Biomedical subjects

Ann M Ferrie

Publications and source records attributed to Ann M Ferrie.

7 recordsLinked to original sources

PubMed2006

Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells.

INTRODUCTION: Screening drugs against G protein-coupled receptors (GPCRs) - the single largest family of drug targets in the human genome - is still a major effort in pharmaceutical and biotech industries. Conventional cell-based assays generally measure a single cellular event, such as the generation of a second messenger or the relocation of a specific protein target. However, manipulation or engineering of cells is often a prerequisite for these technologies to achieve desired sensitivities. The present study is focused on the use of non-invasive and manipulation-free optical biosensors for assaying endogenous GPCRs in adherent cells. METHODS: Resonant waveguide grating (RWG) biosensor was applied to manifest ligand-induced dynamic mass redistribution (DMR) within the bottom portion of adherent cell layer. The DMR signatures mediated through the activation of several endogenous GPCRs in cells were characterized. Endogenous receptor panning was examined at cell system level by using a panel of agonists known to activate many GPCRs, and also at family receptor level by determining the efficacies of a set of family-specific agonists. RESULTS: Three major types of optical signatures were identified; each was correlated with the activation of a class of GPCRs, depending on the G protein with which the receptor is coupled (i.e., G(q), G(s) and G(i)). The characteristics of DMR signals, mostly the amplitude and kinetics of a DMR event, were dependent on the doses of agonists and the expression levels of endogenous receptors. All three classes of endogenous receptors were found in human epidermoid carcinoma A431 cells. Interestingly, the dose-dependent switching from one type of DMR signal to another was observed for several GPCR agonists examined. A small panel of P2Y receptor agonists exhibited distinct efficacies in three cell lines examined. DISCUSSIONS: The RWG biosensors were applicable to study the activation of endogenous GPCRs. Like second messengers or gene expression, the DMR signals obtained could be considered as novel and quantifiable physiological responses of living cells mediated through GPCRs and used for studying receptor biology.

Animals↗

PubMed2006

Resonant waveguide grating biosensor for living cell sensing.

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.

Animals↗

PubMed2006

Cellular functions of cholesterol probed with optical biosensors.

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.

Biosensing Techniques↗

PubMed2006

Air-stable G protein-coupled receptor microarrays and ligand binding characteristics.

This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.

Air↗

PubMed2005

Functional GPCR microarrays.

This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.

Drug Evaluation, Preclinical↗

PubMed2005

Characteristics of dynamic mass redistribution of epidermal growth factor receptor signaling in living cells measured with label-free optical biosensors.

This paper reported the identification of a novel optical signature for epidermal growth factor (EGF) receptor signaling in human epidermoid carcinoma A431 cells mediated by EGF. The optical signature was based on dynamic mass redistribution (DMR) in living cells triggered by EGFR activation, as monitored in real time with resonant waveguide grating biosensors. Analysis of the modulation of the EGF-induced DMR signals by a variety of known modulators provided links of various targets to distinct steps in the cellular responses. Results showed that the dynamic mass redistribution in quiescent A431 cells mediated by EGF required EGFR tyrosine kinase activity, actin polymerization, and dynamin and mainly proceeded through MEK. The DMR signals obtained serve as integrated signatures for interaction networks in the EGFR signaling.

Animals↗

PubMed2005

Probing cytoskeleton modulation by optical biosensors.

This paper reported the use of resonant waveguide grating biosensors for studying the cytoskeleton structure in cells. This was achieved by measuring the changes in mass within the bottom portion of cells upon exposure to saponin in the absence and presence of cytoskeleton modulators. Treatment of Chinese hamster ovary cells with saponin led to a dose-dependent and dynamic mass changes. When a higher concentration of saponin (> 60 microg/ml) was used, a net loss in mass was observed. This is probably resulted from the diffusion of soluble intracellular materials away from the bottom portion of cells after pore formation in the cell plasma membranes by saponin. The pretreatment of cells with actin disruption agents, cytochalasin B and latrunculin A, led to significantly increased loss in cell mass induced by either 75 or 125 microg/ml saponin. These results suggested that optical biosensors provide an attractive means to study the cytoskeleton structure and screen modulators that affect the cytoskeleton structure.

Animals↗