RESUMO
The interactions of proteins, membranes, nucleic acid, and metabolites shape a cell's phenotype. These interactions are stochastic, and each cell develops differently, making it difficult to synchronize cell populations. Consequently, studying biological processes at the single- or few-cell level is often necessary to avoid signal dilution below the detection limit or averaging over many cells. We have developed a method to study metabolites and proteins from a small number of or even a single adherent eukaryotic cell. Initially, cells are lysed by short electroporation and aspirated with a microcapillary under a fluorescent microscope. The lysate is placed on a carrier slide for further analysis using liquid-chromatography mass spectrometry (LC-MS) and/or reverse-phase protein (RPPA) approach. This method allows for a correlative measurement of (i) cellular structures and metabolites and (ii) cellular structures and proteins on the single-cell level. The correlative measurement of cellular structure by light-microscopy, metabolites by LC-MS, and targeted protein detection by RPPA was possible on the few-cell level. We discuss the method, potential applications, limitations, and future improvements.
RESUMO
Based on immunostainings and biochemical analyses, certain post-translationally modified alpha-synuclein (aSyn) variants, including C-terminally truncated (CTT) and Serine-129 phosphorylated (pSer129) aSyn, are proposed to be involved in the pathogenesis of synucleinopathies such as Parkinson's disease with (PDD) and without dementia (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, quantitative information about aSyn proteoforms in the human brain in physiological and different pathological conditions is still limited. To address this, we generated sequential biochemical extracts of the substantia nigra, putamen and hippocampus from 28 donors diagnosed and neuropathologically-confirmed with different synucleinopathies (PD/PDD/DLB/MSA), as well as Alzheimer's disease, progressive supranuclear palsy, and aged normal subjects. The tissue extracts were used to build a reverse phase array including 65 aSyn antibodies for detection. In this multiplex approach, we observed increased immunoreactivity in donors with synucleinopathies compared to controls in detergent-insoluble fractions, mainly for antibodies against CT aSyn and pSer129 aSyn. In addition, despite of the restricted sample size, clustering analysis suggested disease-specific immunoreactivity signatures in patient groups with different synucleinopathies. We aimed to validate and quantify these findings using newly developed immunoassays towards total, 119 and 122 CTT, and pSer129 aSyn. In line with previous studies, we found that synucleinopathies shared an enrichment of post-translationally modified aSyn in detergent-insoluble fractions compared to the other analyzed groups. Our measurements allowed for a quantitative separation of PDD/DLB patients from other synucleinopathies based on higher detergent-insoluble pSer129 aSyn concentrations in the hippocampus. In addition, we found that MSA stood out due to enrichment of CTT and pSer129 aSyn also in the detergent-soluble fraction of the SN and putamen. Together, our results achieved by multiplexed and quantitative immunoassay-based approaches in human brain extracts of a limited sample set point to disease-specific biochemical aSyn proteoform profiles in distinct neurodegenerative disorders.
Assuntos
Doença por Corpos de Lewy , Atrofia de Múltiplos Sistemas , Sinucleinopatias , Idoso , Encéfalo/patologia , Detergentes , Humanos , Doença por Corpos de Lewy/patologia , Atrofia de Múltiplos Sistemas/patologia , alfa-Sinucleína/metabolismoRESUMO
The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K. Collectively, these findings suggest that vascular endothelial inflammation that results from dysregulated insulin signaling (homeostasis) may contribute to coronary artery disease, and that either downregulation or upregulation of the insulin pathway may lead to inflammation of endothelial cells. This suggests that the standard of care for patients must be expanded from control of metabolic parameters to include control of inflammation, such that endothelial dysfunction and cardiovascular disorders can ultimately be prevented.
Assuntos
Células Endoteliais/metabolismo , Edição de Genes , Síndrome Metabólica , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Inflamação/genética , Inflamação/metabolismo , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismoRESUMO
The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Epitopos/química , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Transporte Biológico , Linhagem Celular , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/ultraestrutura , Epitopos/ultraestrutura , Expressão Gênica , Humanos , Lipoproteínas HDL/ultraestrutura , Lipoproteínas LDL/ultraestrutura , Microscopia Eletrônica de Transmissão , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
PURPOSE: Rheumatoid arthritis (RA) is associated with increased cardiovascular risk, mediated in part by elevated circulating interleukin-6 levels and proinflammatory changes in plasma lipoproteins. We hypothesized that RA patients acquire inflammation-induced modifications to the protein cargo of circulating lipoproteins that may be reversed by tocilizumab, an interleukin-6 receptor-alpha inhibitor. EXPERIMENTAL DESIGN: Size-exclusion chromatography and reverse-phase protein arrays using 29 antibodies against 26 proteins were applied at baseline and after tocilizumab treatment to analyze the distributions of apolipoproteins, enzymes, lipid transfer proteins, and other associated proteins in plasma lipoprotein fractions from 20 women with RA. RESULTS: A 30% reduction in high-density lipoprotein (HDL)-associated serum amyloid A4 and complement C4 occurred with tocilizumab. Levels of C-reactive protein, associated or comigrating with HDL and low-density lipoprotein (LDL) peaks, were reduced on treatment by approximately 80% and 24%, respectively. Reductions in lipoprotein-associated phospholipase A2, lipoprotein (a), and cholesteryl ester transfer protein in the LDL fraction suggest reductions in LDL-associated proatherogenic factors. Elevations in very low-density lipoprotein (VLDL) enriched with apolipoprotein E were equally observed. CONCLUSIONS AND CLINICAL RELEVANCE: Tocilizumab treatment led to reductions in proinflammatory components and proatherogenic proteins associated with HDL. Whether changes in the proteome of VLDL, LDL, and HDL induced by anti-inflammatory tocilizumab treatment in RA patients modify cardiovascular disease risk requires further investigation.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Reumatoide/sangue , Artrite Reumatoide/tratamento farmacológico , Subunidade alfa de Receptor de Interleucina-6/antagonistas & inibidores , Lipoproteínas/sangue , Adulto , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Cromatografia em Gel , Método Duplo-Cego , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Serial de ProteínasRESUMO
Trace amine-associated receptor 1 (TAAR1) activation by selective endogenous agonists modulates dopaminergic neurotransmission. This results in antipsychotic-like behavior in vivo which might be initiated by an interaction of TAAR1 and dopamine D2L receptor (D2R). Here we analyzed the functional link between TAAR1 and D2R using highly potent and selective TAAR1 agonists, and newly generated tools such as TAAR1 knock-out and TAAR1 overexpressing rats as well as specific anti-rat TAAR1 antibodies. We provide data from co-immunoprecipitation experiments supporting a functional interaction of the two receptors in heterologous cells and in brain tissue. Interaction of TAAR1 with D2R altered the subcellular localization of TAAR1 and increased D2R agonist binding affinity. Using specific ß-arrestin 2 (ßArr2) complementation assays we show that the interaction of TAAR1 with D2R reduced ßArr2 recruitment to D2R. In addition, we report that besides Gαs-protein signaling TAAR1 also signals via ßArr2. In the presence of D2R, cAMP signaling of TAAR1 was reduced while its ßArr2 signaling was enhanced, resulting in reduced GSK3ß activation. These results demonstrate that ßArr2 signaling may be an important pathway for TAAR1 function and that the activation of the TAAR1-D2R complex negatively modulates GSK3ß signaling. Given that patients with schizophrenia or bipolar disorder show increased GSK3ß signaling, such a reduction of GSK3ß signaling triggered by the interaction of D2R with activated TAAR1 further supports TAAR1 as a target for the treatment of psychiatric disorders.
Assuntos
Arrestinas/metabolismo , Encéfalo/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetulus , AMP Cíclico/metabolismo , Técnicas de Inativação de Genes , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Ratos Transgênicos , Ratos Wistar , Receptores de Dopamina D2/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , beta-Arrestina 2 , beta-ArrestinasRESUMO
The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.
Assuntos
Análise de Célula Única/métodos , Animais , Linhagem Celular , Cricetinae , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Microscopia Eletrônica de Transmissão , Organelas/ultraestruturaRESUMO
Hyperalphalipoproteinemia, as observed in patients who are either homozygous or heterozygous for cholesteryl ester transfer protein (CETP) deficiency, has been shown to be associated with striking changes in apolipoprotein size distribution, namely, of high-density lipoprotein (HDL) and HDL-like particles. We compared the effect of varying degrees of CETP activity on the HDL apolipoprotein profile in Caucasian CETP-deficient subjects and following pharmacological decrease in CETP activity, using Size Exclusion Chromatography followed by Reverse Phase Protein Array (SEC RPA). The main HDL-associated apolipoproteins (Apo), i.e. ApoA-I, ApoA-II, ApoC-I, and ApoC-III, co-eluted with the HDL peak. The presence of a HDL-like peak migrating between the ApoB-LDL and ApoA-I-HDL was identified in a Caucasian patient with homozygosity for a point mutation in exon 2 of the CETP gene (c.109 C > T) resulting in a premature termination codon (R37X) and complete CETP deficiency. This HDL-like peak was not observed either in healthy volunteers treated with the CETP modulator dalcetrapib, patients heterozygous for the same mutation, or in patients heterozygous with G165X mutations. SEC RPA offers the possibility to investigate the distribution of a large number of apolipoproteins simultaneously under non-denaturing separation in normal and dyslipidemic subjects. This is only limited by the availability of antibodies against specific apolipoproteins to be investigated.
Assuntos
Apolipoproteínas/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/deficiência , Metabolismo dos Lipídeos , Lipídeos/sangue , Lipoproteínas HDL/metabolismo , Animais , Apolipoproteínas/química , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/sangue , HDL-Colesterol/metabolismo , Modelos Animais de Doenças , Heterozigoto , Homozigoto , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Lipoproteínas HDL/sangueRESUMO
The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 µl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-ß-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.
Assuntos
Análise Química do Sangue/métodos , Cromatografia em Gel/métodos , Lipoproteínas/sangue , Lipoproteínas/isolamento & purificação , Análise Serial de Proteínas/métodos , Especificidade de Anticorpos , Artefatos , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/farmacologia , Lipoproteínas de Alta Densidade Pré-beta/sangue , Lipoproteínas de Alta Densidade Pré-beta/metabolismo , Humanos , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Quinolinas/farmacologia , Reprodutibilidade dos TestesRESUMO
The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-ß-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-ß-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [³H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [³H]neutral sterols and [³H]bile acids, whereas all compounds increased plasma HDL-[³H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-ß-HDL formation, which may be required to increase reverse cholesterol transport.
Assuntos
Anticolesterolemiantes/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas de Alta Densidade Pré-beta/metabolismo , Amidas , Animais , Ácidos e Sais Biliares/metabolismo , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol/sangue , Cricetinae , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Ésteres , Lipoproteínas de Alta Densidade Pré-beta/sangue , Humanos , Oxazolidinonas/farmacologia , Quinolinas/farmacologia , Compostos de Sulfidrila/farmacologiaRESUMO
Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans.
Assuntos
Úlcera de Buruli/microbiologia , DNA Bacteriano/genética , Doenças Endêmicas , Mutação INDEL , Mycobacterium ulcerans/genética , Polimorfismo Genético , Úlcera de Buruli/epidemiologia , Hibridização Genômica Comparativa , Gana/epidemiologia , Humanos , Mycobacterium ulcerans/classificação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Elucidation of the transmission, epidemiology, and evolution of Mycobacterium ulcerans, the causative agent of Buruli ulcer, is hampered by the striking lack of genetic diversity of this emerging pathogen. However, by using a prototype plasmid-based microarray that covered 10% of the genome, we found multiple genomic DNA deletions among 30 M. ulcerans clinical isolates of diverse geographic origins. Many of the changes appear to have been mediated by insertion sequence (IS) elements IS2404 and IS2606, which have high copy numbers. Classification of the deleted genes according to their biological functions supports the hypothesis that M. ulcerans has recently evolved from the generalist environmental M. marinum to become a niche-adapted specialist. The substantial genomic diversity, along with a prototype microarray that covered a small portion of the genome, suggests that a genome-wide microarray will make available a genetic fingerprinting method with the high resolution required for microepidemiologic studies.
Assuntos
Adaptação Fisiológica , Úlcera de Buruli/microbiologia , Genoma Bacteriano , Mycobacterium ulcerans/genética , Mycobacterium ulcerans/fisiologia , Cromossomos Bacterianos , Elementos de DNA Transponíveis , Evolução Molecular , Variação Genética , Humanos , Dados de Sequência Molecular , Mycobacterium marinum/genética , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da Espécie , Virulência/genéticaRESUMO
Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of catecholamines near the cell surface and not due to slow dissociation from the granular matrix.
Assuntos
Células Cromafins/fisiologia , Exocitose , Microeletrodos , Platina , Análise Serial de Tecidos/métodos , Laranja de Acridina/análise , Laranja de Acridina/metabolismo , Animais , Transporte Biológico , Catecolaminas/análise , Catecolaminas/metabolismo , Bovinos , EletroquímicaAssuntos
Algoritmos , Técnicas Biossensoriais/métodos , Permeabilidade da Membrana Celular/fisiologia , Eletroquímica/métodos , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Vesículas Transportadoras/fisiologia , Animais , Técnicas Biossensoriais/instrumentação , Bovinos , Células Cultivadas , Células Cromafins/fisiologia , Guias como AssuntoRESUMO
We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).
Assuntos
Células Dendríticas/citologia , Análise de Injeção de Fluxo/instrumentação , Imunoensaio de Fluorescência por Polarização/instrumentação , Microquímica/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos/imunologia , Células Dendríticas/metabolismo , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Imunoensaio de Fluorescência por Polarização/métodos , Humanos , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.