Dataset Information

Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis.

ABSTRACT: Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell surface. The analysis demonstrates that under physiological conditions, the EGF receptor exists in a complex equilibrium involving single molecules and clusters of two or more receptors. Acute depletion of cellular cholesterol enhanced EGF receptor clustering whereas cholesterol loading decreased receptor clustering, indicating that receptor aggregation is sensitive to the lipid composition of the membrane.

SUBMITTER: Saffarian S

PROVIDER: S-EPMC1913168 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis.

Saffarian Saveez S Li Yu Y Elson Elliot L EL Pike Linda J LJ

Biophysical journal 20070511 3

Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell ...[more]

PMID: 17496034

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Project description:Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.

Project description:Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand-receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant K(d)=790+/-150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer-receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300+/-190 receptors microm(-2)) and HeLa cervical cancer cells (550+/-90 receptors microm(-2)). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.

Project description:Thermally activated delayed fluorescence (TADF) molecules offer nowadays a powerful tool in the development of novel organic light emitting diodes due to their capability of harvesting energy from non-emissive triplet states without using heavy-metal complexes. TADF emitters have very small energy difference between the singlet and triplet excited states, which makes thermally activated reverse intersystem crossing from the triplet states back to the singlet manifold viable. This mechanism generates a long-lived delayed fluorescence component which can be explored in the sensing of oxygen concentration, local temperature, or used in time-gated optical cell-imaging, to suppress interference from autofluorescence and scattering. Despite this strong potential, until recently the application of TADF outside lighting devices has been hindered due to the low biocompatibility, low aqueous solubility and poor performance in polar media shown by the vast majority of TADF emitters. To achieve TADF luminescence in biological media, careful selection or design of emitters is required. Unfortunately, most TADF molecules are not emissive in polar media, thus complexation with biomolecules or the formation of emissive aggregate states is required, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein, we demonstrate a facile method with great generalization potential that maintains the photophysical properties of solvated dyes by combining luminescent molecules with polymeric nanoparticles. Using an established swelling procedure, two known TADF emitters are loaded onto polystyrene nanoparticles to prepare TADF emitting nanomaterials able to be used in live-cell imaging. The obtained particles were characterized by optical spectroscopy and exhibited the desired TADF emission in aqueous media, due to the polymeric matrix shielding the dye from solvent polarity effects. The prepared nanoparticles were incubated with live human cancer cells and showed very low cytotoxicity and good cellular uptake, thus making fluorescence microscopy imaging possible at low dye concentrations.

Dataset Information

Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis.

Publications

Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis.

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