Measurements of the free luminal ER Ca(2+) concentration with targeted "cameleon" fluorescent proteins.
The free ER Ca(2+) concentration, [Ca(2+)](ER), is a key parameter that determines both the spatio-temporal pattern of Ca(2+) signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca(2+) activity within the ER is thus critical for our understanding of cell signaling. Such measurements, however, are particularly challenging given the highly dynamic nature of Ca(2+) signals, the complex architecture of the ER, and the difficulty of addressing probes specifically into the ER lumen. Prompted by these challenges, a number of ingenious approaches have been developed over the last years to measure ER Ca(2+) by optical means. The two main strategies used to date are Ca(2+)-sensitive synthetic dyes trapped into organelles and genetically encoded probes, based either on the photoprotein aequorin or on the green fluorescent protein (GFP). The GFP-based Ca(2+) indicators comprise the camgaroo and pericam probes based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. Each approach offers unique advantages and suffers from specific drawbacks. In this review, we will discuss the advantages and pitfalls of using the genetically encoded "cameleon" Ca(2+) indicators for ER Ca(2+) measurements.
Demaurex
Nicolas
N
Department of Physiology, University of Geneva Medical Center, 1, Michel-Servet, CH-1211 4, Geneva, Switzerland. nicolas.demaurex@medecine.unige.ch
Frieden
Maud
M
D000331:Aequorin; D000818:Animals; D002118:Calcium / Q000032:analysis*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005307:Fertilization in Vitro; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008958:Models, Molecular; D014162:Transfection
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000331:Aequorin; D002118:Calcium