26 ANALYTICAL AND LABORATORY EQUIPMENT
HomoFRET fluorescence polarization
Dr Tobias Pusterla details a novel approach to monitor intracellular hormone packaging
A
berrant secretion of hormones and growth factors is related to
diseases such as diabetes and cancer. Terefore, it is essential to find drugs that interfere with this secretory process. In preparation for the release into the extracellular matrix, molecules are tightly packed into granules. As it is a cellular process that needs to be recorded in a cellular background, common biochemical assays fail to record changes in packaging. A novel approach uses homoFRET fluorescence polarization (FP) for identification of drugs that act on insulin packaging in live cells.
HomoFRET FP detects granular hormone accumulation FP measurements established in high-throughput screenings due to numerous advantages including cost-effectiveness, homogeneity, sensitivity and a huge diversity of applications. During homoFRET FP a
fluorophore excited by polarized light transfers its emission light to a fluorophore of the same type if found in close proximity. Upon energy transfer, the final emission light gets depolarized. Consequently, FP decreases with proximity of the fluorophores. Tis change in FP value can be used to study cellular protein accumulation or dimerization events. Advantages of this
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method are the capability to measure intracellular processes and to monitor the events in real time. Based on homoFRET events,
researchers at North Carolina Central University in the USA developed a cell based assay to measure insulin packaging.1 Te group used rat insulinoma cells (INS-1) that expressed the biosensor: an mCherry-coupled preproinsulin. Whereas discrete mCherry-coupled insulin will not change polarization of the emitted signal, insulin that is tightly packed into granules will transfer energy between mCherry molecules leading to decreased fluorescence polarization (Fig 1.). Te scientists validated
the assay by detecting FP on a Pherastar microplate reader from BMG Labtech. Bafilomycin, an agent that blocks granule formation, was used to modulate insulin packaging. Te unperturbed insulin- producing cells exhibited a low FP value representing granules with densely packed insulin and accordingly high homoFRET. Bafilomycin dose-dependently increased the FP value due to loss of tight packaging. Acceptable Z factors as well as the possibility
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Fig. 2. Pilot screen. Scatter plot of test compounds (circles), hits (asterisks), negative control (red triangle) and positive control (Bafilomycin – blue triangle)
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Dr Tobias Pusterla is with BMG Labtech.
www.bmglabtech.com
A Free Insulin-mCherry B Insulin-mCherry Granule
Polarized Excitation
High Polarization value
to automate and run the assay in 384 well format proves it suitable for HTS. Tis is underlined by the identification of drugs that act on insulin processing out of 1,782 compounds (Fig. 2). To reliably detect the small changes in FP originating from different packaging states, a microplate reader requires stable measurements. By simultaneously detecting the emission in parallel and perpendicular to the excitation light, the Pherastar FSX not only offers low data variation but also fast read times. Te highly sensitive reader for measuring FP can be easily integrated into robotic systems and its software accelerates data analysis such as calculation of IC50 values.
For more information ✔ at
www.scientistlive.com/eurolab Low Polarization value
Fig. 1. HomoFRET fluorescence polarization decreases in granules with tightly packed biosensor
REFERENCE 1
Yi NY et al. (2015),
Development of a Cell- Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics. Assay Drug. Dev. Technol. 13(9): 558- 569
% Inhibition
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