Application Note


Simultaneous detection of GPCR second messengers in living cells


L


ive cell assays with genetically-encod- ed, fluorescent biosensors provide advantages over end-point assays in cell lysates because functional information about the timing and location of cellular responses can be monitored in cells that are relevant to disease. Fluorescent biosensors are tools in basic research and can be detect- ed on automated plate readers1. Here we


show how second messengers relevant to Gq signalling can be detected in living cells using assays for diacylglycerol (DAG), phos-


phatidylinositol 4,5-bisphosphate (PIP2) and Ca2+. By combining spectrally distinct sen- sors in a single assay, we show that two responses can be detected simultaneously. Sensitive and simultaneous detection of two sensors is enabled by the BMG LABTECH multi-mode plate readers PHERAstar® FSX or CLARIOstar®.


Assay principle


The biosensors achieve optimal expression due to packaging in a modified baculovirus (BacMam). Figure 1A shows the mechanism for the DAG sensor in which a circularly-per- muted fluorescent protein is inserted near the DAG binding domain of protein kinase C.


Similar sensors are used for Ca2+ and PIP2. Each sensor comes in a spectrally-resolv- able red or green version that can be com- bined in a single transduction step to detect changes in two second messengers simulta- neously. The DAG biosensors are available as decreasing in fluorescence (Downward DAG) or increasing in fluorescence (Upward DAG). Both versions indicate increases in diacylglycerol in living cells.


The suite of DAG, PIP2 and Ca2+ biosen- sors unambiguously detects Gq signalling2,3 as it is initiated by Carbachol stimulation of muscarinic receptors, for instance.


Materials and methods l HEK293 cells.


l DAG sensor, PIP2 sensor, R-GECO Ca2+ sensor packaged in BacMam (Montana Molecular).


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l Greiner black microplates. l Microplate reader from BMG LABTECH. HEK293 cells were transduced in suspen- sion with the indicated sensor 24-36h prior to the experiment. Thirty minutes before measurement media was exchanged for PBS and cells rested at room temperature.


Results and discussion


Performance of the biosensors was assessed using HEK293 expressing the DAG sensor and stimulating them with Carbachol while mock-treating negative controls with PBS. The Z’ value was calculated to be 0.8 which is indicative for robust assays (Figure 1B).


Next, a duplex real-time assay was mea- sured with cells co-expressing the red R- GECO Ca2+ and the green DAG sensor. Upon Carbachol stimulation using the readers' on-board injector, calcium was rapidly released whereas DAG was pro- duced slightly slower but persisted over the period of measurement (200s) (Figure 1C).


Conclusion


BMG LABTECH multi-mode microplate readers CLARIOstar and PHERAstar FSX offer high sensitivity detection of the genet- ically encoded Montana Molecular sen- sors. Fast cellular responses can be moni- tored using on-board reagent injectors. The spectrally-resolved sensor variants indicate simultaneous changes in multiple second messengers following Gq activation. DDW


References 1Tewson, P et al (2016). J. Biomol. Screen. 21:298-305. 2 Ding, Y et al (2015). Nature Methods 12:195-198. 3Tewson, P et al (2012). Simultaneous Detection of Ca2+ and Diacylglycerol Signaling in Living Cells.


Figure 1: Genetically-encoded biosensors for


studying Gq signalling. (A)The DAG sensor undergoes conformational changes upon binding of DAG leading to a change in fluorescence intensity. (B) Cabachol changes the fluorescence intensity of the DAG sensor. 14 replicates are shown and Z’ value was calculated. (C) R-GECO (red) calcium sensor and green DAG (green) sensor were multiplexed. Traces depict average response to 30µM Carbachol (n=16) expressed as 100% response. Carbachol was dispensed using on- board reagent injectors at the 30-second time point as indicated


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