Epidermal Growth Factor Receptor Clustering Research Articles

A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model.

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells.

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR–HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR–EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Expeditious Generation of Biparatopic Common Light Chain Antibodies via Chicken Immunization and Yeast Display Screening.

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings.

Correction to "Characterizing Large-Scale Receptor Clustering on the Single Cell Level: A Comparative Plasmon Coupling and Fluorescence Superresolution Microscopy Study".

Spatial clustering of cell membrane receptors has been indicated to play a regulatory role in signal initiation, and the distribution of receptors on the cell surface may represent a potential biomarker. To realize its potential for diagnostic purposes, scalable assays capable of mapping spatial receptor heterogeneity with high throughput are needed. In this work, we use gold nanoparticle (NP) labels with an average diameter of 72.17 ± 2.16 nm as bright markers for large-scale epidermal growth factor receptor (EGFR) clustering in hyperspectral plasmon coupling microscopy and compare the obtained clustering maps with those obtained through fluorescence superresolution microscopy (direct stochastic optical reconstruction microscopy, dSTORM). Our dSTORM experiments reveal average EGFR cluster sizes of 172 ± 99 and 150 ± 90 nm for MDA-MB-468 and HeLa, respectively. The cluster sizes decrease after EGFR activation. Hyperspectral imaging of the NP labels shows that differences in the EGFR cluster sizes are accompanied by differences in the average separations between electromagnetically coupled NPs. Because of the distance dependence of plasmon coupling, changes in the average interparticle separation result in significant spectral shifts. For the experimental conditions investigated in this work, hyperspectral plasmon coupling microscopy of NP labels identified the same trends in large-scale EGFR clustering as dSTORM, but the NP imaging approach provided the information in a fraction of the time. Both dSTORM and hyperspectral plasmon coupling microscopy confirm the cortical actin network as one structural component that determines the average size of EGFR clusters.

Leader cell PLCγ1 activation during keratinocyte collective migration is induced by EGFR localization and clustering.

Re‐epithelialization is a critical step in wound healing and results from the collective migration of keratinocytes. Previous work demonstrated that immobilized, but not soluble, epidermal growth factor (EGF) resulted in leader cell‐specific activation of phospholipase C gamma 1 (PLCγ1) in HaCaT keratinocytes, and that this PLCγ1 activation was necessary to drive persistent cell migration. To determine the mechanism responsible for wound edge‐localized PLCγ1 activation, we examined differences in cell area, cell–cell interactions, and EGF receptor (EGFR) localization between wound edge and bulk cells treated with vehicle, soluble EGF, or immobilized EGF. Our results support a multistep mechanism where EGFR translocation from the lateral membrane to the basolateral/basal membrane allows clustering in response to immobilized EGF. This analysis of factors regulating PLCγ1 activation is a crucial step toward developing therapies or wound dressings capable of modulating this signal and, consequently, cell migration.

Reactive oxygen species mediates 50-Hz magnetic field-induced EGF receptor clustering via acid sphingomyelinase activation

Purpose: Exposure to extremely low frequency electromagnetic fields (ELF-EMFs) could elicit biological effects including carcinogenesis. However, the detailed mechanisms by which these ELF-EMFs interact with biological system are currently unclear. Previously, we found that a 50-Hz magnetic field (MF) exposure could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell membranes. In the present experiment, the possible roles of reactive oxygen species (ROS) in MF-induced EGFR clustering were investigated.Materials and methods: Human amnion epithelial (FL) cells were exposed to a 50-Hz MF with or without N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). EGFR clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining. The intracellular ROS level and acid sphingomyelinase (ASMase) activity were detected using an ROS assay kit and an Amplex® Red Sphingomyelinase Assay Kit, respectively.Results: Results showed that exposure of FL cells to a 50-Hz MF at 0.4 mT for 15 min significantly enhanced the ROS level, induced EGFR clustering and increased ASMase activity. However, pretreatment with NAC or PDTC, the scavenger of ROS, not only counteracted the effects of a 50-Hz MF on ROS level and AMS activity, but also inhibited the EGFR clustering induced by MF exposure.Conclusions: The present and previous data suggest that ROS mediates the MF-induced EGFR clustering via ASMase activation.

The Effect of Nanoparticles on the Cluster Size Distributions of Activated EGFR Measured with Photobleaching Image Correlation Spectroscopy.

The epidermal growth factor receptor (EGFR) is an important cell surface receptor in normal physiology and disease. Recent work has shown that EGF-gold nanoparticle conjugates can influence cell behaviour, but the underlying mechanism at the receptor quaternary structural level remains poorly understood.In the present work, the cluster density and cluster size of activated (phosphorylated) EGFR clusters in HeLa cells were determined with photobleaching image correlation spectroscopy. EGFR activation was probed via immunofluorescence-detected phosphorylation of tyrosines (pY-mAb) located in the kinase domain of EGFR (Y845) and at the EGFR cytoplasmic tail (Y1173). Cell activation was probed via nuclear extracellular-regulated kinase (ERK) phosphorylation. The cluster size of activated EGFR was 1.3-2.4 pY-mAb/cluster in unstimulated HeLa cells. EGF or nanorod treatment led to an increase in EGFR oligomers containing multiple phosphotyrosines (>2 phosphotyrosines per EGFR oligomer, average cluster size range = 3-5 pY-mAb/cluster) which paralleled increases in nuclear p-ERK. In contrast, EGF-nanorods decreased the contribution from higher-order phospho-clusters and decreased nuclear p-ERK relative to the nanorod control. These studies provide direct evidence that targeted nanotechnology can manipulate receptor organization and lead to changes in receptor activation and subsequent signalling processes.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) promotes EGF receptor signaling of oral squamous cell carcinoma metastasis via the complex N-glycosylation.

Aberrant protein glycosylation could be a distinct surface-marker of cancer cells that influences cancer progression and metastasis because glycosylation can regulate membrane protein folding which alters receptor activation and changes epitope exposure for antibody (Ab) recognition. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a glycophosphoinositol-anchored protein, is a heavily glycosylated tumor antigen. However, the clinical significance and biological effect of CEACAM6 glycosylation has not been addressed in cancers. We recently developed an anti-CEACAM6 Ab (TMU) from an immune llama library which can be engineered to a single-domain (sd)Ab or a heavy-chain (HC)Ab. The TMU HCAb specifically recognized glycosylated CEACAM6 compared to the conventional antibodies. Using the TMU HCAb, we found that glycosylated CEACAM6 was a tumor marker associated with recurrence in early-stage OSCC (oral squamous cell carcinoma) patients. CEACAM6 promoted OSCC cell invasion, migration, cytoskeletal rearrangement, and metastasis via interaction with epidermal growth factor (EGF) receptor (EGFR) and enhancing EGFR activation, clustering and intracellular signaling cascades. These functions were modulated by N-acetylglucosaminyltransferase 5 (MGAT5) which mediated N-glycosylation at Asn256 (N256) of CEACAM6. Finally, the TMU sdAb and HCAb treatment inhibited the migration, invasion and EGF-induced signaling in CEACAM6-overexpressing cells. In conclusion, the complex N-glycosylation of CEACAM6 is critical for EGFR signaling of OSCC invasion and metastasis. Targeting glycosylated CEACAM6 with the TMU sdAb or TMU HCAb could be a feasible therapy for OSCC.

Abstract 2919: Quantitative mapping of epidermal growth factor receptor activation in single breast cancer cells treated with targeted therapies

Abstract Twenty percent of breast cancers are triple-negative, lacking estrogen receptor, progesterone receptor, and HER2. There is currently no specific FDA-approved targeted therapy for triple-negative breast cancer patients. Approximately half of triple-negative breast cancers overexpress epidermal growth factor receptor (EGFR), however phase II trials have only shown a 5% response rate with targeted inhibition. A quantitative analysis of EGFR signaling pathway dynamics of single cells at the single molecule level has the potential to shed light on the mechanism of lack of response to targeted therapy, as well as the cell-to-cell heterogeneity of response. Here, we combine single-molecule 3D imaging of EGFR using fluorescent quantum dots (QDs) with cell geometry normalization by micro-patterning to quantitatively map EGFR endocytosis in single cancer cells. Triple negative MDA-MB-231 cells conformed to defined geometries (rectangular with different aspect ratios) were grown on soft polyacrylamide gels after micro-contact printing islands of fibronectin. Two color QD conjugates of EGFR and its ligand, EGF, are used to track ligand binding and receptor internalization upon activation in the absence and presence of different families and dosages of pharmaceutical EGFR inhibitors. We demonstrated that microcontact-printing normalizes cell geometry while maintaining EGFR localization so that EGFR signaling response can be quantitatively assessed on a single-cell basis and that QD conjugates can specifically label EGFR and EGF. Quantitative single-molecule imaging at single-cell level revealed high level of heterogeneity in the amount and spatial localization of EGF binding and EGFR endocytosis among the cell population in the presence and absence of EGFR inhibitor, gefitinib. Using this platform, we will investigate if spatial organization of EGFR clusters within individual cells contribute to single-cell heterogeneous response to EGFR inhibitors. We anticipate quantitative mapping of EGFR activity with drug treatment will help to elucidate why EGFR drug treatments fail and provide a means to develop combination therapy to address heterogeneity in triple negative-breast cancer. Citation Format: Phuong Le, Kristopher Kilian, Andrew Smith. Quantitative mapping of epidermal growth factor receptor activation in single breast cancer cells treated with targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2919. doi:10.1158/1538-7445.AM2017-2919

Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy.

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ≥ 3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells.

NADPH oxidase-produced superoxide mediated a 50-Hz magnetic field-induced epidermal growth factor receptor clustering

Purpose: A 50-Hz magnetic field (MF) was found to induce epidermal growth factor receptor (EGFR) clustering in our previous study. The aim of this work was to investigate the molecular mechanisms that mediated MF-induced EGFR clustering.Materials and methods: Human amniotic epithelial (FL) cells were exposed to a 50-Hz MF. Total reactive oxygen species (ROS), cytoplasmic and mitochondrial superoxide production were detected by DCFH-DA, DHE and MitoSOX, respectively. EGFR clustering was analyzed using confocal microscopy after indirect immunofluorescence staining.Results: Results showed that exposing FL cells to MF at intensity higher than 0.2 mT for 15 min enhanced total ROS production. Additionally, enhanced total ROS and cytoplasmic superoxide production were observed after exposing cells to MF at 0.4 mT for 5, 15, or 30 min, while mitochondrial superoxide production for 15 or 30 min. Pretreatment with Nox inhibitor, DPI, effectively inhibited MF-induced cytoplasmic superoxide production and subsequent EGFR clustering while mitochondrial superoxide production was not affected.Conclusions: Nox-produced superoxide mediated a 50-Hz magnetic field-induced EGFR clustering.

Acid sphingomyelinase mediates 50-Hz magnetic field-induced EGF receptor clustering on lipid raft

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell surface. In order to explore the possible mechanisms, the roles of acid sphingomyelinase (ASMase) and lipid raft in MF-induced EGFR clustering were investigated in the present study. Human amnion epithelial (FL) cells were exposed to a 50-Hz MF at 0.4 mT for different durations. Intracellular ASMase activity was detected using the Amplex® Red Sphingomyelinase Assay Kit. EGFR clustering, ASMase, and lipid rafts on cell membrane were analyzed using confocal microscopy after indirect immunofluorescence staining. Results showed that disturbing lipid rafts with nystatin could inhibit MF-induced EGFR clustering, indicating that it was dependent on intact lipid raft. Exposure of FL cells to MF significantly enhanced ASMase activity and induced ASMase translocation to membrane that co-localized with lipid rafts. Treatment with imipramine, an ASMase inhibitor, inhibited the MF-induced EGFR clustering. This inhibitory effect could be blocked by the addition of C2-ceramide in the culture medium. It suggested that ASMase mediated the 50-Hz MF-induced EGFR clustering via ceramide which was produced from hydrolyzation on lipid rafts.

Probing the Non-Genetic Cell-To-Cell Heterogeneity in EGFR-Mediated MAPK Activation by Super-Resolution Microscopy

Understanding the biological consequences of cellular heterogeneity is an unmet challenge. For example, heterogeneity of mitogen-activated protein kinase phosphorylation (pMAPK) in response to epidermal growth factor receptor (EGFR) activation is poorly understood in genetically identical cells. We used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intra-cluster distances (d). From this data, we predicted the extent of pMAPK with 85% accuracy on a cell-by-cell basis with control data returning 54% accuracy (P 50nm) were most predictive. Electron microscopy revealed that they were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many smaller dimers/multimers (d<50nm) were found on intraluminal vesicles within MVBs, providing a likely explanation for reduced pMAPK in those cells.

Influenza a Viruses use Multivalent Sialic Acid Clusters for Cell Binding and Receptor Activation

Binding of influenza A virus to the host cell is mediated by the major viral surface protein hemagglutinin (HA). HA recognises sialic acid (SA), a plasma membrane glycan that functions as the specific primary virus attachment factor. Since, sialic acid alone can not fulfil a signalling function, the virus needs to activate downstream factors in order to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase (RTK) family, was shown to be involved and transmit virus entry signals. However, how influenza viruses engage with and activate EGFR is largely unknown. Using quantitative super-resolution microscopy (STORM and PALM), we show that SA and EGFR are organized in submicrometer clusters in the apical plasma membrane of different permissive cell lines. We found that the local SA concentration, a parameter that directly influences virus-cell binding, strongly increases towards the cluster center, thereby representing a multivalent virus-binding platform. By using 2-color super-resolution microscopy, we show that SA and EGFR clusters co-localize, eventually allowing SA-mediated virus-EGFR interaction. Finally, we show that virus binding activates EGFR and that this process occurs without a major EGFR redistribution, suggesting the activation of preformed clusters. Further, our quantitative characterization of SA distribution on the host cell membrane, enabled us to simulate 2D virus trajectories, which could be confirmed by live cell single-virus tracking. Taken together, our results provide a quantitative functional model of the initial events of influenza virus infection and provide insights into how the virus finds and activates EGFR.

The Dynamics and Localization of the Epidermal Growth Factor Receptor (EGFR) on Live Cell Plasma Membrane Studied by ITIR-FCS

The Epidermal Growth Factor Receptor (EGFR), a prototypical receptor tyrosine kinase, plays an important role in cellular processes such as proliferation, migration and apoptosis. EGFR is known to be strongly regulated by its localization and the state of activation on the plasma membrane. However, the spatial distribution of EGFR on the plasma membrane has not been resolved in detail.Here, we use Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) to investigate EGFR behavior on live cells before and after stimulation. ITIR-FCS allows monitoring EGFR dynamics on cell membranes and in combination with the FCS diffusion laws provides information on the nanoscopic EGFR organization.Our results indicate that EGFR is partly trapped in cholesterol sensitive domains and also couples to the cytoskeleton. Stimulation by EGF activates EGFR in a dose dependent manner. At low-dose stimulation (10 ng/ml), the membrane diffusion and confinement of EGFR remain unchanged and EGFR internalization is cholesterol independent and EGFR recycles back to the membrane. At high-dose EGF treatment (100 ng/ml), EGFR endocytosis is cholesterol dependent and cholesterol-depletion impairs receptor internalization. In addition, cholesterol-depletion with concomitant EGF stimulation leads to stronger confinement of the receptor, independent of the EGF dose, as clusters accumulate on the membrane. Furthermore, the actin cytoskeleton promotes EGFR internalization after ligand stimulation. Disruption of the actin cytoskeleton inhibits EGFR endocytosis and induces clustering of EGFR on the cell membrane. This study demonstrates the capabilities of ITIR-FCS to follow receptor stimulation events on live cells. It demonstrates not only that EGFR activation is cholesterol and cytoskeleton dependent but also that the activation pathway is EGF dose dependent, thus providing new insights into receptor dynamics and organization on live cells.

Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States

The spatial arrangement of the epidermal growth factor receptor (EGFR) on the cellular plasma membrane is one of the prime factors that control its downstream signaling pathways and related functions. However, the molecular organization, which spans the scale from nanometers to micrometer-size clusters, has not been resolved in detail, mainly due to a lack of techniques with the required spatiotemporal resolution. Therefore, we used imaging total internal reflection-fluorescence correlation spectroscopy to investigate EGFR dynamics on live CHO-K1 plasma membranes in resting and ligand-bound states. In combination with the fluorescence correlation spectroscopy diffusion law, this provides information on the subresolution organization of EGFR on cell membranes. We found that overall EGFR organization is sensitive to both cholesterol and the actin cytoskeleton. EGFR in the resting state is partly trapped in cholesterol-containing domains, whereas another fraction exhibits cholesterol independent trapping on the membrane. Disruption of the cytoskeleton leads to a broader range of EGFR diffusion coefficients and a reduction of hop diffusion. In the ligand-bound state we found a dose-dependent behavior. At 10 ng/mL EGF the EGFR is endocytosed and recycled to the membrane, whereas diffusion and organization do not change significantly. At 100 ng/mL EGF the EGFR forms clusters, which are subsequently internalized, whereas outside the clusters diffusivity increases and the organization of the receptor remains unchanged. After disruption of cholesterol-containing domains or actin cytoskeleton, EGF induces microscopic EGFR clusters on the membrane and endocytosis is inhibited.

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.

The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

Microenvironmental stiffness enhances glioma cell proliferation by stimulating epidermal growth factor receptor signaling.

The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling.

Single Molecule Imaging Reveals that Activating Kinase Domain Mutations Reduce EGFR Mobility and Enhance Dimerization

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases which play an important role in a number of physiological responses and have also been identified as a common signaling component in several cancer types. Physiological signaling is initiated by ligand binding which stabilizes EGFR dimers, resulting in auto-phosphorylation of the intracellular kinase domain. Mutation of EGFR is associated with tumor progression due to constitutive activity resulting from several known kinase domain variants. Our previous work has shown that ligand-induced dimerization of wild type EGFR results in decreased receptor mobility, and that this reduction in mobility is dependent on kinase activity. Therefore, we wondered whether ligand-independent activity of EGFR mutants correlates with differences in receptor mobility or dimerization kinetics.Here, we employ multi-color, single particle tracking (SPT) of quantum dots to visualize and quantify the dynamics and interactions of individual receptors within the membrane. Using a novel, high-speed hyperspectral microscope, we are able to track up to eight spectrally distinct quantum dots for visualization and quantification of receptor interactions. From this data, we quantify receptor dimerization kinetics using a Hidden Markov Model. We find that unliganded EGFR mutants exhibit a reduced mobility and longer dimer lifetimes when compared to wild type. These results are consistent with constitutive activity of the EGFR mutants and suggest that the active kinase domain facilitates dimer formation in the absence of ligand. Moreover, we test the effects of clinical tyrosine kinase inhibitors on the single molecule behavior of EGFR mutants. We further examined the mobility and clustering of EGFR within the cellular actin cytoskeleton by hyperspectral tracking over GFP-actin, as well as two-color super-resolution microscopy.

Exploring higher-order EGFR oligomerisation and phosphorylation--a combined experimental and theoretical approach.

The epidermal growth factor receptor (EGFR) kinase is generally considered to be activated by either ligand-induced dimerisation or a ligand-induced conformational change within pre-formed dimers. Ligand-induced higher-order EGFR oligomerisation or clustering has been reported but it is not clear how EGFR oligomers, as distinct from EGFR dimers, influence signaling outputs. To address this question, we combined measures of receptor clustering (microscopy; image correlation spectroscopy) and phosphorylation (Western blots) with modelling of mass-action chemical kinetics. A stable BaF/3 cell-line that contains a high proportion (>90%) of inactive dimers of EGFR-eGFP but no secreted ligand and no other detectable ErbB receptors was used as the model cell system. EGF at concentrations of greater than 1 nM was found to cluster EGFR-eGFP dimers into higher-order complexes and cause parallel increases in EGFR phosphorylation. The kinetics of EGFR clustering and phosphorylation were both rapid, plateauing within 2 minutes after stimulation with 30 nM EGF. A rule-based model was formulated to interpret the data. This model took into account ligand binding, ligand-induced conformational changes in the cytosolic tail, monomer-dimer-trimer-tetramer transitions via ectodomain- and kinase-mediated interactions, and phosphorylation. The model predicts that cyclic EGFR tetramers are the predominant phosphorylated species, in which activated receptor dimers adopt a cyclic side-by-side orientation, and that receptor kinase activation is stabilised by the intramolecular interactions responsible for cyclic tetramerization.