Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics

Abstract

Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. direct and indirect healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Therefore, innovative and efficacious therapies for diabetes are urgently needed. Herein we describe a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic b-cells with utility for highcontent assessment of real-time insulin dynamics. Additionally, we report a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cellbased fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system. We observed that bafilomycin, a vacuolar H + ATPase inhibitor and inhibitor of insulin granule formation, significantly increased mCherry FP in INS-1 cells with PPI-mCherry. Partial least squares regression analysis demonstrated that an increase of FP by bafilomycin is significantly correlated with a decrease in granularity of PPI-mCherry signal in the cells. The increased FP by bafilomycin is due to inhibition of self-Fo¨rster resonant energy transfer (homo-FRET) caused by the increased mCherry intermolecular distance. FP substantially decreases when insulin is tightly packaged in the granules, and the homo-FRET decreases when insulin granule packaging is inhibited, resulting in increased FP. We performed pilot HTS of 1782 FDAapproved small molecules and natural products from Prestwick and Enzo chemical libraries resulting in an overall Z 0 -factor of 0.52 -0.03, indicating the suitability of this biosensor for HTS. This novel biosensor enables live-cell assessment of protein-protein interaction/protein aggregation in live cells and is compatible with conventional FP plate readers.

Fluorescence polarization immunoassay for the determination and screening of medicines.

“…Одним из распространенных методов тестирования флуоресцентно меченных соединений является поляризация флуоресценции (FP). Благодаря универсальности FP и наличию высококачественных поляризаторов, стабильных детекторов и источников возбуждения, этот метод нашел широкое применение в различных биологических приложениях: исследование клеточного микроокружения [2] и мониторинг клеточных сигнальных путей [3], до 2D и 3D-визуализация, [4], оценка систем доставки лекарств [5], обнаружение органических веществ и лекарственных средств [6][7][8].…”

Ca2+-dependent liquid-liquid phase separation underlies intracellular Ca2+ stores

“…To understand the influence of FAM20C phosphorylation on CASQ1 oligomerization in live cells, we developed a method to quantify oligomerization in real time. Previous studies monitored secretory pathway oligomerization processes like insulin granule packing using homologous Förster Resonance Energy Transfer (homo-FRET) biosensors (Yi et al, 2015). These biosensors rely on FRET between identical fluorophores, and fluorophore proximity is indicated by decreased relative fluorescent intensity of light at incident polarization (Supplementary Figure 6A).…”

Monitoring of Insulin Granule Packaging in Live Cells Using Homofret-FP Detection

“…Furthermore, if polarized light is applied as the excitation light it will become randomized as homoFRET occurs between adjacent fluorophores. It was reasoned that a homoFRET-FP approach would monitor the extent of packing of mature insulin into dense core granules 1 (Figure 1). The assay is based on the preproinsulin-mCherry (PPI-mCherry) system that uses mCherry as a fluorophore coupled to the precursor preproinsulin.…”

“…A) Free insulin-mCherry with polarized excitation will exhibit conserved polarization and relatively high MP signal. B) Within dense core granule polarized light will exhibit homoFRET, randomized polarization and a decrease in MP signal 1.…”