RESUMO
Ezrin, radixin, and moesin (ERM) family proteins regulate cytoskeletal responses by tethering the plasma membrane to the underlying actin cortex. Mutations in ERM proteins lead to severe combined immunodeficiency, but the function of these proteins in T cells remains poorly defined. Using mice in which T cells lack all ERM proteins, we demonstrate a selective role for these proteins in facilitating S1P-dependent egress from lymphoid organs. ERM-deficient T cells display defective S1P-induced migration in vitro, despite normal responses to standard protein chemokines. Analysis of these defects revealed that S1P promotes a fundamentally different mode of migration than chemokines, characterized by intracellular pressurization and bleb-based motility. ERM proteins facilitate this process, controlling directional migration by limiting blebbing to the leading edge. We propose that the distinct modes of motility induced by S1P and chemokines are specialized to allow T cell migration across lymphatic barriers and through tissue stroma, respectively.
Assuntos
Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/fisiologia , Linfócitos/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Esfingosina/análogos & derivados , Animais , Membrana Celular , Proteínas do Citoesqueleto/genética , Feminino , Linfócitos/citologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Fosforilação , Esfingosina/metabolismoRESUMO
Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.
Assuntos
Actinas/metabolismo , Giardia lamblia/metabolismo , Plaquinas/metabolismo , Tubulina (Proteína)/metabolismo , Actinas/química , Sequência de Aminoácidos , Animais , Anquirinas/química , Sequência de Bases , Western Blotting , Biologia Computacional , Sequência Consenso , Citoplasma/química , Citoesqueleto/química , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Dinaminas/análise , Feminino , Imunofluorescência , Giardia lamblia/química , Giardia lamblia/ultraestrutura , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Plaquinas/química , Alinhamento de Sequência , Tubulina (Proteína)/químicaRESUMO
The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.
Assuntos
Movimento Celular/fisiologia , Conexinas/fisiologia , Células Dendríticas/fisiologia , Leucócitos/fisiologia , Fagócitos/fisiologia , Nucleotídeos de Adenina/fisiologia , Envelhecimento/imunologia , Envelhecimento/fisiologia , Animais , Astrócitos/fisiologia , Polaridade Celular , Quimiotaxia de Leucócito/fisiologia , Citoesqueleto/fisiologia , Fibroblastos/fisiologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Queratinócitos/fisiologia , Mecanotransdução Celular/fisiologia , Neoplasias/imunologia , Degeneração Neural/imunologia , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Purinérgicos/fisiologiaRESUMO
Cell motility is a central process involved in fundamental biological phenomena during embryonic development, wound healing, immune surveillance, and cancer spreading. Cell movement is complex and dynamic and requires the coordinated activity of cytoskeletal, membrane, adhesion and extracellular proteins. Cellular prion protein (PrPC) has been implicated in distinct aspects of cell motility, including axonal growth, transendothelial migration, epithelial-mesenchymal transition, formation of lamellipodia, and tumor migration and invasion. The preferential location of PrPC on cell membrane favors its function as a pivotal molecule in cell motile phenotype, being able to serve as a scaffold protein for extracellular matrix proteins, cell surface receptors, and cytoskeletal multiprotein complexes to modulate their activities in cellular movement. Evidence points to PrPC mediating interactions of multiple key elements of cell motility at the intra- and extracellular levels, such as integrins and matrix proteins, also regulating cell adhesion molecule stability and cell adhesion cytoskeleton dynamics. Understanding the molecular mechanisms that govern cell motility is critical for tissue homeostasis, since uncontrolled cell movement results in pathological conditions such as developmental diseases and tumor dissemination. In this review, we discuss the relevant contribution of PrPC in several aspects of cell motility, unveiling new insights into both PrPC function and mechanism in a multifaceted manner either in physiological or pathological contexts.
Assuntos
Movimento Celular/fisiologia , Proteínas Priônicas/metabolismo , Animais , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , HumanosRESUMO
BACKGROUND AND OBJECTIVE: Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. METHODS: Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test. RESULTS: Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. CONCLUSION: Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.
Assuntos
Proliferação de Células , Senescência Celular , Citoesqueleto/fisiologia , Fibroblastos/citologia , Envelhecimento , Células Cultivadas , Gengiva/citologia , HumanosRESUMO
Lonomia obliqua is a caterpillar of potential therapeutic interest whose venom is able to induce severe blood leakage and modulate leukocyte migration. Since both phenotypes are associated with changes in cytoskeleton dynamics and cell adhesion properties, the aim of this study was to analyze the effects of Lonomia obliqua bristle extract (LOBE) in cell adhesion and migration signaling. Proteomic analysis revealed that epithelial cells (CHO-K1) exposed to LOBE (30⯵g/mL, 30â¯min) exhibited changes in levels of actin regulatory proteins, including RhoGTPases. These changes correlated with an increase in the activity of the RhoGTPase family member Rac as measured by Förster resonance energy transfer (FRET). When plated in migration promoting conditions, CHO-K1 cells exposed to LOBE (10⯵g/mL) showed an increase in membrane ruffling after short (30â¯min) period of incubation that was accompanied by changes in the distribution of the adhesion markers paxillin, vinculin and an increase of focal adhesion kinase autophosphorylation levels (Y397), suggesting changes in cell-extracellular matrix (ECM) adhesion properties and signaling. These data suggest that LOBE possesses bioactive molecules that are capable to modulated cell migration signaling, cytoskeletal dynamics and cell-ECM properties of several cell types.
Assuntos
Venenos de Artrópodes/toxicidade , Adesão Celular/efeitos dos fármacos , Mariposas/química , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Células CHO , Movimento Celular/efeitos dos fármacos , Cricetulus , Citoesqueleto/fisiologia , Larva/química , Paxilina/metabolismo , Fosforilação , Proteoma/análise , Vinculina/metabolismoRESUMO
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
Trypanosoma cruzi, the causative agent of Chagas disease, is a public health concern in Latin America. Epigenetic events, such as histone acetylation, affect DNA topology, replication and gene expression. Histone deacetylases (HDACs) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. HDAC inhibitors, like trichostatin A (TSA), inhibit tumour cell proliferation and promotes ultrastructural modifications. In the present study, TSA effects on cell proliferation, viability, cell cycle and ultrastructure were evaluated, as well as on histone acetylation and tubulin expression of the T. cruzi epimastigote form. Protozoa proliferation and viability were reduced after treatment with TSA. Quantitative proteomic analyses revealed an increase in histone acetylation after 72 h of TSA treatment. Surprisingly, results obtained by different microscopy methodologies indicate that TSA does not affect chromatin compaction, but alters microtubule cytoskeleton dynamics and impair kDNA segregation, generating polynucleated cells with atypical morphology. Confocal fluorescence microscopy and flow cytometry assays indicated that treated cell microtubules were more intensely acetylated. Increases in tubulin acetylation may be directly related to the higher number of parasites in the G2/M phase after TSA treatment. Taken together, these results suggest that deacetylase inhibitors represent excellent tools for understanding trypanosomatid cell biology.
Assuntos
Divisão Celular/fisiologia , Citoesqueleto/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Histonas/química , Ácidos Hidroxâmicos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Tubulina (Proteína)/química , Acetilação , Microtúbulos/fisiologiaRESUMO
BACKGROUND: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. RESULTS: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. CONCLUSION: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.
Assuntos
Proteínas 14-3-3/metabolismo , Aedes/imunologia , Imunidade Celular , Proteínas de Insetos/metabolismo , Mosquitos Vetores/imunologia , Fagocitose , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/genética , Actinas/metabolismo , Aedes/citologia , Animais , Linhagem Celular , Citoplasma/química , Citoesqueleto/fisiologia , Escherichia coli/imunologia , Inativação Gênica , Proteínas de Insetos/deficiência , Proteínas de Insetos/genética , Mosquitos Vetores/citologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Staphylococcus aureus/imunologiaRESUMO
The cytoskeleton (CSK) is a tensed fiber framework that supports, shapes and stabilizes the cell. The CSK is in a constant state of remodeling, moreover, which is an active non-equilibrium thermodynamic process. We report here that cytoskeletal remodeling involves reconfigurations that are not only sudden but also are transmitted to great distances within the cell in a fashion reminiscent of quakes in the Earth's crust. Remarkably, these events in the cell conform both qualitatively and quantitatively to empirical laws typical of earthquakes, including hierarchical fault structures, cumulative energy distributions following the Gutenberg-Richter law, and rate of after-shocks following Omori's law. While it is well-established that remodeling and stabilization of the cytoskeleton are non-equilibrium process, these new unanticipated observations establish that these processes are also remarkably non-local and strongly cooperative.