RESUMO
P21 is an immunomodulatory protein expressed throughout the life cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease. In vitro and in vivo studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization. Furthermore, in vivo studies have shown that P21 inhibits angiogenesis, induces inflammation and fibrosis, and regulates intracellular amastigote replication. In this study, we used the CRISPR/Cas9 system for P21 gene knockout and investigated whether the ablation of P21 results in changes in the phenotypes associated with this protein. Ablation of P21 gene resulted in a lower growth rate of epimastigotes and delayed cell cycle progression, accompanied by accumulation of parasites in G1 phase. However, P21 knockout epimastigotes were viable and able to differentiate into metacyclic trypomastigotes, which are infective to mammalian cells. In comparison with wild-type parasites, P21 knockout cells showed a reduced cell invasion rate, demonstrating the role of this protein in host cell invasion. However, there was a higher number of intracellular amastigotes per cell, suggesting that P21 is a negative regulator of amastigote proliferation in mammalian cells. Here, for the first time, we demonstrated the direct correlation between P21 and the replication of intracellular amastigotes, which underlies the chronicity of T. cruzi infection.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Citoesqueleto de Actina/fisiologia , Animais , Doença de Chagas/parasitologia , Técnicas de Inativação de Genes , Estágios do Ciclo de Vida/fisiologia , Mamíferos/genética , Camundongos , Trypanosoma cruzi/fisiologiaRESUMO
Chagas' disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this pathogen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for maintenance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell's actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages-a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to preserve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomastigotes may trigger sudden egress. IMPORTANCE Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite's lytic cycle: how parasites egress and thereby sustain round after round of infection. Our results show that once in the host cell cytosol and having undergone amastigogenesis, T. cruzi begins to alter the host cell cytoskeleton, remodeling normal F-actin meshworks into encapsulating spheroidal cages. Filamentous actin diminishes over the course of the lytic cycle, and just prior to egress, the filaments comprising the cages are severely degraded where adjacent to the parasites. We conclude that sudden egress follows breach of the containment afforded by the actin cytoskeleton and subsequent plasma membrane rupture-a process that when understood in molecular detail may serve as a target for future novel therapeutic interventions.
Assuntos
Citoesqueleto de Actina/fisiologia , Membrana Celular/patologia , Citoesqueleto/metabolismo , Citoesqueleto/parasitologia , Interações Hospedeiro-Parasita , Trypanosoma cruzi/fisiologia , Actinas/metabolismo , Animais , Membrana Celular/parasitologia , Doença de Chagas/parasitologia , Chlorocebus aethiops , Células VeroRESUMO
BACKGROUND: UV-B signaling in plants is mediated by UVR8, which interacts with transcriptional factors to induce root morphogenesis. However, research on the downstream molecules of UVR8 signaling in roots is still scarce. As a wide range of functional cytoskeletons, how actin filaments respond to UV-B-induced root morphogenesis has not been reported. The aim of this study was to investigate the effect of actin filaments on root morphogenesis under UV-B and hydrogen peroxide exposure in Arabidopsis. RESULTS: A Lifeact-Venus fusion protein was used to stain actin filaments in Arabidopsis. The results showed that UV-B inhibited hypocotyl and root elongation and caused an increase in H2O2 content only in the root but not in the hypocotyl. Additionally, the actin filaments in hypocotyls diffused under UV-B exposure but were gathered in a bundle under the control conditions in either Lifeact-Venus or uvr8 plants. Exogenous H2O2 inhibited root elongation in a dose-dependent manner. The actin filaments changed their distribution from filamentous to punctate in the root tips and mature regions at a lower concentration of H2O2 but aggregated into thick bundles with an abnormal orientation at H2O2 concentrations up to 2 mM. In the root elongation zone, the actin filament arrangement changed from lateral to longitudinal after exposure to H2O2. Actin filaments in the root tip and elongation zone were depolymerized into puncta under UV-B exposure, which showed the same tendency as the low-concentration treatments. The actin filaments were hardly filamentous in the maturation zone. The dynamics of actin filaments in the uvr8 group under UV-B exposure were close to those of the control group. CONCLUSIONS: The results indicate that UV-B inhibited Arabidopsis hypocotyl elongation by reorganizing actin filaments from bundles to a loose arrangement, which was not related to H2O2. UV-B disrupted the dynamics of actin filaments by changing the H2O2 level in Arabidopsis roots. All these results provide an experimental basis for investigating the interaction of UV-B signaling with the cytoskeleton.
Assuntos
Citoesqueleto de Actina/fisiologia , Arabidopsis/crescimento & desenvolvimento , Peróxido de Hidrogênio/farmacologia , Raízes de Plantas/crescimento & desenvolvimento , Raios Ultravioleta , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis , Proteínas Cromossômicas não HistonaRESUMO
BACKGROUND: UV-B signaling in plants is mediated by UVR8, which interacts with transcriptional factors to induce root morphogenesis. However, research on the downstream molecules of UVR8 signaling in roots is still scarce. As a wide range of functional cytoskeletons, how actin filaments respond to UV-B-induced root morphogenesis has not been reported. The aim of this study was to investigate the effect of actin filaments on root morphogenesis under UV-B and hydrogen peroxide exposure in Arabidopsis. RESULTS: A Lifeact-Venus fusion protein was used to stain actin filaments in Arabidopsis. The results showed that UV-B inhibited hypocotyl and root elongation and caused an increase in H2O2 content only in the root but not in the hypocotyl. Additionally, the actin filaments in hypocotyls diffused under UV-B exposure but were gathered in a bundle under the control conditions in either Lifeact-Venus or uvr8 plants. Exogenous H2O2 inhibited root elongation in a dose-dependent manner. The actin filaments changed their distribution from filamentous to punctate in the root tips and mature regions at a lower concentration of H2O2 but aggregated into thick bundles with an abnormal orientation at H2O2 concentrations up to 2 mM. In the root elongation zone, the actin filament arrangement changed from lateral to longitudinal after exposure to H2O2. Actin filaments in the root tip and elongation zone were depolymerized into puncta under UV-B exposure, which showed the same tendency as the low-concentration treatments. The actin filaments were hardly filamentous in the maturation zone. The dynamics of actin filaments in the uvr8 group under UV-B exposure were close to those of the control group. CONCLUSIONS: The results indicate that UV-B inhibited Arabidopsis hypocotyl elongation by reorganizing actin filaments from bundles to a loose arrangement, which was not related to H2O2. UV-B disrupted the dynamics of actin filaments by changing the H2O2 level in Arabidopsis roots. All these results provide an experimental basis for investigating the interaction of UV-B signaling with the cytoskeleton.
Assuntos
Raios Ultravioleta , Citoesqueleto de Actina/fisiologia , Arabidopsis/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Peróxido de Hidrogênio/farmacologia , Proteínas Cromossômicas não Histona , Arabidopsis/efeitos da radiação , Proteínas de ArabidopsisRESUMO
Echinococcus granulosus is a cestode parasite whose cytoskeleton plasticity allows it to enter and develop inside its hosts, completing thus its life cycle. We focused our attention on F-actin organization and distribution in E. granulosus protoscoleces (PSC) in order to contribute to the knowledge of the parasite cytoskeleton. In particular, we addressed some aspects of F-actin rearrangements in PSC at different stages of the evagination/invagination process. The use of light microscopy allowed us to identify different PSC structures and phalloidin staining displayed a parasite's highly organized F-actin cytoskeleton. Suckers exhibit an important musculature composed of a set of radial fibers. At the rostellum, the F-actin filaments are arranged in a bulbar shape with perforations that appear to be the attachment places for the hooks. Also, "circular" structures of F-actin were identified, which remind the flame cells. Furthermore, parasite F-actin filaments, unevenly distributed, seem to have remained substantially unchanged during the evagination/invagination process. Finally, we showed that the scolex of an evaginated E. granulosus PSC reinvaginates in vitro without any treatment.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/análise , Echinococcus granulosus/anatomia & histologia , Echinococcus granulosus/fisiologia , Matadouros , Citoesqueleto de Actina/ultraestrutura , Animais , Bovinos , Echinococcus granulosus/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Pulmão/parasitologia , Microscopia de Fluorescência , Faloidina , Coloração e Rotulagem , UruguaiRESUMO
Although redox processes closely interplay with mechanoresponses to control vascular remodeling, redox pathways coupling mechanostimulation to cellular cytoskeletal organization remain unclear. The peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) supports postinjury vessel remodeling. Using distinct models, we investigated whether pecPDIA1 could work as a redox-dependent organizer of cytoskeletal mechanoresponses. In vascular smooth muscle cells (VSMCs), pecPDIA1 immunoneutralization impaired stress fiber assembly in response to equibiaxial stretch and, under uniaxial stretch, significantly perturbed cell repositioning perpendicularly to stretch orientation. During cyclic stretch, pecPDIA1 supported thiol oxidation of the known mechanosensor ß1-integrin and promoted polarized compartmentalization of sulfenylated proteins. Using traction force microscopy, we showed that pecPDIA1 organizes intracellular force distribution. The net contractile moment ratio of platelet-derived growth factor-exposed to basal VSMCs decreased from 0.90 ± 0.09 (IgG-exposed controls) to 0.70 ± 0.08 after pecPDI neutralization ( P < 0.05), together with an enhanced coefficient of variation for distribution of force modules, suggesting increased noise. Moreover, in a single cell model, pecPDIA1 neutralization impaired migration persistence without affecting total distance or velocity, whereas siRNA-mediated total PDIA1 silencing disabled all such variables of VSMC migration. Neither expression nor total activity of the master mechanotransmitter/regulator RhoA was affected by pecPDIA1 neutralization. However, cyclic stretch-induced focal distribution of membrane-bound RhoA was disrupted by pecPDI inhibition, which promoted a nonpolarized pattern of RhoA/caveolin-3 cluster colocalization. Accordingly, FRET biosensors showed that pecPDIA1 supports localized RhoA activity at cell protrusions versus perinuclear regions. Thus, pecPDI acts as a thiol redox-dependent organizer and noise reducer mechanism of cytoskeletal repositioning, oxidant generation, and localized RhoA activation during a variety of VSMC mechanoresponses. NEW & NOTEWORTHY Effects of a peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) during mechanoregulation in vascular smooth muscle cells (VSMCs) were highlighted using approaches such as equibiaxial and uniaxial stretch, random single cell migration, and traction force microscopy. pecPDIA1 regulates organization of the cytoskeleton and minimizes the noise of cell alignment, migration directionality, and persistence. pecPDIA1 mechanisms involve redox control of ß1-integrin and localized RhoA activation. pecPDIA1 acts as a novel organizer of mechanoadaptation responses in VSMCs.
Assuntos
Adaptação Fisiológica/fisiologia , Citoesqueleto/fisiologia , Miócitos de Músculo Liso/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Fenômenos Biomecânicos , Movimento Celular , Células Cultivadas , Inativação Gênica , Integrina beta1/metabolismo , Músculo Liso Vascular/metabolismo , Oxidantes/metabolismo , Pressorreceptores , Isomerases de Dissulfetos de Proteínas/genética , Coelhos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in ß2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in human neutrophils. However, inhibition of the major RhoA effector protein, Rho kinase (ROCK), by Y-27632 failed to inhibit TNF-induced neutrophil adhesion. In contrast, the formin FH2 domain inhibitor, SMIFH2, abolished TNF-induced human neutrophil adhesion and diminished leukocyte recruitment in vivo. SMIFH2 also inhibited TNF-induced cytoskeletal reorganization in human neutrophils and abolished the alterations in ß2-integrin expression elicited by TNF stimulation. As such, Rho GTPase/mDia formin-mediated cytoskeletal reorganization appears to participate in the orchestration of TNF-induced neutrophil-adhesive interactions, possibly mediated by formin-mediated actin nucleation and subsequent modulation of ß2-integrin activity on the neutrophil surface. This pathway may represent a pharmacologic target for reducing leukocyte recruitment in inflammatory diseases.
Assuntos
Citoesqueleto de Actina/fisiologia , Antígenos CD18/metabolismo , Adesão Celular , Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neutrófilos/fisiologia , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Adolescente , Adulto , Animais , Antígenos CD18/genética , Células Cultivadas , Proteínas Fetais/genética , Forminas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Proteínas Nucleares/genética , Transdução de Sinais , Adulto Jovem , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.
Assuntos
Colo/fisiologia , Enterócitos/fisiologia , Células Epiteliais/fisiologia , Rim/fisiologia , Microtúbulos/fisiologia , Microvilosidades/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Polaridade Celular , Centrômero/fisiologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/ultraestrutura , Cães , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Rim/efeitos dos fármacos , Rim/ultraestrutura , Células Madin Darby de Rim Canino , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Nocodazol/farmacologia , Fatores de Tempo , Moduladores de Tubulina/farmacologiaRESUMO
The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²âº/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Citoesqueleto/fisiologia , Hormônios de Invertebrado/metabolismo , Ovário/metabolismo , Palaemonidae/fisiologia , Pigmentos Biológicos/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Transporte Biológico/efeitos dos fármacos , Brasil , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/fisiologia , Extensões da Superfície Celular/ultraestrutura , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Feminino , Toxinas Marinhas/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Miosinas/antagonistas & inibidores , Miosinas/metabolismo , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Miosina não Muscular Tipo IIB/metabolismo , Oligopeptídeos/metabolismo , Ovário/efeitos dos fármacos , Ovário/ultraestrutura , Palaemonidae/efeitos dos fármacos , Palaemonidae/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo , Rios , Moduladores de Tubulina/farmacologiaRESUMO
Mechanical pressure plays an important role in many physiological and pathological processes. Mimicking the mechanical pressure present in vitro is necessary for related research, but usually requires expensive and complicated equipment. In this study we created a simple pressure culture system based on the transwell culture system. By cutting off the top rim of the transwell insert, the cells were compressed between the insert membrane and the well floor. The new pressure culture system was proven effective in that it induced cell morphological change, integrin ß1 upregulation, actin polymerization and growth change in rat retinal ganglion cells, human nasopharyngeal carcinoma cells and mice embryonic fibroblasts. Though the pressure value is immeasurable and inhomogeneous, the easily available culture system still provides a choice for the laboratories that do not have access to the better, but much more expensive pressure culture equipment.
Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Integrina beta1/genética , Citoesqueleto de Actina/fisiologia , Análise de Variância , Animais , Carcinoma , Linhagem Celular/fisiologia , Fibroblastos/fisiologia , Imunofluorescência/métodos , Humanos , Pressão Hidrostática , Metilaminas , Camundongos Endogâmicos C57BL , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Cultura Primária de Células , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estresse MecânicoRESUMO
Mechanical pressure plays an important role in many physiological and pathological processes. Mimicking the mechanical pressure present in vitro is necessary for related research, but usually requires expensive and complicated equipment. In this study we created a simple pressure culture system based on the transwell culture system. By cutting off the top rim of the transwell insert, the cells were compressed between the insert membrane and the well floor. The new pressure culture system was proven effective in that it induced cell morphological change, integrin β1 upregulation, actin polymerization and growth change in rat retinal ganglion cells, human nasopharyngeal carcinoma cells and mice embryonic fibroblasts. Though the pressure value is immeasurable and inhomogeneous, the easily available culture system still provides a choice for the laboratories that do not have access to the better, but much more expensive pressure culture equipment.
Assuntos
Animais , Humanos , Ratos , /genética , Proliferação de Células , Técnicas de Cultura de Células/métodos , Análise de Variância , Citoesqueleto de Actina/fisiologia , Linhagem Celular/fisiologia , Fibroblastos/fisiologia , Imunofluorescência/métodos , Pressão Hidrostática , Metilaminas , Neoplasias Nasofaríngeas/patologia , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estresse MecânicoRESUMO
In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.
Assuntos
Citoesqueleto de Actina/fisiologia , Músculo Liso/citologia , Miosinas/fisiologia , Túbulos Seminíferos/citologia , Animais , Endotelina-1/fisiologia , Masculino , Músculo Liso/fisiologia , Ratos , Túbulos Seminíferos/fisiologiaRESUMO
The retinal pigment epithelium (RPE) plays an essential role in the function of the neural retina and the maintenance of vision. Most of the functions displayed by RPE require a dynamic organization of the acto-myosin cytoskeleton. Myosin II, a main cytoskeletal component in muscle and non-muscle cells, is directly involved in force generation required for organelle movement, selective molecule transport within cell compartments, exocytosis, endocytosis, phagocytosis, and cell division, among others. Contractile processes are triggered by the phosphorylation of myosin II light chains (MLCs), which promotes actin-myosin interaction and the assembly of contractile fibers. Considerable evidence indicates that non-muscle myosin II activation is critically involved in various pathological states, increasing the interest in studying the signaling pathways controlling MLC phosphorylation. Particularly, recent findings suggest a role for non-muscle myosin II-induced contraction in RPE cell transformation involved in the establishment of numerous retinal diseases. This review summarizes the current knowledge regarding myosin function in RPE cells, as well as the signaling networks leading to MLC phosphorylation under pathological conditions. Understanding the molecular mechanisms underlying RPE dysfunction would improve the development of new therapies for the treatment or prevention of different ocular disorders leading to blindness.
Assuntos
Células Epiteliais/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Células Epiteliais/fisiologia , Humanos , Modelos Biológicos , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/fisiologia , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/fisiologia , Fosforilação/genética , Epitélio Pigmentado da Retina/fisiologiaRESUMO
PURPOSE: The transplant of retinal pigment epithelium (RPE) cells on supports may well be an effective therapeutic approach to improve the visual results of patients with age-related macular degeneration. In this study, two biodegradable polyurethanes were investigated as supports for human RPE cells (ARPE-19). METHODS: Polyurethane aqueous dispersions based on poly(caprolactone) and/or poly(ethylene glycol) as soft segments, and isophorone diisocyanate and hydrazine as hard segments were prepared. Polyurethane films were produced by casting the dispersions and allowing them to dry at room temperature for one week. The ARPE-19 cells were seeded onto the polyurethane films and they were investigated as supports for in vitro adhesion, proliferation, and uniform distribution of differentiated ARPE-19 cells. Additionally, the in vivo ocular biocompatibility of the polyurethane films was evaluated. RESULTS: The RPE adhered to and proliferated onto the polyurethane supports, thus establishing cell-PUD surface interactions. Upon confluence, the cells formed an organized monolayer, exhibited a polygonal appearance, and displayed actin filaments which ran along the upper cytoplasm. At 15 days of seeding, the occluding expression was confirmed between adjacent cells, representing the barrier functionality of epithelial cells on polymeric surfaces and the establishment of cell-cell interactions. Results from the in vivo study indicated that polyurethanes exhibited a high degree of short-term intraocular biocompatibility. CONCLUSIONS: Biodegradable polyurethane films display the proper mechanical properties for an easy transscleral-driven subretinal implantation and can be considered as biocompatible supports for a functional ARPE-19 monolayer.
Assuntos
Materiais Biocompatíveis , Proliferação de Células , Células Epiteliais/fisiologia , Poliuretanos/química , Epitélio Pigmentado da Retina/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Citoesqueleto de Actina/fisiologia , Animais , Adesão Celular , Diferenciação Celular , Linhagem Celular , Células Epiteliais/transplante , Feminino , Humanos , Hidrazinas/química , Imuno-Histoquímica , Isocianatos/química , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Poliésteres/química , Polietilenoglicóis/química , Ratos , Ratos Endogâmicos BN , Epitélio Pigmentado da Retina/transplante , Fatores de TempoRESUMO
To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Proteínas do Citoesqueleto/fisiologia , Matriz Extracelular/fisiologia , Filamentos Intermediários/fisiologia , Músculo Esquelético/embriologia , Citoesqueleto de Actina/ultraestrutura , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Ensaios de Migração Celular , Desmina/metabolismo , Matriz Extracelular/ultraestrutura , Filamentos Intermediários/ultraestrutura , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Plectina/metabolismo , Peixe-ZebraRESUMO
Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a highly prevalent vector-borne disease in Latin America. Chagas' disease is a major public health problem in endemic regions with an estimated 18 million people are infected with T. cruzi and another 100 million at risk (http://www.who.int/ctd/chagas/disease.htm). During its life cycle, T. cruzi alternates between triatomine insect vectors and mammalian hosts. While feeding on host's blood, infected triatomines release in their feces highly motile and infective metacyclic trypomastigotes that may initiate infection. Metacyclic trypomastigotes promptly invade host cells (including gastric mucosa) and once free in the cytoplasm, differentiate into amastigotes that replicate by binary fission. Just before disruption of the parasite-laden cell, amastigotes differentiate back into trypomastigotes which are then released into the tissue spaces and access the circulation. Circulating trypomastigotes that disseminate the infection in the mammalian host may be taken up by feeding triatomines and may also transform, extracellularly, into amastigote-like forms. Unlike their intracellular counterparts, these amastigote-like forms, henceforth called amastigotes, are capable of infecting host cells. Studies in which the mechanisms of amastigote invasion of host cells have been compared to metacyclic trypomastigote entry have revealed interesting differences regarding the involvement of the target cell actin microfilament system.
Assuntos
Actinas/metabolismo , Estágios do Ciclo de Vida/fisiologia , Trypanosoma cruzi/fisiologia , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Chlorocebus aethiops , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Trypanosoma cruzi/ultraestrutura , Células VeroRESUMO
Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca(2+)-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca(2+) in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca(2+). Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca(2+) with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca(2+) from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.
Assuntos
Citoesqueleto de Actina/fisiologia , Cálcio/metabolismo , Exocitose/fisiologia , Mastócitos/metabolismo , Polímeros/metabolismo , Rianodina/farmacologia , Citoesqueleto de Actina/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cafeína/farmacologia , Degranulação Celular , Citoplasma/metabolismo , Exocitose/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Peritônio/citologia , Ratos , Ratos Sprague-Dawley , Tiazolidinas/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Physical and functional association between the beta2-integrin Mac-1 (CD11b/CD18) and receptors of immunoglobulin G (IgG) (FcgammaRs) has been previously reported. In this study, we examined the modulation of Mac-1 expression by IgG in different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate the expression of Mac-1 after overnight exposure to surface-bound IgG. This effect, which requires at least 6 h of incubation, is not associated with a general downmodulation of membrane antigens, and is selectively induced by immobilized IgG (iIgG), as the stimulation of monocytes with N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha) or soluble IgG did not modify the Mac-1 expression after 18 h in culture. The loss of Mac-1 was completely prevented by blocking antibodies (Abs) directed to FcgammaRII (CD32) or CD18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac-1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac-1. However, there was no inhibition of TNF-alpha production induced by zymosan, suggesting that Mac-1-dependent responses require different levels of its expression to be fully activated.
Assuntos
Imunoglobulina G/farmacologia , Antígeno de Macrófago 1/metabolismo , Monócitos/metabolismo , Citoesqueleto de Actina/fisiologia , Antígenos CD18/fisiologia , Células Cultivadas , Regulação para Baixo , Humanos , Neutrófilos/metabolismo , Fagocitose , Receptores Fc/fisiologia , Receptores de IgG/fisiologia , Serina Endopeptidases/metabolismo , Zimosan/metabolismoRESUMO
In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.
Assuntos
Axônios/fisiologia , Proteínas/fisiologia , Células Piramidais/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Polaridade Celular/fisiologia , Células Cultivadas , Imunofluorescência , Cones de Crescimento/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Microtúbulos/fisiologia , Proteínas de Neoplasias , Ratos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
The electric eel Electrophorus electricus is a fresh water teleost showing an electrogenic tissue that produces electric discharges. This electrogenic tissue is distributed in three well-defined electric organs which may be found symmetrically along both sides of the eel. These electric organs develop from muscle and exhibit several biochemical properties and morphological features of the muscle sarcolema. This review examines the contribution of the cytoskeletal meshwork to the maintenance of the polarized organization of the electrocyte, the cell that contains all electric properties of each electric organ. The cytoskeletal filaments display an important role in the establishment and maintenance of the highly specialized membrane model system of the electrocyte. As a muscular tissue, these electric organs expresses actin and desmin. The studies that characterized these cytoskeletal proteins and their implications on the electrophysiology of the electric tissues are revisited.