RESUMO
Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2-HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2-HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.
Assuntos
Inibidores de Proteínas Quinases , Receptor ErbB-3 , Regulação Alostérica , Animais , Células CHO , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estudo de Prova de Conceito , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/química , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismoRESUMO
The challenge of determining the architecture and geometry of oligomers of the epidermal growth factor receptor (EGFR) on the cell surface has been approached using a variety of biochemical and biophysical methods. This review is intended to provide a narrative of how key concepts in the field of EGFR research have evolved over the years, from the origins of the prevalent EGFR signalling dimer hypothesis through to the development and implementation of methods that are now challenging the conventional view. The synergy between X-ray crystallography and cellular fluorescence microscopy has become particularly important, precisely because the results from these two methods diverged and highlighted the complexity of the challenge. We illustrate how developments in super-resolution microscopy are now bridging this gap. Exciting times lie ahead where knowledge of the nature of the complexes can assist with the development of a new generation of anti-cancer drugs.
Assuntos
Membrana Celular/ultraestrutura , Cristalografia por Raios X/métodos , Receptores ErbB/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Regulação Alostérica , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Drosophila melanogaster/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Multimerização Proteica , Transdução de SinaisRESUMO
Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.
Assuntos
Receptores ErbB/química , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Fotodegradação , Animais , Cricetinae , Feminino , Humanos , Substâncias MacromolecularesRESUMO
Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.
Assuntos
Receptores ErbB/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Receptores ErbB/química , Receptores ErbB/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Fosforilação , Estabilidade Proteica , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismoRESUMO
There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5-50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.
Assuntos
Fator de Crescimento Epidérmico/química , Receptores ErbB/química , Substâncias Macromoleculares/química , Corantes Fluorescentes/química , Humanos , Multimerização ProteicaRESUMO
A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Nicotiana/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Microdomínios da Membrana/genética , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Nicotiana/citologia , Nicotiana/genéticaRESUMO
Dimerization and higher-order oligomerization are believed to play an important role in the activation of the EGFR (epidermal growth factor receptor). Understanding of the process has been limited by the lack of availability of suitable methods for the measurement in cells of distances in the range 10-100 nm, too short for imaging methods and too long for spectroscopic methods such as FRET. In the present article, we review the current state of our knowledge of EGFR oligomerization, and describe results from a new single-molecule localization method that has allowed the quantitative characterization of the distribution of EGFR-EGFR distances in cells. Recent data suggest the involvement of cortical actin in regulating the formation of EGFR complexes.
Assuntos
Receptores ErbB/fisiologia , Membrana Celular/metabolismo , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ligantes , Receptores ErbB/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.
Assuntos
Moléculas de Adesão Celular , Glicoproteínas de Membrana , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Dimerização , Glicoproteínas de Membrana/química , Transdução de SinaisRESUMO
Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.
Assuntos
Microscopia de Fluorescência/métodos , Algoritmos , Automação , Teorema de Bayes , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
The dependence on model-fitting to evaluate particle trajectories makes it difficult for single particle tracking (SPT) to resolve the heterogeneous molecular motions typical of cells. We present here a global spatiotemporal sampler for SPT solutions using a Metropolis-Hastings algorithm. The sampler does not find just the most likely solution but also assesses its likelihood and presents alternative solutions. This enables the estimation of the tracking error. Furthermore the algorithm samples the parameters that govern the tracking process and therefore does not require any tweaking by the user. We demonstrate the algorithm on synthetic and single molecule data sets. Metrics for the comparison of SPT are generalised to be applied to a SPT sampler. We illustrate using the example of the diffusion coefficient how the distribution of the tracking solutions can be propagated into a distribution of derived quantities. We also discuss the major challenges that are posed by the realisation of a SPT sampler.
Assuntos
Algoritmos , Modelos Teóricos , Movimento (Física) , Imagem Individual de MoléculaRESUMO
Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.
RESUMO
Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens (superSIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples.
Assuntos
Microscopia Crioeletrônica/métodos , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Microscopia de Fluorescência/métodos , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Maleimidas/química , Reprodutibilidade dos TestesRESUMO
Epidermal growth factor (EGF) receptor (EGFR) modulates mitosis and apoptosis through signaling by its high-affinity (HA) and low-affinity (LA) EGF-binding states. The prevailing model of EGFR activation-derived from x-ray crystallography-involves the transition from tethered ectodomain monomers to extended back-to-back dimers and cannot explain these EGFR affinities or their different functions. Here, we use single-molecule Förster resonant energy transfer analysis in combination with ensemble fluorescence lifetime imaging microscopy to investigate the three-dimensional architecture of HA and LA EGFR-EGF complexes in cells by measuring the inter-EGF distances within discrete EGF pairs and the vertical distance from EGF to the plasma membrane. Our results show that EGFR ectodomains form interfaces resulting in two inter-EGF distances ( approximately 8 nm and < 5.5 nm), different from the back-to-back EGFR ectodomain interface ( approximately 11 nm). Distance measurements from EGF to the plasma membrane show that HA EGFR ectodomains are oriented flat on the membrane, whereas LA ectodomains stand proud from it. Their flat orientation confers on HA EGFR ectodomains the exclusive ability to interact via asymmetric interfaces, head-to-head with respect to the EGF-binding site, whereas LA EGFRs must interact only side-by-side. Our results support a structural model in which asymmetric EGFR head-to-head interfaces may be relevant for HA EGFR oligomerization.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Microscopia de Fluorescência/métodos , Técnicas de Sonda Molecular , Frações Subcelulares/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Ligação ProteicaRESUMO
Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Multimerização Proteica , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Matriz Extracelular/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ligantes , Modelos Biológicos , Modelos Moleculares , Fotodegradação , Polímeros/química , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/metabolismoRESUMO
Optical aberrations degrade image quality in fluorescence microscopy, including for single-molecule based techniques. These depend on post-processing to localize individual molecules in an image series. Using simulated data, we show the impact of optical aberrations on localization success, accuracy and precision. The peak intensity and the proportion of successful localizations strongly reduces when the aberration strength is greater than 1.0 rad RMS, while the precision of each of those localisations is halved. The number of false-positive localisations exceeded 10% of the number of true-positive localisations at an aberration strength of only ~0.6 rad RMS when using the ThunderSTORM package, but at greater than 1.0 rad RMS with the Radial Symmetry package. In the presence of coma, the localization error reaches 100 nm at ~0.6 rad RMS of aberration strength. The impact of noise and of astigmatism for axial resolution are also considered. Understanding the effect of aberrations is crucial when deciding whether the addition of adaptive optics to a single-molecule microscope could significantly increase the information obtainable from an image series.
RESUMO
Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.
Assuntos
Receptores ErbB/química , Receptores ErbB/metabolismo , Animais , Artefatos , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Fator de Crescimento Epidérmico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ligantes , Simulação de Dinâmica Molecular , Fosforilação , Domínios Proteicos , Multimerização Proteica , Transdução de SinaisRESUMO
Dimerisation, oligomerisation, and clustering of receptor molecules are important for control of the signalling process. There has been a lack of suitable methods for the study and quantification of these processes in cells. Here we describe a protocol for a method that we have named "fluorophore localisation imaging with photobleaching" (FLImP), which uses single molecule localisation and single-step photobleaching to determine the separation of two fluorophores with a resolution of 7 nm or better. We describe the procedures required for the collection of FLImP data, and point out some of the pitfalls that must be avoided for the collection of high resolution data. We also present recent data obtained using FLImP, showing that the intracellular domain of the Epidermal Growth Factor Receptor is not required in the basal state for the receptor to form ordered inactive oligomers in the plasma membrane.
Assuntos
Receptores ErbB/química , Imagem Óptica/métodos , Multimerização Proteica , Animais , Receptores ErbB/metabolismo , Humanos , Espaço Intracelular/metabolismo , Fotodegradação , Estrutura Terciária de ProteínaRESUMO
The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Receptores ErbB/metabolismo , Proteólise , Receptor ErbB-4/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Feminino , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Estrutura Terciária de Proteína , Transporte Proteico/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptor ErbB-4/genéticaRESUMO
Electron multiplication charge-coupled devices (EMCCD) are widely used for photon counting experiments and measurements of low intensity light sources, and are extensively employed in biological fluorescence imaging applications. These devices have a complex statistical behaviour that is often not fully considered in the analysis of EMCCD data. Robust and optimal analysis of EMCCD images requires an understanding of their noise properties, in particular to exploit fully the advantages of Bayesian and maximum-likelihood analysis techniques, whose value is increasingly recognised in biological imaging for obtaining robust quantitative measurements from challenging data. To improve our own EMCCD analysis and as an effort to aid that of the wider bioimaging community, we present, explain and discuss a detailed physical model for EMCCD noise properties, giving a likelihood function for image counts in each pixel for a given incident intensity, and we explain how to measure the parameters for this model from various calibration images.