title: Förster resonance energy transfer-based biosensors for multiparameter ratiometric imaging of Ca2+ dynamics and caspase-3 activity in single cells.
abstract: As one of the principal cytoplasmic second messengers, the calcium ion (Ca(2+)) is central to a variety of intracellular signal transduction pathways. Accordingly, there is a sustained interest in methods for spatially- and temporally resolved imaging of the concentration of Ca(2+) in live cells using noninvasive methods such as genetically encoded biosensors based on Förster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca(2+) biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca(2+) affinity for various applications. We now report the development and systematic optimization of a pair of spectrally distinct FRET-based biosensors that enable the simultaneous imaging of Ca(2+) in two compartments of a single cell without substantial spectral crosstalk between emission channels. Furthermore, we demonstrate that these new biosensors can be used in conjunction with previously reported caspase-3 substrates based on the same set of FRET pairs.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Yidan
authors.initials: Y
authors.lastname: Ding
authors.forename: Hui-wang
authors.initials: HW
authors.lastname: Ai
authors.forename: Hiofan
authors.initials: H
authors.lastname: Hoi
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D007477:Ions / Q000737:chemistry; D015398:Signal Transduction
chemical_list: D007477:Ions; D053148:Caspase 3; D002118:Calcium
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