantly affected (p < 0.05) after treatment with paclitaxel or nocodazole due to changes in the MTs network. Interestingly, disassembly of MFs with cytochalasin D, at early stage of PIXV replication cycle, significantly increased the virus yields in the extracellular medium (p < 0.005). Furthermore, the stabilization of actin network with jasplakinolide had no effect on virus yields. Our results demonstrate that PIXV relies not only on intact MTs for the efficient production of virus, but also on a dynamic actin network during the early steps of viral replication.
Asunto(s)
Alphavirus/fisiología , Citoesqueleto/virología , Microtúbulos/virología , Replicación Viral , Alphavirus/efectos de los fármacos , Animales , Chlorocebus aethiops , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Depsipéptidos/farmacología , Interacciones Huésped-Patógeno , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Paclitaxel/farmacología , Factores de Tiempo , Moduladores de Tubulina/farmacología , Células VeroRESUMEN
The fungus Xylaria arbuscula was isolated as an endophyte from Cupressus lusitanica and has shown to be a prominent producer of cytochalasins, mainly cytochalasins C, D and Q. Cytochalasins comprise an important class of fungal secondary metabolites that have aroused attention due to their uncommon molecular structures and pronounced biological activities. Due to the few published studies on the ESI-MS/MS fragmentation of this important class of secondary metabolites, in the first part of our work, we studied the cytochalasin D fragmentation pathways by using an ESI-Q-ToF mass spectrometer coupled with liquid chromatography. We verified that the main fragmentation routes were generated by hydrogen and McLafferty rearrangements which provided more ions than just the ones related to the losses of H2 O and CO as reported in previous studies. We also confirmed the diagnostic ions at m/z 146 and 120 as direct precursor derived from phenylalanine. The present work also aimed the production of structurally diverse cytochalasins by varying the culture conditions used to grow the fungus X. arbuscula and further insights into the biosynthesis of cytochalasins. HPLC-MS analysis revealed no significant changes in the metabolic profile of the microorganism with the supplementation of different nitrogen sources but indicated the ability of X. arbuscula to have access to inorganic and organic nitrogen, such as nitrate, ammonium and amino acids as a primary source of nitrogen. The administration of 2-13 C-glycine showed the direct correlation of this amino acid catabolism and the biosynthesis of cytochalasin D by X. arbuscula, due to the incorporation of three labeled carbons in cytochalasin chemical structure. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Citocalasina D/química , Xylariales/metabolismo , Isótopos de Carbono , Cromatografía Líquida de Alta Presión/métodos , Citocalasina D/metabolismo , Fermentación , Marcaje Isotópico , Estructura Molecular , Peso Molecular , Isótopos de Nitrógeno , Fenilalanina/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity.
Asunto(s)
Polaridad Celular/fisiología , Cinesinas/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Receptor IGF Tipo 1/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Citocalasina D/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Microtúbulos/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Nocodazol/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Paclitaxel/farmacología , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratas , Moduladores de Tubulina/farmacologíaRESUMEN
Mechanical properties of cells are known to be influenced by the actin cytoskeleton. In this article, the action of drugs that interact with the actin cortex is investigated by tether extraction and rheology experiments using optical tweezers. The influences of Blebbistatin, Cytochalasin D and Jasplakinolide on the cell mechanical properties are evaluated. The results, in contradiction to current views for Jasplakinolide, show that all three drugs and treatments destabilize the actin cytoskeleton, decreasing the cell membrane tension. The cell membrane bending modulus increased when the actin cytoskeleton was disorganized by Cytochalasin D. This effect was not observed for Blebbistatin and Jasplakinolide. All drugs decreased by two-fold the cell viscoelastic moduli, but only Cytochalasin D was able to alter the actin network into a more fluid-like structure. The results can be interpreted as the interplay between the actin network and the distribution of myosins as actin cross-linkers in the cytoskeleton. This information may contribute to a better understanding of how the membrane and cytoskeleton are involved in cell mechanical properties, underlining the role that each one plays in these properties.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Citocalasina D/farmacología , Depsipéptidos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Miosinas/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Animales , Fenómenos Biomecánicos , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Elasticidad/efectos de los fármacos , Humanos , Ratones , Células 3T3 NIH , Pinzas Ópticas , Reología , Viscosidad/efectos de los fármacosRESUMEN
The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.(AU)
Asunto(s)
Estructuras Fúngicas , Trichosporon , Colorantes Fluorescentes , Citocalasina D , EndocitosisRESUMEN
Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO2 NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry. TiO2 NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO2 NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO2 NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed. In conclusion, glial cells are able to internalize TiO2 NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO2 NPs internalization and their accumulation in brain cells could be dangerous to human health.
Asunto(s)
Actinas/metabolismo , Endocitosis , Nanopartículas del Metal/administración & dosificación , Neuroglía/fisiología , Neuroglía/ultraestructura , Titanio/administración & dosificación , Amilorida/farmacología , Animales , Caveolina 1/metabolismo , Línea Celular , Cisteína/metabolismo , Citocalasina D/farmacología , Endocitosis/efectos de los fármacos , Humanos , Nanopartículas del Metal/química , Neuroglía/efectos de los fármacos , RatasRESUMEN
BACKGROUND: Granulation tissue remodeling and myofibroblastic differentiation are critically important events during wound healing. Tobacco smoking has a detrimental effect in gingival tissue repair. However, studies evaluating the effects of cigarette smoke on these events are lacking. MATERIAL AND METHODS: We used gingival fibroblasts cultured within free-floating and restrained collagen gels to simulate the initial and final steps of the granulation tissue phase during tissue repair. Collagen gel contraction was stimulated with serum or transforming growth factor-ß1. Cigarette smoke condensate (CSC) was used to evaluate the effects of tobacco smoke on gel contraction. Protein levels of alpha-smooth muscle actin, ß1 integrin, matrix metalloproteinase-3 and connective tissue growth factor were evaluated through Western blot. Prostaglandin E(2) (PGE(2)) levels were determined through ELISA. Actin organization was evaluated through confocal microscopy. RESULTS: CSC reduced collagen gel contraction induced by serum and transforming growth factor-ß1 in restrained collagen gels. CSC also altered the development of actin stress fibers in fibroblasts cultured within restrained collagen gels. PGE(2) levels were strongly diminished by CSC in three-dimensional cell cultures. However, other proteins involved in granulation tissue remodeling and myofibroblastic differentiation such as alpha-smooth muscle actin, ß1 integrin, matrix metalloproteinase-3 and connective tissue growth factor, were unmodified by CSC. CONCLUSIONS: CSC may alter the capacity of gingival fibroblasts to remodel and contract a collagen matrix. Inhibition of PGE(2) production and alterations of actin stress fibers in these cells may impair proper tissue maturation during wound healing in smokers.
Asunto(s)
Dinoprostona/biosíntesis , Fibroblastos/metabolismo , Encía/citología , Nicotiana , Humo/efectos adversos , Actinas/análisis , Sangre , Supervivencia Celular/fisiología , Células Cultivadas , Colágeno Tipo I/efectos de los fármacos , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/análisis , Citocalasina D/farmacología , Dinoprostona/análisis , Fibroblastos/efectos de los fármacos , Geles , Encía/efectos de los fármacos , Humanos , Integrina beta1/análisis , Masculino , Metaloproteinasa 3 de la Matriz/análisis , Nicotina/efectos adversos , Técnicas de Cultivo de Tejidos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.
Asunto(s)
Actinas/metabolismo , Células Cromafines/metabolismo , Familia-src Quinasas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Animales , Calcio/farmacología , Bovinos , Células Cultivadas , Células Cromafines/citología , Células Cromafines/efectos de los fármacos , Citocalasina D/farmacología , Exocitosis/efectos de los fármacos , Cinética , Pirazoles/farmacología , Pirimidinas/farmacología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidad , Lisina/metabolismo , Acetilación/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Aspirina/farmacología , Sitios de Unión , Cricetinae , Citocalasina D/farmacología , Entamoeba histolytica/crecimiento & desarrollo , Entamoeba histolytica/ultraestructura , Masculino , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Movimiento/efectos de los fármacos , Parásitos/efectos de los fármacos , Parásitos/crecimiento & desarrollo , Polimerizacion/efectos de los fármacos , Trofozoítos/efectos de los fármacos , Trofozoítos/crecimiento & desarrollo , Trofozoítos/ultraestructura , VirulenciaRESUMEN
Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca(2+) concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca(2+) spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca(2+) release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca(2+) ionophore, suggesting that the Ca(2+) source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca(2+) rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.
Asunto(s)
Membrana Celular/metabolismo , Fertilización/fisiología , Óvulo/fisiología , Fosfatidilserinas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Anexina A5/metabolismo , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/deficiencia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Membrana Celular/efectos de los fármacos , Citocalasina D/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Femenino , Fluoresceína-5-Isotiocianato/metabolismo , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Óvulo/citología , Óvulo/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermatozoides/citología , Espermatozoides/fisiología , Cigoto/metabolismoRESUMEN
In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO2 molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Glucólisis , Metaboloma , Complejos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Actinas/agonistas , Actinas/antagonistas & inhibidores , Actinas/química , Anticuerpos Antifúngicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citocalasina D/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/metabolismo , Estabilidad de Enzimas/efectos de los fármacos , Fermentación/efectos de los fármacos , Glucólisis/efectos de los fármacos , Cinética , Metaboloma/efectos de los fármacos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Faloidina/farmacología , Polimerizacion/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Tiazolidinas/farmacología , Trehalosa/farmacología , Moduladores de Tubulina/farmacología , ViscosidadRESUMEN
Toxoplasma gondii is a protozoan parasite that can infect the nucleated cells of all warm-blooded animals. Despite its medical and veterinary importance, the egress of T. gondii from host cells has not been fully elucidated. This process is usually studied with calcium ionophores, which artificially trigger T. gondii egress. Among the diverse signaling events that take place during egress, kinases appear to play a crucial role. In this work we employed several kinase inhibitors to examine their role in egress: although parasite egress was only slightly impaired by treatment with the PI3K and PKC inhibitors wortmannin and staurosporine, the addition of the tyrosine kinase-specific inhibitor genistein efficiently blocked the exit of parasites by more than 50%. IPA-3, a non-ATP-competitive inhibitor of p21-activated kinases, which play a role in actin cytoskeleton remodeling inhibited egress of T. gondii by only 15%. The myosin motor inhibitor blebbistatin and the actin polymerization inhibitor cytochalasin D also blocked the egress of T. gondii. Nevertheless, dynasore, which is known to block the GTPase activity of dynamin, had little or no effect on T. gondii egress.
Asunto(s)
Androstadienos/farmacología , Citocalasina D/farmacología , Células Epiteliales/parasitología , Genisteína/farmacología , Estaurosporina/farmacología , Toxoplasma/fisiología , Actinas/antagonistas & inhibidores , Animales , Línea Celular , Dinaminas/antagonistas & inhibidores , Células Epiteliales/efectos de los fármacos , Macaca mulatta , Ratones , Miosinas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , WortmaninaRESUMEN
We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.
Asunto(s)
Acuaporina 2/metabolismo , Riñón/citología , Canales Catiónicos TRPV/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Capsaicina/farmacología , Tamaño de la Célula , Células Cultivadas , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Expresión Génica , Presión Osmótica , Forboles/farmacología , Unión Proteica , Transporte de Proteínas , Ratas , Rojo de Rutenio/farmacología , Estrés Fisiológico , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/genética , Moduladores de Tubulina/farmacologíaRESUMEN
OBJECTIVES: Although platelet-rich plasma (PRP) has been proposed as a therapeutic tool to enhance wound repair, the cellular and molecular mechanisms stimulated by this agent are still not completely understood. The present study was designed to characterize the effects of PRP and platelet-poor plasma (PPP) supernatants on cell responses involved in gingival tissue repair. METHODS: We studied the response of human gingival fibroblasts (HGF) to PRP and PPP fractions on: matrix contraction, cell migration, myofibroblastic differentiation, production of matrix components and proteolytic enzymes. PRP and PPP were obtained from donors using a commercial kit. Matrix contraction was evaluated by means of collagen lattices in the presence of matrix metalloproteinase (MMP) and actin polymerization inhibitors. The production of matrix molecules and proteinases was assessed through Western-blot. RhoA activity was evaluated through a pull-down assay. Actin distribution and focal adhesions were assessed through immunofluorescence. Transforming growth factor-beta (TGF-ß) was quantified through ELISA. RESULTS: Both PRP and PPP stimulated human gingival fibroblasts-populated collagen gel contraction and Ilomastat and cytochalasin D inhibited this response. PRP and PPP also stimulated MT1-MMP and TIMP-2 production, RhoA activation and actin cytoskeleton remodeling, cell migration/invasion and myofibroblastic differentiation. TGF-ß1 was found at significantly higher concentrations in PRP than in PPP. CONCLUSIONS: Both PRP and PPP promote wound tissue remodeling and contraction through the stimulation of actin remodeling, the activity of MMPs, promotion of cell migration, and myofibroblastic differentiation. The similar biological responses induced by PRP and PPP suggest that both platelet-derived fractions may exert a positive effect on gingival repair.
Asunto(s)
Fibroblastos/efectos de los fármacos , Encía/citología , Encía/efectos de los fármacos , Plasma Rico en Plaquetas/fisiología , Cicatrización de Heridas/efectos de los fármacos , Actinas/análisis , Adulto , Western Blotting , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citocalasina D/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Ácidos Hidroxámicos , Indoles/farmacología , Péptidos y Proteínas de Señalización Intercelular/análisis , Masculino , Metaloproteinasas de la Matriz/farmacología , Estadísticas no Paramétricas , Factor de Crecimiento Transformador beta/análisisRESUMEN
Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011.
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Citoesqueleto/metabolismo , Cristalino/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Amidas/farmacología , Animales , Toxinas Botulínicas/genética , Toxinas Botulínicas/metabolismo , Embrión de Pollo , Pollos , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Ectodermo/embriología , Ectodermo/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Inmunohistoquímica , Cristalino/embriología , Miosina Tipo II/antagonistas & inhibidores , Miosina Tipo II/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.
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Axones/enzimología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Cinética , Quinasas Lim/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , Modelos Biológicos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Extensive evidence supports the notion that the cytoskeleton participates in the immobilization and membrane clustering of the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Stimulated emission depletion fluorescence microscopy has revealed the supramolecular organization of AChR nanoclusters at the surface of CHO-K1/A5 cells with subdiffraction resolution (Kellner et al., Neuroscience 144:135-143 2007). We studied the effect of two cytoskeletal-disrupting drugs (cytochalasin D and jasplakinolide) on the nanoscale distribution of muscle-type AChR expressed in these cells by means of mathematical and statistical analysis of images obtained with the same high-resolution microscopy. AChR nanoclusters were found to be randomly distributed in both controls and cells treated with either drug for distances larger than 500 nm. Treatments altered the distribution of AChR nanoclusters according to their brightness/size. Cytochalasin D and jasplakinolide produced a statistically significant increase in the proportion of medium-size nanoclusters and a diminution of small nanoclusters, indicating higher disrupting activity on the latter. This was further corroborated by the diminution of the brightness/diameter ratio of nanoclusters (a measure of the intracluster density of AChR molecules) and by Ripley's analysis applied to simulated patterns with intracluster aggregation of AChR molecules. The combined analytical tools bring out subtle changes in the two-dimensional organization of the AChR nanoaggregates on disruption of the cytoskeletal network and throw light on the possible link between the cytoskeleton and the distribution of the AChR at the cell surface.
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Receptores Nicotínicos/efectos de los fármacos , Animales , Células CHO , Cricetinae , Cricetulus , Citocalasina D/farmacología , Depsipéptidos/farmacología , Microscopía Confocal , Microscopía FluorescenteRESUMEN
BACKGROUND: Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. METHODOLOGY AND PRINCIPAL FINDINGS: By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%). CONCLUSIONS AND SIGNIFICANCE: All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression.