New Fluorescent Tools to Monitor Cell Stress and Toxicity

This biosensor allows real-time monitoring of genetically or chemically induced cell stress and drug induced cellular toxicity in under 24 hours. It can be combined with second messenger biosensors to simultaneously monitor cell signaling in stressed and healthy cells.

The Cell Stress sensor produced a strong response compared to a control in healthy cells exposed to a panel of cancer drugs that are known to be very toxic. We can imagine that the sensor could be used in live cardiomyocytes as a way to identify better cancer drugs that do not stress healthy cells.Another example would be to identify drugs that can reverse the stress response in degenerative diseases. Parkinson’s or cystic fibrosis are examples.

Identify stressed cells and interrogate second messenger signaling in neurodegeneration.

Neurodegenerative diseases are slow processes that only involve a few cells at a time. Imagine being able to find these cells, while they are still alive and undergoing stress. One could ask: are the cells still responsive to neurotransmitters and drugs? Are their second messenger signaling systems still functional? There are good reasons to ask these questions because Ca2+ and cAMP signaling appear to be affected in Alzheimer’s, Parkinson’s, and Huntington’s disease. To address these questions we developed two-color, multiplex sensor systems that indicate stressed cells in one color and in another color, report second messenger signals in both stressed and healthy cells.

To detect cellular stress we designed synthetic transcripts based on the non-canonical splicing of the XBP1 intron. Stressed cells splice the XBP1 intron, introduce a frameshift in the coding region, and produce a very bright mNeon green fluorescent protein sensor that is easily detected in automated plate readers. These sensors are sensitive enough to detect the difference between normal rhodopsin and the P23H mutant that causes retinitis pigmentosa as well as chemical stress induced by thapsigargin and tunicamycin. We combined this bright mNeon green stress sensor with R-GECO, a red fluorescent Ca2+ indicator and also with red cADDis, our red sensor for cAMP. The P23H mutant rhodopsin raised the resting level of cytosolic Ca2+ in the stressed cells, and blunted the amplitude of Ca2+ release from their intracellular stores in response to activation of a Gq pathway. Interestingly, the resting levels of cAMP in the stressed cells was reduced, though the absolute amplitude of the response to Gs stimulation (?F/F) was not altered. Copyright 2017 Society of Biomolecular Imaging and Informatics powered