Researchers at MUSC have configured a robust, platform-independent algorithm for use of real-time quantification of receptor endosome formation, agonist mobilization, co-localization, areas, and shapes. The algorithm works by segmenting the cells, localizing fluorescently stained nuclei to the segmented outlines, counting the number of small objects within the cells on 2 wavelengths, and then counting the number of co-localizations between the objects. The algorithm contains several improvements over its first iteration including the ability to select a region of interest of cells, faster performance, platform-independent and adjustable parameters with smart defaults.
This technology has successfully discriminated between Class A and B G protein-coupled receptor (GPCR) internalization characteristics with respect to interactions with β-arrestin2. The applicability of this platform is broad, however, such that the method provides an ability to correlate real-time GPCR signaling kinetics with protein mobilization kinetics.
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Overview: One shortcoming of current microscopy and quantification of microscopic data is the inability to present quantitative data for an entire dataset. Images are typical presented to represent an entire cell population. Manual scoring, counting, classification methods, and low-throughput are subjected to sampling rates and observer bias. The two assays currently employed for evaluating GPCR have shortcomings such that they observe whole well changes of cells, as opposed to cell-by cell changes, and cannot differentiate agonist profiles among different cell types or class of GPCR.
Applications: Drug discovery, drug development, quantification of real-time cell analysis
Advantages: Real-time analysis of receptor internalization following ligand mediated activation with real-time protein tracking. High kinetic resolution allows for analytic flexibility that can eliminate the need for cell fixation.
Key Words: high content, GPCR, adrenergic, parathyroid, arrestin, co-localization, kinetics, image processing, microscopy
Publications: Leonard, Anthony P., et al. “A High-Content, Live-Cell, and Real-Time Approach to the Quantitation of Ligand-Induced β-Arrestin2 and Class A/Class B GPCR Mobilization.“ Microscopy and Microanalysis 19.01 (2013): 150-170.
Wilson, P.C., et al. “The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.” Journal of Biological Chemistry 288(26) (2013): 18872-84.
DotQuanta Citations: Lee, M.H., et al. “S1P in HDL Promotes Interaction Between SR-BI and S1PR1 and Activates S1PR1-Mediated Biological Functions: Calcium Flux and S1PR1 Internalization.” Journal of Lipid Research M070706 (2016): 1-45
Author: Yuri Peterson
Patent Status: Copyrighted
MUSC-FRD Technology ID: P1406C
TTO Home Page: http://musc.technologypublisher.com
Name: Kaitlyn Crobar
Title: Technology Transfer Associate