In drug discovery screening campaigns as well as in fundamental research activities, cellular parameters have to be measured and monitored in living cells regularly. Often fluorescent dyes are used to detect and follow some of the parameters. These methods are powerful, especially in screening large numbers of compounds, but have their limitations e.g. when it comes to measuring more than one parameter in the same living cell, or when specific locations in the cell shall be targeted.
To overcome these limitations and to provide tools to especially look into GPCR related signalling in the most comprehensive and detailed way currently possible, Montana Molecular have developed genetically encoded fluorescent biosensors to measure parameters such as cAMP, DAG, PIP2, Ca2+and voltage changes in living cells.
Montana Molecular’s fluorescent biosensors: How do these live cell assays work?
The principle of the assays is essentially based on single Red and Green Fluorescent Protein sensors, meaning the genetic information for either GFP or RFP linked to an analyte specific sensor protein domain is introduced into the cells to be investigated. Upon analyte binding to the sensor the fluorescent intensity of GFP or RFP changes which can be easily detected.
The genetic transfer is done with BacMam Vectors based on a modified baculovirus system. In mammalian cells, the baculovirus genome is silent, and it cannot replicate to produce new virus in mammalian cells. For the workflow of the assay see Figure 1.
Which analytes can be measured with the Montana Molecular fluorescent biosensors?
Kits are available for live cell measurements of various cellular parameters:
- cAMP
- DAG
- PIP2
- Ca2+ and voltage changes
The sensors are partly available with either GFP or RFP and with different promotors optimized for different target cells (ArcLight Voltage Sensor either with CMV or synapsin promoters).
Fluorescent biosensors: which applications?
Multiplexing in a single live-cell assay shows when an activated receptor signals through two different pathways
Figure 2 shows how combining red cAMP sensor with green DAG sensor, and co-expressed with the Calcitonin receptor in HEK293 cells, can give you a more detailed insight into GPCR mediated pathways. While signaling through G protein Gs results in the increase of cAMP (red), Gq mediated signalling results in DAG increase (green) – meaning that both Gq and Gs pathways have been activated in this experiment.
Red sensors can be combined with green sensors to make simultaneous measurement in a single assay
An example shown in Figure 3 is the green DAG sensor, coexpressed in HEK293 cells with R-GECO, a red Ca2+ sensor and P2Y11, a purinergic GPCR. If only Ca2+ (shown in red) is measured ATP and UTP responses seem very similar. By measuring DAG in the green channel, we see that ATP produces an increase in DAG, indicating signaling via Gq while UTP does not lead to Gq mediated DAG increase.
Simultaneous measurements of GPCR signaling can be visualized in living cells
The video below shows an example for a simultaneous kinetic visualization of different parameters in HEK 293 cells. M1 muscarinic receptor has been activated by carbachol. The same cells are shown in the top and bottom panels of the columns, green sensors in the top panels, red sensors in the bottom panels. In the first column you can see the kinetics of both the decrease of phospholipase C substrate (PIP2) with a corresponding increase in DAG, one of the products of PIP2 hydrolysis.
The BacMam system works with most cell types
A number of cell lines (e.g. HEK293, CHO, NIH3T3) as well as relevant primary cells like pancreatic islets, neurons, cardiomyocytes, osteoblasts, and fibroblasts have been successfully transduced with the BacMam system. Furthermore, successful experiments have been run with iPSC-derived cells.
Furthermore, the assays…
- show robust and reliable signals on automated plate readers (with Z’ values of > 0.7)
- enable genetic targeting of specific location in the cell (cilia vs membranes vs nucleus)
- enable specific expression in rare cells or mixed populations — for example: neurons vs glia
- allow detection of Gi mediated signalling without the use of forskolin or IBMX
- avoid interference by interaction with compounds as is the case with luciferase based reporter systems
Montana Molecular have recently chosen tebu-bio to market their products in Europa. Their products are used for drug discovery and to observe and measure cell signaling processes. Their scientific team is focused on creating breakthrough innovations that empower both basic research scientists and drug discovery teams.
Interested in more information about these highly innovative kits? Don’t hesitate to contact me through the form below for further information.
Thanks to our friends from Montana Molecular who provided the material for this blog.
