Live-cell transforms between Ca2+ transients and FRET responses for a troponin-C-based Ca2+ sensor.
Genetically encoded Ca(2+) sensors promise sustained in vivo detection of Ca(2+) signals. However, these sensors are sometimes challenged by inconsistent performance and slow/uncertain kinetic responsiveness. The former challenge may arise because most sensors employ calmodulin (CaM) as the Ca(2+)-sensing module, such that interference via endogenous CaM may result. One class of sensors that could minimize this concern utilizes troponin C as the Ca(2+) sensor. Here, we therefore probed the reliability and kinetics of one representative of this class (cyan fluorescence protein/yellow fluorescent protein-fluorescence resonance energy transfer (FRET) sensor TN-L15) within cardiac ventricular myocytes. These cells furnished a pertinent live-cell test environment, given substantial endogenous CaM levels and fast reproducible Ca(2+) transients for testing sensor kinetics. TN-L15 was virally expressed within myocytes, and Indo-1 acutely loaded to monitor "true" Ca(2+) transients. This configuration permitted independent and simultaneous detection of TN-L15 and Indo-1 signals within individual cells. The relation between TN-L15 FRET responses and Indo-1 Ca(2+) transients appeared reproducible, though FRET signals were delayed compared to Ca(2+) transients. Nonetheless, a three-state mechanism sufficed to map between measured Ca(2+) transients and actual TN-L15 outputs. Overall, reproducibility of TN-L15 dynamics, coupled with algorithmic transforms between FRET and Ca(2+) signals, renders these sensors promising for quantitative estimation of Ca(2+) dynamics in vivo.
Tay
Lai Hock
LH
Calcium Signals Laboratory, Department of Biomedical Engineering, and Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Griesbeck
Oliver
O
Yue
David T
DT
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D032383:Myocytes, Cardiac / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019209:Troponin C / Q000378:metabolism*
D019209:Troponin C; D002118:Calcium
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