RESUMO
Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.
Assuntos
Movimento Celular , Entamoeba histolytica , Fibronectinas , Entamoeba histolytica/metabolismo , Entamoeba histolytica/fisiologia , Fibronectinas/metabolismo , Humanos , Movimento Celular/fisiologia , Entamebíase/parasitologia , Entamebíase/metabolismo , Actinas/metabolismo , Podossomos/metabolismo , Adesão Celular/fisiologia , Proteínas de Protozoários/metabolismoRESUMO
The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared "public" clones. We previously reported a strong public IgHµ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates.
Assuntos
Formação de Anticorpos/imunologia , Peixes/imunologia , Mamíferos/imunologia , Animais , Linfócitos B/imunologia , Células Clonais/imunologia , Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/imunologia , Recombinação V(D)J/imunologia , Vacinação/métodosRESUMO
Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Citoesqueleto/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de Interleucina-7/metabolismo , Citoesqueleto de Actina/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/ultraestrutura , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Toxina da Cólera/imunologia , Citoesqueleto/ultraestrutura , Humanos , Janus Quinase 1/metabolismo , Janus Quinase 3/metabolismo , Cinética , Ativação Linfocitária , Microdomínios da Membrana/ultraestrutura , Microtúbulos/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Receptores de Interleucina-7/fisiologia , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
One of the most common approaches for large-scale protein identification is LC, followed by MS. If more than a few proteins are to be identified, the additional fragmentation of individual peptides has so far been considered as indispensable, and thus, the associated costs, in terms of instrument time and infrastructure, as unavoidable. Here, we present evidence to the contrary. Using a combination of (i) highly accurate and precise mass measurements, (ii) modern retention time prediction, and (iii) a robust scoring algorithm, we were able to identify 257 proteins of Francisella tularensis from a single LC-MS experiment in a fragmentation-free approach (i.e. without experimental fragmentation spectra). This number amounts to 59% of the number of proteins identified in a standard fragmentation-based approach, when executed with the same false discovery rate. Independent evidence supports at least 27 of a set of 31 proteins that were identified only in the fragmentation-free approach. Our results suggest that additional developments in retention time prediction, measurement technology, and scoring algorithms may render fragmentation-free approaches an interesting complement or an alternative to fragmentation-based approaches.
Assuntos
Cromatografia Líquida de Alta Pressão , Biologia Computacional , Espectrometria de Massas em Tandem/métodos , Francisella tularensis/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismoRESUMO
Adenomatous polyposis coli (Apc) is a cell polarity regulator and a tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Apc involvement in T lymphocyte functions and antitumor immunity remains poorly understood. Investigating Apc-depleted human CD8 T cells and CD8 T cells from ApcMin/+ mutant mice, we found that Apc regulates actin and microtubule cytoskeleton remodeling at the immunological synapse, controlling synapse morphology and stability and lytic granule dynamics, including targeting and fusion at the synapse. Ultimately, Apc tunes cytotoxic T cell activity, leading to tumor cell killing. Furthermore, Apc modulates early TCR signaling and nuclear translocation of the NFAT transcription factor with mild consequences on the expression of some differentiation markers. In contrast, no differences in the production of effector cytokines were observed. These results, together with our previous findings on Apc function in regulatory T cells, indicate that Apc mutations may cause a dual damage, first unbalancing epithelial cell differentiation and growth driving epithelial neoplasms and, second, impairing T cell-mediated antitumor immunity at several levels.
Assuntos
Actinas/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Sinapses Imunológicas/metabolismo , Microtúbulos/imunologia , Fatores de Transcrição NFATC/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/imunologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/ultraestrutura , Mutação , Fatores de Transcrição NFATC/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4+ T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response.
Assuntos
Adesão Celular/fisiologia , Fibronectinas/metabolismo , Análise Espectral/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Adesão Celular/genética , Matriz Extracelular/metabolismo , Humanos , Interleucina-7/metabolismo , Cinética , Ativação Linfocitária/fisiologiaRESUMO
The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5'-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5'-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (-3299/-3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (-220/-61) located in the long 5'-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.
Assuntos
Éxons/fisiologia , Regulação da Expressão Gênica/fisiologia , Receptores 5-HT4 de Serotonina/genética , Regiões 5' não Traduzidas/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Átrios do Coração/metabolismo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Receptores 5-HT4 de Serotonina/biossíntese , Sequências Reguladoras de Ácido Nucleico , Sítio de Iniciação de Transcrição , Transcrição Gênica/fisiologiaRESUMO
Serotonin 5-HT4 receptors are present in human atrial myocytes and have been proposed to contribute to the generation of atrial fibrillation. However, 5-HT4 receptors have so far been only found in human and pig atria and are absent from the heart of small laboratory animals, such as rat, guinea pig, rabbit and frog, which limits the experimental settings for studying their functional properties. In this study, we developed an adenovirus expression system to examine the properties of two human 5-HT4 receptor splice variants, h5-HT4(b) and h5-HT4(d), expressed in adult cardiomyocytes devoid of native 5-HT4 receptors. When expressed in the HL-1 murine cell line of atrial origin, both receptors caused specific binding of the 5-HT4 selective antagonist GR113808 and activated adenylyl cyclase in the presence of serotonin (5-HT, 1 microM). When expressed in freshly isolated adult rat ventricular cardiomyocytes, a stimulation of the L-type Ca2+ current (ICa,L) by 5-HT (100 nM) was revealed. Both effects were blocked by GR113808. In HL-1 cells, the h5-HT4(d) receptor was found to be more efficiently coupled to adenylyl cyclase than the h5-HT4(b). Pertussis toxin treatment (250 ng/ml for 5 h) potentiated the stimulatory effect of 5-HT on ICa,L in rat myocytes expressing the h5-HT4(b) but not the h5-HT4(d) receptor, indicating a likely coupling of the (b) isoform to both Gs and Gi/o proteins. Adenoviral expression of h5-HT4 receptor isoforms in adult cardiac myocytes provides a valuable means for the exploration of the receptor signaling cascades in normal and pathological situations.
Assuntos
Miócitos Cardíacos/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Adenoviridae/genética , Adenilil Ciclases/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Humanos , Indóis/farmacologia , Isoproterenol/farmacologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Toxina Pertussis/farmacologia , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Antagonistas do Receptor 5-HT4 de Serotonina , Sulfonamidas/farmacologiaRESUMO
HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K(+) channel. Here, we examined the presence of a hyperpolarization-activated I(f) current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (C(m)) ranged from 5 to 53 pF. I(f) was detected in about 30% of the cells and its occurrence was independent of the stage of the culture. I(f) maximal slope conductance was 89.7 +/- 0.4 pS pF(-1) (n = 10). I(f) current in HL-1 cells showed typical characteristics of native cardiac I(f) current: activation threshold between -50 and -60 mV, half-maximal activation potential of -83.1 +/- 0.7 mV (n = 50), reversal potential at -20.8 +/- 1.5 mV (n = 10), time-dependent activation by hyperpolarization and blockade by 4 mM Cs(+). In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 microM) induced both an approximately 6 mV positive shift of the half-activation potential and an approximately 37 % increase in the fully activated I(f) current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to I(f) current. Cytosolic Ca(2+) oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mM Cs(+). Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of -69 mV, i.e. more negative than the threshold potential for I(f) activation. In conclusion, HL-1 cells display a hyperpolarization-activated I(f) current which might contribute to the spontaneous contractile activity of these cells.