Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus.
Genetically encoded sensors are becoming a powerful tool for investigating cellular signaling pathways and, potentially, signaling in vivo. Many sensors use changes in fluorescence resonance energy transfer (FRET) between donor and acceptor variants of GFP separated by a ligand binding domain sensitive to a particular signaling pathway. Accurate measurements require that sensors be insensitive to extraneous intracellular environmental factors. We have found that the responsiveness of the Ca(2+) sensor, cameleon YC6.1, varies linearly with the resting YFP/CFP emission ratio in the cell. However, cells expressing responsive or non-responsive sensor can easily be segregated by determining a resting YFP/CFP ratio cutoff for the sensor. This environmental sensitivity has been eliminated by replacing EYFP with Venus to produce a new cameleon we have designated VC6.1. Measurements show that VC6.1 has a greater dynamic range than YC6.1 and better environmental resistance. We also show that YC6.1 is inactivated by persistent activation of the IP(3) pathway following expression of constitutively active G(q), while VC6.1 is not. The stability of VC6.1 may make it well suited to studies utilizing mixed cell populations such as those encountered in vivo.
Evanko
Daniel S
DS
Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, Philadelphia, PA 19104-6074, USA.
Haydon
Philip G
PG
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D008164:Luminescent Proteins; D008853:Microscopy; D011993:Recombinant Fusion Proteins; D014162:Transfection
D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C503160:VC6.1 cameleon protein, recombinant; C503159:cameleon YC6.1; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium