RESUMO
In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation.
Assuntos
Apoptose , Caspases Efetoras/análise , Microscopia de Fluorescência/métodos , Técnicas Biossensoriais/métodos , Caspases Efetoras/metabolismo , Ativação Enzimática , Polarização de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Transdução de SinaisRESUMO
Cells communicate with their environment via proteins, located at the plasma membrane separating the interior of a cell from its surroundings. The spatial distribution of these proteins in the plasma membrane under different physiological conditions is of importance, since this may influence their signal transmission properties. In this study, the authors compare different methods such as hierarchical clustering, extensible Markov models and the gammics method for analysing such a spatial distribution. The methods are examined in a simulation study to determine their optimal use. Afterwards, they analyse experimental imaging data and extend these methods to simulate dual colour data.
Assuntos
Comunicação Celular , Membrana Celular , Cor , Proteínas , Análise por Conglomerados , Proteínas/fisiologiaRESUMO
Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Análise por Conglomerados , Humanos , Ligantes , Células MCF-7 , Microscopia Confocal , Fosforilação , Transdução de SinaisRESUMO
Well-defined human epidermal growth factor (hEGF) constructs featuring selectively addressable labels are urgently needed to address outstanding questions regarding hEGF biology. A protein-engineering approach was developed to provide access to hEGF constructs that carry two cysteine-based site-specific orthogonal labeling sites in multi-milligram quantities. Also, a site-selective (de)protection and labeling approach was devised, which allows selective modification of these hEGF constructs. The hEGF, featuring three native disulfide bonds, was expressed featuring additional sulfhydryl groups, in the form of cysteine residues, as orthogonal ligation sites at both the N and Câ termini. Temporary protection of the N-terminal cysteine unit, in the form of a thiazolidine ring, avoids interference with protein folding and enables sequential labeling in conjunction with the cysteine residue at the Câ terminus. Based on thus-generated hEGF constructs, sequential and site-specific labeling with a variety of molecular probes could be demonstrated, thus leading to a biological fully functional hEGF with specifically incorporated fluorophores or protein cargo and native cellular targeting and uptake profiles. Thus, this novel strategy provides a robust entry to high-yielding access of hEGF and rapid and easy site-specific and multifunctional dual labeling of this growth factor.
Assuntos
Cisteína/química , Fator de Crescimento Epidérmico/administração & dosagem , Fator de Crescimento Epidérmico/química , Tiazolidinas/química , Cisteína/genética , Diagnóstico por Imagem , Sistemas de Liberação de Medicamentos , Fator de Crescimento Epidérmico/genética , Humanos , Modelos Moleculares , Sondas Moleculares/química , Engenharia de Proteínas , Dobramento de Proteína , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.
Assuntos
Proteínas do Citoesqueleto/análise , Microscopia de Fluorescência/métodos , Neurofibromina 2/análise , Algoritmos , Células HeLa , Humanos , LasersRESUMO
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência/métodosRESUMO
Interest in imaging of Förster resonance energy transfer (FRET) in biological systems has been steadily increasing in the last 30 years. The ability to transduce a near-field interaction into a far-field signal has provided a unique optical tool to assess biological phenomena well below the resolution of standard optical microscopy. In recent years, sub-diffraction microscopy techniques have achieved maturation and are increasingly used in biological applications. As the resolution of these methods increases they will slowly encroach on the domains where FRET is now dominant. Herein we review the major applications in biological FRET imaging and we discuss the possibilities and challenges in the super-resolution era.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Biologia Computacional , Humanos , Microscopia de Fluorescência , Neoplasias/metabolismo , Mapeamento de Interação de Proteínas , Proteoma , RNA Interferente Pequeno/metabolismoRESUMO
Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in vitro and in vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.
Assuntos
Adenocarcinoma/patologia , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias Pulmonares/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Adenocarcinoma/metabolismo , Animais , Dimerização , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Transplante de Neoplasias , Estrutura Terciária de Proteína , Transdução de Sinais , Transplante Heterólogo , Triazóis/farmacologiaRESUMO
Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain. Mutation of Cys(257) abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75(NTR)/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75(NTR). FRET experiments revealed a close association of p75(NTR) intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys(257) did not alter the oligomeric state of p75(NTR), the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75(NTR) by a mechanism involving rearrangement of disulphide-linked receptor subunits.
Assuntos
Multimerização Proteica/fisiologia , Receptor de Fator de Crescimento Neural/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Chlorocebus aethiops , Cisteína/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Mutação/genética , NF-kappa B/metabolismo , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligopeptídeos/genética , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptor de Fator de Crescimento Neural/genética , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/genética , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/citologia , Transfecção/métodos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data. With this approach, the fluorescent lifetimes and relative concentrations of free and interacting molecules can be reliably estimated, even if the SNR is low. The required calibration values of the impulse response function are directly estimated from the data, eliminating the need for reference samples. The proposed method is efficient and robust, and can be routinely applied to analyze FRET-FLIM data acquired in intact cells.
Assuntos
Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Aumento da Imagem/métodos , Fótons , Doses de Radiação , Radiometria/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Understanding cellular function requires studying the spatially resolved dynamics of protein networks. From the isolated proteins we can only learn about their individual properties, but by investigating their behavior in their natural environment, the cell, we obtain information about the overall response properties of the network module in which they operate. Fluorescence microscopy methods provide currently the only tools to study the dynamics of molecular processes in living cells with high temporal and spatial resolution. Combined with computational approaches they allow us to obtain insights in the reaction-diffusion processes that determine biological function on the scale of cells.
Assuntos
Microscopia de Fluorescência , Técnicas de Sonda Molecular , Biologia de Sistemas , Animais , Humanos , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
We report that single (or selective) plane illumination microscopy (SPIM), combined with a new deconvolution algorithm, provides a three-dimensional spatial resolution exceeding that of confocal fluorescence microscopy in large samples. We demonstrate this by imaging large living multicellular specimens obtained in a three-dimensional cell culture. The ability to rapidly image large samples at high resolution with minimal photodamage provides new opportunities especially for the study of subcellular processes in large living specimens.
Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Algoritmos , Animais , Células Cultivadas , Humanos , Sensibilidade e EspecificidadeRESUMO
To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Fase G1 , Ciclo Celular , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Recuperação de Fluorescência Após Fotodegradação/métodos , Geminina , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Microscopia de Fluorescência/métodos , Modelos Biológicos , Método de Monte Carlo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The mitogen-activated protein kinase (MAPK) network is a conserved signalling module that regulates cell fate by transducing a myriad of growth-factor signals. The ability of this network to coordinate and process a variety of inputs from different growth-factor receptors into specific biological responses is, however, still not understood. We investigated how the MAPK network brings about signal specificity in PC-12 cells, a model for neuronal differentiation. Reverse engineering by modular-response analysis uncovered topological differences in the MAPK core network dependent on whether cells were activated with epidermal or neuronal growth factor (EGF or NGF). On EGF stimulation, the network exhibited negative feedback only, whereas a positive feedback was apparent on NGF stimulation. The latter allows for bi-stable Erk activation dynamics, which were indeed observed. By rewiring these regulatory feedbacks, we were able to reverse the specific cell responses to EGF and NGF. These results show that growth factor context determines the topology of the MAPK signalling network and that the resulting dynamics govern cell fate.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Citometria de Fluxo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Método de Monte Carlo , Fator de Crescimento Neural/farmacologia , Células PC12 , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Interferente Pequeno/genética , Ratos , Receptor trkA/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission. Conversely, fluorescence emission tends to tail into the red part of the spectrum, causing donor fluorescence bleed-through into the acceptor detection channel. Corrections for these effects involve the acquisition of fluorescence images of samples containing the donor, the acceptor, and both of these for three different filter settings. The result is an estimation of the sensitized emission, i.e., the emission induced by FRET from the donor to the acceptor alone. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor fluorescence and increased emission from the acceptor (sensitized emission). This can be normalized using the acceptor emission, measured after specific excitation of the acceptor, to define apparent energy transfer efficiency in each pixel of the image. It is also proportional to the fraction of acceptor molecules that is bound to a donor-tagged molecule. Alternatively, an apparent energy transfer efficiency can also be defined that is proportional to the bound fraction of donor-tagged molecules.
RESUMO
This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region. Because of mass movement of protein during the extended time required for photobleaching (typically 1-20 min), it is preferable to perform this type of FRET determination on fixed cell samples. Live-cell FRET measurements based only on donor fluorescence are more feasible using fluorescence lifetime imaging (FLIM), because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.
RESUMO
This image acquisition protocol is a basic plan for taking a fluorescence lifetime imaging (FLIM) series. FLIM makes live-cell FRET measurements based only on donor fluorescence more feasible, because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.
RESUMO
This protocol provides a method for labeling proteins, such as antibodies and purified recombinant proteins, with succinimide esters of sulfoindocyanine (Cy) dyes. Cy dyes covalently bind to free amino groups (the α-amino-terminal or ε-amino groups on lysine side chains).
RESUMO
The following protocol describes the preparation of cells for FRET analysis on live and fixed cells. The reagents used have been optimized to minimize the quenching of GFP mutants and fluorescent dyes.