ABSTRACT
Liquid-phase electron microscopy is highly desirable for observing biological samples in their native liquid state at high resolution. We developed liquid imaging approaches for biological cells using scanning electron microscopy. Novel approaches included scanning transmission electron imaging using a liquid-cell apparatus (LC-STEM), as well as correlative cathodoluminescence and electron microscopy (CCLEM) imaging. LC-STEM enabled imaging at a â¼2 nm resolution and excellent contrast for the precise recognition of localization, distribution, and configuration of individually labeled membrane proteins on the native cells in solution. CCLEM improved the resolution of fluorescent images down to 10 nm. Liquid SEM technologies will bring unique and wide applications to the study of the structure and function of cells and membrane proteins in their near-native states at the monomolecular level.
Subject(s)
Membrane Proteins/ultrastructure , Microscopy, Electron, Scanning , Cell Line, Tumor , ErbB Receptors/ultrastructure , Fluorescence , HumansABSTRACT
Single-pass membrane receptors contain extracellular domains that respond to external stimuli and transmit information to intracellular domains through a single transmembrane (TM) α-helix. Because membrane receptors have various roles in homeostasis, signaling malfunctions of these receptors can cause disease. Despite their importance, there is still much to be understood mechanistically about how single-pass receptors are activated. In general, single-pass receptors respond to extracellular stimuli via alterations in their oligomeric state. The details of this process are still the focus of intense study, and several lines of evidence indicate that the TM domain (TMD) of the receptor plays a central role. We discuss three major mechanistic hypotheses for receptor activation: ligand-induced dimerization, ligand-induced rotation, and receptor clustering. Recent observations suggest that receptors can use a combination of these activation mechanisms and that technical limitations can bias interpretation. Short peptides derived from receptor TMDs, which can be identified by screening or rationally developed on the basis of the structure or sequence of their targets, have provided critical insights into receptor function. Here, we explore recent evidence that, depending on the target receptor, TMD peptides cannot only inhibit but also activate target receptors and can accommodate novel, bifunctional designs. Furthermore, we call for more sharing of negative results to inform the TMD peptide field, which is rapidly transforming into a suite of unique tools with the potential for future therapeutics.
Subject(s)
Integrins/ultrastructure , Peptides/genetics , Receptors, Antigen, T-Cell/chemistry , Amino Acid Sequence/genetics , ErbB Receptors/chemistry , ErbB Receptors/ultrastructure , Humans , Integrins/chemistry , Peptides/chemistry , Protein Conformation , Protein Conformation, alpha-Helical/genetics , Protein Interaction Maps , Protein Multimerization , Receptors, Antigen, T-Cell/ultrastructure , Signal Transduction/geneticsABSTRACT
The combinatorial dimerization of the ErbB growth factor receptors (ErbB1- ErbB4) are critical for their function. Here, we have characterized the conformational dynamics of ErbB transmembrane homo-dimers and hetero-dimers by using a coarse-grain simulation framework. All dimers, except ErbB4-4 and ErbB1-4, exhibit at least two conformations. The reported NMR structures correspond to one of these conformations, representing the N-terminal active state in ErbB1-1 (RH2), ErbB2-2 (RH1) and ErbB4-4 (RH) homo-dimers and the LH dimer in ErbB3-3 homo-dimer, validating the computational approach. Further, we predict a right-handed ErbB3-3 dimer conformer that warrants experimental testing. The five hetero-dimers that have not yet been experimentally resolved display prominent right-handed dimers associating by the SmXXXSm motif. Our results provide insights into the constitutive signaling of ErbB4 after cleavage of the extracellular region. The presence of the inactive-like dimer conformers leading to symmetric kinase domains gives clues on the autoinhibition of the receptor dimers. The dimer states characterized here represent an important step towards understanding the combinatorial cross associations in the ErbB family.
Subject(s)
Amino Acid Sequence/genetics , ErbB Receptors/ultrastructure , Protein Multimerization , Amino Acid Motifs/genetics , ErbB Receptors/chemistry , Humans , Molecular Dynamics Simulation , Protein Binding/genetics , Protein Conformation , Signal Transduction/geneticsABSTRACT
Protein kinases are an important class of enzymes that play an essential role in virtually all major disease areas. In addition, they account for approximately 50% of the current targets pursued in drug discovery research. In this work, we explore the generation of structure-based quantum mechanical (QM) quantitative structure-activity relationship models (QSAR) as a means to facilitate structure-guided optimization of protein kinase inhibitors. We explore whether more accurate, interpretable QSAR models can be generated for a series of 76 N-phenylquinazolin-4-amine inhibitors of epidermal growth factor receptor (EGFR) kinase by comparing and contrasting them to other standard QSAR methodologies. The QM-based method involved molecular docking of inhibitors followed by their QM optimization within a ~ 300 atom cluster model of the EGFR active site at the M062X/6-31G(d,p) level. Pairwise computations of the interaction energies with each active site residue were performed. QSAR models were generated by splitting the datasets 75:25 into a training and test set followed by modelling using partial least squares (PLS). Additional QSAR models were generated using alignment dependent CoMFA and CoMSIA methods as well as alignment independent physicochemical, e-state indices and fingerprint descriptors. The structure-based QM-QSAR model displayed good performance on the training and test sets (r2 ~ 0.7) and was demonstrably more predictive than the QSAR models built using other methods. The descriptor coefficients from the QM-QSAR models allowed for a detailed rationalization of the active site SAR, which has implications for subsequent design iterations.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinases/ultrastructure , Quantitative Structure-Activity Relationship , Catalytic Domain , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , ErbB Receptors/ultrastructure , Humans , Molecular Docking Simulation , Molecular Structure , Protein Kinases/chemistry , Quantum TheoryABSTRACT
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.
Subject(s)
Cell Membrane/ultrastructure , Crystallography, X-Ray/methods , ErbB Receptors/ultrastructure , Fluorescence Resonance Energy Transfer/methods , Microscopy, Fluorescence/methods , Allosteric Regulation , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Drosophila melanogaster/metabolism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Humans , Molecular Dynamics Simulation , Phosphorylation , Protein Multimerization , Signal TransductionABSTRACT
The discovery of genetic drivers of lung cancer in patient sub-groups has led to their use as predictive biomarkers and as targets for selective drug therapy. Some of the most important lung cancer drivers are mutations in the EGFR gene, for example, the exon 19 deletions and the L858R variant that confer sensitivity to the front line drugs erlotinib and gefitinib; the acquired T790M variants confer drug resistance and a poor prognosis. A challenge then in targeting EGFR is to produce drugs that inhibit both sensitising variants and resistance variants, leaving wild type protein in healthy cells unaffected. One such agent is AstraZeneca's "breakthrough" AZD9291 molecule that shows a 200-fold selectivity for T790M/L858R over wild type EGFR. Our X-ray crystal structure reveals the binding mode of AZD9291 to the kinase domain of wild type EGFR.
Subject(s)
Acrylamides/pharmacology , Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Lung Neoplasms/genetics , Protein Kinase Inhibitors/pharmacology , Acrylamides/metabolism , Aniline Compounds/metabolism , Crystallography, X-Ray , Drug Resistance, Neoplasm/genetics , ErbB Receptors/ultrastructure , Erlotinib Hydrochloride/pharmacology , Gefitinib , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Protein Binding/physiology , Quinazolines/pharmacologyABSTRACT
BACKGROUND: The epidermal growth factor receptor (EGFR) is a member of the ErbB family that is involved in a number of processes responsible for cancer development and progression such as angiogenesis, apoptosis, cell proliferation and metastatic spread. Malfunction in activation of protein tyrosine kinases has been shown to result in uncontrolled cell growth. The EGFR TK domain has been identified as suitable target in cancer therapy and tyrosine kinase inhibitors such as erlotinib have been used for treatment of cancer. Mutations in the region of the EGFR gene encoding the tyrosine kinase (TK) domain causes altered responses to EGFR TK inhibitors (TKI). In this paper we perform molecular dynamics simulations and PCA analysis on wild-type and mutant (T854A) structures to gain insight into the structural changes observed in the target protein upon mutation. We also report two novel inhibitors identified by combined approach of QSAR model development. RESULTS: The wild-type and mutant structure was observed to be stable for 26 ns and 24 ns respectively. In PCA analysis, the mutant structure proved to be more flexible than wild-type. We developed a 3D-QSAR model using 38 thiazolyl-pyrazoline compounds which was later used for prediction of inhibitory activity of natural compounds of ZINC library. The 3D-QSAR model was proved to be robust by the statistical parameters such as r2 (0.9751), q2(0.9491) and pred_r2(0.9525). CONCLUSION: Analysis of molecular dynamics simulations results indicate stability loss and increased flexibility in the mutant structure. This flexibility results in structural changes which render the mutant protein drug resistant against erlotinib. We report two novel compounds having high predicted inhibitory activity to EGFR TK domain with both wild-type and mutant structure.
Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/ultrastructure , Erlotinib Hydrochloride/pharmacology , Humans , Indoles/pharmacology , Isoflavones/pharmacology , Lung Neoplasms/drug therapy , Molecular Dynamics Simulation , Mutation/genetics , Naphthyridines/pharmacology , Principal Component Analysis , Pyrazoles/chemistry , Quantitative Structure-Activity RelationshipABSTRACT
Formation of an asymmetric dimer by the EGFR (epidermal growth factor receptor) kinase domains results in allosteric activation. Since this dimer does not readily form in solution, the EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, and we sought to understand the basis for this. Peptide C was found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C probably underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization by an aggregation-mediated mechanism.
Subject(s)
ErbB Receptors/metabolism , Peptides/metabolism , Chromatography, High Pressure Liquid , Dimerization , ErbB Receptors/ultrastructure , Immunoblotting , Microscopy, Electron, Transmission , Phosphorylation , Substrate SpecificityABSTRACT
Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR-528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.
Subject(s)
Cetuximab/metabolism , Epitopes/metabolism , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Animals , Binding, Competitive , CHO Cells , Cetuximab/chemistry , Cetuximab/ultrastructure , Cricetulus , Epidermal Growth Factor/metabolism , Epitopes/chemistry , ErbB Receptors/ultrastructure , Hot Temperature , Models, Molecular , Protein Binding , Protein DomainsABSTRACT
The molecular mechanism underlying epidermal growth factor receptor (EGFR) localization in mitochondria remains largely unknown. Using immune electron microscopy, we validated that EGFR could be localized on either the outer or the inner membrane of mitochondria. Mutant receptor lacked amino acids 646-660 was flawed in migration onto the organelles, whereas the mutated receptor with a defective endocytosis showed a greater capability of moving onto mitochondria upon stimulation of epidermal growth factor (EGF). Gefitinib, an inhibitor of EGFR kinase, inhibited the receptor endocytosis after short time of treatment, yet, only reduced cell viability as well as the amount of mitochondrial EGFR after longer time of exposure. Moreover, the content of mitochondrial EGFR transfer was decreased when the cells were exposed to the apoptotic inducer etoposide. EGF-induced programmed cell death usually coincided with a decline in mitochondrial EGFR. These data indicated that the mitochondrial-localized EGFR is independent of its internalization and may be correlated with cell survival and participate in the ligand-induced programmed cell death.
Subject(s)
Endocytosis , ErbB Receptors/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Epidermal Growth Factor/pharmacology , ErbB Receptors/genetics , ErbB Receptors/ultrastructure , Etoposide/pharmacology , Gefitinib , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunoblotting , Microscopy, Immunoelectron , Mitochondria/ultrastructure , Mitochondrial Proteins/genetics , Mitochondrial Proteins/ultrastructure , Mutation , Protein Transport/drug effects , Quinazolines/pharmacologyABSTRACT
BACKGROUND: According to WHO, in 2017, about 90.5 million people suffered from cancer and about 8.8 million deaths occurred due to disease. Although the chemotherapeutic agents have decreased the mortality among the cancer patients but high toxicity and non-specific targets are still major drawbacks. Many researchers have identified linomide, a 4-hydroxy-2-quinolone derivative, as a lead molecule for the development of anticancer agents. With this background, we thought of the following objective. OBJECTIVE: The objective of this research work involves the synthesis of a series of N-(2-(4- hydroxy-2-oxo-1-phenyl-1,2-dihydroquinolin-3-yl)-2-oxoethyl)-N-alkyl substituted benzene sulfonamides IVa-d (1-3) by replacing the anilide moiety at the third position of linomide with sulfamoylacyl and also N-methyl by N-phenyl functionality. To perform in silico anticancer activity by using Molegro Virtual Docker (MVD-2013, 6.0) software and in vitro anticancer activity by MTT assay. METHODS: The starting material 4-hydroxy-1-phenylquinolin-2(1H)-one was treated with N-bromosuccinamide to yield compound II. Condensation of compound II with primary amines resulted in compounds IIIa-d, which, on coupling with substituted aromatic sulfonyl chlorides yield the title compounds IVa-d (1-3). RESULTS: All the synthesized compounds were satisfactorily characterized by spectral data. The results of docking revealed that the synthesized compounds exhibited well-conserved hydrogen bonds with one or more amino acid residues in the active pocket of EGFRK tyrosine kinase domain (PDB ID: 1m17). The MolDock Score of compound IVd-1 (-115.503) was the highest amongst those tested. The in vitro anticancer activity results showed that compound IVc-1 (R= - (CH2) 2-CH3 ; R'= -H) and IV d-1 (R= -CH2-C6H5; R'= -H) were found to be most potent against K562 cell line with an IC50 of 0.451 µM/ml and 0.455 µM/ml respectively. Compound IVd-1 also showed better potency against A549 cell line with IC50 value of 0.704 µM/ml. CONCLUSION: The results of in silico and in vitro anticancer activity are in agreement with each other. Compound IV d-1 was found to be most active of the series.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Hydroxyquinolines/pharmacology , Neoplasms/drug therapy , A549 Cells , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Catalytic Domain/drug effects , Cell Proliferation , Crystallography, X-Ray , Drug Screening Assays, Antitumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , ErbB Receptors/ultrastructure , Humans , Hydrogen Bonding , Hydroxyquinolines/chemistry , Hydroxyquinolines/therapeutic use , Inhibitory Concentration 50 , K562 Cells , Molecular Docking Simulation , Structure-Activity RelationshipABSTRACT
EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.
Subject(s)
Acetylcysteine/analogs & derivatives , Cysteine Endopeptidases/metabolism , Cytoplasmic Vesicles/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Intracellular Membranes/metabolism , Multienzyme Complexes/metabolism , Protein Transport/physiology , Ubiquitin-Protein Ligases , Ubiquitins/metabolism , Acetylcysteine/pharmacology , Ammonium Chloride/pharmacology , Animals , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cysteine Endopeptidases/drug effects , Cysteine Endopeptidases/ultrastructure , Cysteine Proteinase Inhibitors/pharmacology , Cytoplasmic Vesicles/ultrastructure , Endocytosis/drug effects , Endocytosis/physiology , Endopeptidases/metabolism , ErbB Receptors/drug effects , ErbB Receptors/ultrastructure , Humans , Immunohistochemistry , Intracellular Membranes/ultrastructure , Leupeptins/pharmacology , Microscopy, Confocal , Microscopy, Electron , Multienzyme Complexes/drug effects , Multienzyme Complexes/ultrastructure , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins c-cbl , Transforming Growth Factor alpha/metabolismABSTRACT
Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of beta-sheets and beta-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-alpha (TGF-alpha), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min(-1) (Tyr), 0.09 min(-1) (Arg, beta-turns), and 1.88 x 10(-3) min(-1) (beta-sheets and 3(10)-helix); and for TGF-alpha 0.91 min(-1) (Tyr), 0.27 min(-1) (Arg, beta-turns), and 1.41 x 10(-4) min(-1) (beta-sheets). The time constants for AR 0.47 min(-1) (Tyr), 0.04 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (buried 3(10)-helix, beta-turns, and beta-sheets); for HB-EGF 0.89 min(-1) (Tyr), 0.14 min(-1) (Arg and 3(10)-helix), and 1.00 x 10(-3) min(-1) (buried 3(10)-helix, beta-sheets, and beta-turns); and for epiregulin 0.16 min(-1) (Tyr), 0.03 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (3(10)-helix and beta-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.
Subject(s)
Deuterium Exchange Measurement/methods , ErbB Receptors/chemistry , ErbB Receptors/ultrastructure , Models, Chemical , Models, Molecular , Computer Simulation , Kinetics , Ligands , Protein ConformationABSTRACT
We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Cell Membrane/metabolism , Crystallography/methods , ErbB Receptors/metabolism , ErbB Receptors/ultrastructure , Fluorescence Resonance Energy Transfer/instrumentation , Molecular Probe Techniques/instrumentation , Cell Line, Tumor , Equipment Design , Equipment Failure Analysis , Humans , Protein ConformationABSTRACT
Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monoclonal anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF binding and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.
Subject(s)
Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Animals , Binding Sites , Cell Membrane/metabolism , Cell Membrane/ultrastructure , ErbB Receptors/ultrastructure , Freeze Fracturing , Tumor Cells, CulturedABSTRACT
Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of â¼15 µm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 µm. The system has a response time of 1 ms with a temporal resolution down to 50 µs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of â¼100 µm in tumour spheroids.
Subject(s)
ErbB Receptors/metabolism , Imaging, Three-Dimensional , Optical Imaging , Protein Transport , Cell Line, Tumor , ErbB Receptors/ultrastructure , Humans , Models, Molecular , Nonlinear DynamicsABSTRACT
The â¼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first â¼80 residues of the tail are inhibitory, as demonstrated previously. The entire â¼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.
Subject(s)
ErbB Receptors/genetics , ErbB Receptors/ultrastructure , Amino Acid Sequence , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Crystallography, X-Ray , Enzyme Activation/genetics , ErbB Receptors/metabolism , Flow Cytometry , HEK293 Cells , Humans , Molecular Dynamics Simulation , Phosphorylation , Protein Structure, Tertiary , Sequence Deletion/geneticsABSTRACT
We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.
Subject(s)
ErbB Receptors/metabolism , Biotin/metabolism , Cells, Cultured , Epidermal Growth Factor/metabolism , ErbB Receptors/ultrastructure , Fluorescence , Lasers , Microscopy, Electron/methodsABSTRACT
Day-14 lower incisors and day-18 first lower molars of mouse embryos produced in vitro transforming activities for non-confluent NRK cells co-cultured in agar, and mitogenic activities for exponentially growing NRK and BHK cells. The patterns of distribution of TGF beta 1 and EGF receptor, both known to regulate cell proliferation, differentiation and transformation in vitro and suspected to play important roles in developmental processes, were studied during mouse odontogenesis by means of indirect immunofluorescence on fixed or frozen fixed sections. TGF beta 1 epitopes were detected in the stellate reticulum of day-13 to day-16 incisors and of molars from day-17 onwards. Dental mesenchyme of day-14 incisors and postnatal molars, and peridental mesenchyme of bud and cap stage molars and incisors were also stained by TGF beta 1 antibodies. EGF receptor was localized in the enamel organs of incisors and molars; the inner dental epithelium and later the outer dental epithelium rapidly became negative while the stellate reticulum remained stained. Incisor apical mesenchyme showed an intense reaction with EGF receptor antibodies after birth.
Subject(s)
Epidermal Growth Factor , Epitopes/analysis , ErbB Receptors/analysis , Odontogenesis , Receptors, Cell Surface/analysis , Tooth Germ/chemistry , Transforming Growth Factor beta , Animals , Cell Differentiation , Cell Line , Enamel Organ/chemistry , Enamel Organ/ultrastructure , ErbB Receptors/ultrastructure , Fibroblasts , Fluorescent Antibody Technique , Incisor , Mesoderm/chemistry , Mesoderm/ultrastructure , Mice , Mitogens , Molar , Receptors, Cell Surface/ultrastructure , Receptors, Transforming Growth Factor beta , Tooth Germ/ultrastructureABSTRACT
Epidermal growth factor receptors (EGFR) were labeled with 10 nm immunogold and examined on uncoated specimens of A431 human epidermoid carcinoma cells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (Vo). At Vo = 2 kV, the SE and BSE signals were too weak to identify all markers, while at Vo = 3-7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increasing the Vo to above 7 kV sometimes provokes instability of the specimens. A Vo of > or = 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.