Dr Tobias Pusterla details a novel approach to monitor intracellular hormone packaging
Aberrant secretion of hormones and growth factors is related to diseases such as diabetes and cancer. Therefore, 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 fluorescence polarization 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. This change in FP value can be used to study cellular protein accumulation or dimerization events. Advantages of this 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) The 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.
The 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. The 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 to automate and run the assay in 384 well format proves it suitable for HTS. This is underlined by the identification of drugs that act on insulin processing out of 1,782 compounds.
Detection of homoFRET fluorescence polarization
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. The 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.
Dr Tobias Pusterla is with BMG Labtech.
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
