Fluorescent Genetically Encoded Calcium Indicators and Their In Vivo Application

Gensch, Thomas; Kaschuba, Dagmar

doi:10.1007/4243_2011_29

Cited by 7 publications

(6 citation statements)

References 274 publications

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“…It is almost impossible to compare different GECI based on the literature because their properties have not been determined by standardized methods. 16 Many parameters, such as Ca 2+ sensitivity, potential toxicity, and targeting, may depend on the experimental conditions and, in -sensing domain of troponin-C. 64,65 Interestingly, for unknown reasons, we were unable to measure TN-XXL-dependent fluorescence changes in cardiac myocytes, despite the sufficient expression of the probe and visible stimulation-dependent contractions of the cells (compare Figure 3A). This result was puzzling given that the viral entry vector for TN-XXL, when expressed in human embryonic kidney cells, was able to display appropriate fluorescence changes in response to ATP stimulation (data not shown).…”

Section: Properties Of Geci In Cardiac Myocytes Whenmentioning

confidence: 98%

“…Furthermore, a recent report highlighted that some indicators may display cell-type-specific behaviors 16 ; therefore, results obtained in one cell system may not necessarily be transferable to other cell types. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in GECI, with particular emphasis on applications in cardiac cells and heart tissue.…”

Section: Conceptual Considerationsmentioning

confidence: 99%

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Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Kaestner

Scholz

Tian

et al. 2014

Circulation Research

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Section: Properties Of Geci In Cardiac Myocytes Whenmentioning

confidence: 98%

Section: Conceptual Considerationsmentioning

confidence: 99%

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Kaestner

Scholz

Tian

et al. 2014

Circulation Research

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“…Unimolecular FRET is the read-out mode in a large number of biosensors that employ a donor and acceptor fluorescent protein, predominantly cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) or improved derivatives thereof (4)(5)(6). Genetically encoded calcium indicators (GECIs) enable observation of intracellular signaling in multicellular tissues and neuronal activity in living organisms (7,8). The currently available GECIs can be subdivided into single-wavelength indicators like the GCaMPs (9) and GECOs (10) on the one hand and dual-wavelength indicators based on FRET on the other hand.…”

Section: Introductionmentioning

confidence: 99%

Correlating Calcium Binding, Förster Resonance Energy Transfer, and Conformational Change in the Biosensor TN-XXL

Geiger

Russo

Gensch

et al. 2012

Biophysical Journal

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Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.

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“…Until today, new versions of both groups were developed to enable better temporal resolution and imaging in living organisms [ 31 , 32 ]. Members of both groups of sensors were frequently applied at the cellular level as well as in transgenic organisms and facilitated Ca 2+ measurements under physiological conditions [ 33 , 34 , 35 , 36 , 37 ]. Since GECI are ideally suited for additional genetic engineering, some sensors have been modified to target the protein to subcellular domains allowing locally confined Ca 2+ measurements [ 38 , 39 , 40 , 41 , 42 , 43 ].…”

Section: Introductionmentioning

confidence: 99%

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

Kempmann

Gensch

Offenhäusser

et al. 2022

IJMS

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Calcium (Ca2+) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca2+ concentration ([Ca2+]i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca2+]i are ideally suited to modulate Ca2+-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca2+, the introduction and the ongoing development of genetically encoded Ca2+ indicators (GECI) opened a new era to study Ca2+-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca2+ signals at the (sub)-cellular level with pronounced temporal resolution.

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Fluorescent Genetically Encoded Calcium Indicators and Their In Vivo Application

Cited by 7 publications

References 274 publications

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Correlating Calcium Binding, Förster Resonance Energy Transfer, and Conformational Change in the Biosensor TN-XXL

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

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Fluorescent Genetically Encoded Calcium Indicators and Their In Vivo Application

Cited by 7 publications

References 274 publications

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

Correlating Calcium Binding, Förster Resonance Energy Transfer, and Conformational Change in the Biosensor TN-XXL

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

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Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes