Green Fluorescent Protein Calcium Biosensors: Calcium Imaging with GFP Cameleons

Váradi, Anikó; Rutter, Guy A.

doi:10.1385/1-59259-280-5:255

Cited by 5 publications

(5 citation statements)

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“…The yellow cameleon fluorescent calcium indicators are the most suitable reporters for in vivo detection of calcium because they do not require any cofactors and can be targeted to specific intracellular locations (Miyawaki et al 1997(Miyawaki et al , 1999. Several different variants of yellow cameleons have been constructed but all consist of a tandem fusion of ECFP, a calmodulin domain having four calcium binding sites, a calmodulin binding peptide M13 and EYFP (Miyawaki et al 1997;Varadi and Rutter 2002). The level of intramolecular FRET is dependent on the extent of Ca 2?…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

et al. 2009

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Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

et al. 2009

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“…Between steps, cells were intensively washed with PBS. Imaging was performed using a Leica TCS-MT laser-scanning confocal microscope, fitted with a ×65, 1.4 numerical aperture oil-immersion objective lens, as described previously [39,40].…”

Section: Immunocytochemistrymentioning

confidence: 99%

Importin beta1 mediates the glucose-stimulated nuclear import of pancreatic and duodenal homeobox-1 in pancreatic islet beta-cells (MIN6)

Guillemain

Xavier

Rafiq

et al. 2004

Biochemical Journal

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The transcription factor PDX-1 (pancreatic and duodenal homeobox-1) is essential for pancreatic development and the maintainence of expression of islet beta-cell-specific genes. In an previous study [Rafiq, Kennedy and Rutter (1998) J. Biol. Chem. 273, 23241-23247] we demonstrated that PDX-1 may be activated at elevated glucose concentrations by translocation from undefined binding sites in the cytosol and nuclear membrane into the nucleoplasm. In the present study, we show that PDX-1 interacts directly and specifically in vitro with the nuclear import receptor family member, importin beta1, and that this interaction is mediated by the PDX-1 homeodomain (amino acids 146-206). Demonstrating the functional importance of the PDX-1-importin beta1 interaction, microinjection of MIN6 beta-cells with anti-(importin beta1) antibodies blocked both the nuclear translocation of PDX-1, and the activation by glucose (30 mM versus 3 mM) of the pre-proinsulin promoter. However, treatment with extracts from pancreatic islets incubated at either low or high glucose concentrations had no impact on the ability of PDX-1 to interact with importin beta1 in vitro. Furthermore, importin beta1 also interacted with SREBP1c (sterol-regulatory-element-binding protein 1c) in vitro, and microinjection of importin beta1 antibodies blocked the activation by glucose of SREBP1c target genes. Since the subcellular distribution of SREBP1c is unaffected by glucose, these findings suggest that a redistribution of importin beta1 is unlikely to explain the glucose-stimulated nuclear uptake of PDX-1. Instead, we conclude that the uptake of PDX-1 into the nucleoplasm, as glucose concentrations increase, may be mediated by release of the factor both from sites of retention in the cytosol and from non-productive complexes with importin beta1 at the nuclear membrane.

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“…Low molecular weight fluorescent calcium sensors do make their way to intracellular compartments (Silver et al, 1992) and can be used to measure calcium there, but they are difficult to target precisely (Varadi and Rutter Guy, 2002b). One of the two major advantages of genetically-encoded calcium sensors is that chimeric constructs and signalling tags can target them specifically to subcellular locations.…”

Section: Applications Of Genetically-encoded Sensorsmentioning

confidence: 99%

“…More recently, a construct encoding recombinant aequorin has been used to express the aequorin apoprotein in cells directly [see chapter by Miller in this volume]. Aequorin is a luminescent molecule and at the concentrations used inside cells emits relatively few photons when compared to fluorescent molecules at appropriate excitation intensities (Varadi and Rutter Guy, 2002b). However, proteins that are fluorescent at the visible wavelengths best suited to fluorescence imaging are relatively rare.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Probes for Measurement of Intracellular Calcium

Whitaker

2010

Methods in Cell Biology

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Small, fluorescent, calcium-sensing molecules have been enormously useful in mapping intracellular calcium signals in time and space, as chapters in this volume attest. Despite their widespread adoption and utility, they suffer some disadvantages. Genetically-encoded calcium sensors that can by expressed inside cells by transfection or transgenesis are desirable. The last ten years have been marked by a rapid evolution in the laboratory of genetically encoded calcium sensors two families both figuratively and literally, resulting in11distinct configurations of fluorescent proteins and their attendant calcium sensor modules. Here, I described the design logic and performance of this abundant collection of sensors and describe their use and performance in intro and in vivo. Genetically-encoded calcium sensors have proved valuable in the measurement of calcium concentration in cellular organelles, for the most part in single cells in vitro. Their success as quantitative calcium sensors in tissues in vitro and in vivo is qualified, but they have proved valuable in imaging the pattern of calcium signals within tissues in whole animals. Some branches of the calcium sensor evolutionary tree continue to evolve rapidly and the steady progress in optimising sensor parameters leads to the certain hope that these drawbacks will eventually be overcome by further genetic engineering.

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Green Fluorescent Protein Calcium Biosensors: Calcium Imaging with GFP Cameleons

Cited by 5 publications

References 10 publications

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Importin beta1 mediates the glucose-stimulated nuclear import of pancreatic and duodenal homeobox-1 in pancreatic islet beta-cells (MIN6)

Genetically Encoded Probes for Measurement of Intracellular Calcium

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