Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

Direnberger, Stephan; Mues, Marsilius; Micale, Vincenzo; Wotjak, Carsten T.; Dietzel, Steffen; Schubert, Michaël; Scharr, Andreas; Hassan, Sami; Wahl‐Schott, Christian; Biel, Martin; Krishnamoorthy, Gurumoorthy; Griesbeck, Oliver

doi:10.1038/ncomms2035

Cited by 44 publications

(52 citation statements)

References 47 publications

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“…Some other biosensors, such as the A-kinase activity reporter fused to PLN have recently been introduced into adult cardiomyocytes via adenoviral vectors to monitor local kinase activity 21 . Although we and others have successfully generated TG mice expressing some of the cytosolic FRET biosensors 19,[22][23][24] , it remained unclear whether targeted, microdomain-specific biosensors, which act in the functionally relevant subcellular locations, are also compatible with in vivo animal models. Here, we describe the generation and validation of the first TG mouse model, which expresses a targeted cAMP biosensor designed to dissect the cAMP signalling in the vicinity of an important calcium-handling protein SERCA2a.…”

Section: Discussionmentioning

confidence: 99%

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

et al. 2015

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3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced b-adrenergic receptor-cAMP signalling to SERCA.

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Section: Discussionmentioning

confidence: 99%

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

et al. 2015

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“…Thus, currently available GECIs are highly nonlinear sensors binding up to four calcium ions per sensor. Identification of a smaller calciumbinding domain with fewer binding sites could help to reduce buffering during long-term chronic GECI expression 26 , make the sensor smaller and further minimize the risk of cytotoxicity. It may also help to simplify response properties and facilitate the biophysical modeling of sensor behavior.…”

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confidence: 99%

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

et al. 2014

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Imaging with GECIs has become a widely used method in physiology and neuroscience [1][2][3] . According to readout mode, the design of the sensors has followed two different pathways, leading to single-wavelength sensors and FRET-based ratiometric sensors [4][5][6][7][8] . Among the most popular single-wavelength sensors are the G-CaMPs 9-13 , R-CaMPs 14 and GECOs 15 . FRET sensors include yellow cameleon 3.60 (refs. 16,17), D3cpv 18 , yellow cameleon Nano 19 and TN-XXL 20 .Quantification by ratiometric FRET imaging is more accurate than single-channel measurements and may be more suitable for long-term functional imaging studies, as it is less influenced by changes in optical path length, excitation light intensity and indicator expression level and by tissue movement and growth during development. In addition, FRET indicators are substantially brighter than single-wavelength sensors at rest, allowing better identification of expressing cells and their subcellular structures. Another practical feature of FRET-based indicators is their ability to measure basal Ca 2+ levels within cells, for example, to distinguish between resting and continuously spiking neuronssomething that cannot easily be achieved with single-wavelength indicators 21 . Increased basal Ca 2+ levels within the brain are also observed at the onset of neurodegenerative processes, and ratiometric FRET calcium imaging has been used in these conditions to monitor disease progression 22,23 . Moreover, ratiometric indicators are advantageous for monitoring calcium in motile cells.Both calmodulin and troponin C (TnC), the calcium binding proteins within the various GECIs, consist of two globular domains connected by a central linker 24,25 . Each domain possesses two calcium-binding EF hand motifs. Thus, currently available GECIs are highly nonlinear sensors binding up to four calcium ions per sensor. Identification of a smaller calciumbinding domain with fewer binding sites could help to reduce buffering during long-term chronic GECI expression 26 , make the sensor smaller and further minimize the risk of cytotoxicity. It may also help to simplify response properties and facilitate the biophysical modeling of sensor behavior.Here we report several improvements of FRET-based calcium sensors for in vivo imaging. First, we identified a minimal calcium binding motif based on the C-terminal domain of TnC with only two or one remaining calcium binding sites per sensor molecule, thus reducing the overall calcium buffering of the sensors. Second, by sampling TnCs from various species we identified a new TnC variant from the toadfish Opsanus tau, which offered the possibility of generating minimal domains with high-affinity calcium binding. Third, we used a large-scale, two-step functional screen to optimize the FRET changes in the sensor by linker diversification. This approach allowed us to identify Twitch sensors with a superior FRET change and may become useful for optimizing other types of FRET sensors. Finally, we improved brightness and photostability o...

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“…Although AM-ester-based Ca 2+ indicators have been useful for investigating Ca 2+ dynamics in isolated granulosa cells [9,10,12] and isolated oocytes [23,24], their poor permeation into multilayer tissues precludes their use in isolated follicles. Mice expressing an early generation 50 genetically encoded sensor, TN-XXL [25], had insufficient sensitivity to be useful (our unpublished results), but recently, mice expressing high-affinity optical sensors for Ca 2+ have been developed [26,27]. Using mouse lines expressing the sensors Twitch-2B and GCaMP6s, we show that FSH and LH both elevate Ca 2+ in the granulosa cells of intact follicles.…”

Section: Introductionmentioning

confidence: 86%

Follicle-stimulating hormone and luteinizing hormone increase Ca²⁺ in the granulosa cells of mouse ovarian follicles¹

Egbert

Fahey

Reimer

et al. 2019

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Running title: FSH and LH-induced Ca 2+ rises in ovarian follicles 2 Summary sentence: Both FSH and LH increase Ca 2+ in the granulosa cells of intact ovarian follicles from mice expressing genetically encoded sensors. ABSTRACTIn mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein G s to elevate cAMP, but both of these hormones can also elevate Ca 2+ under some conditions. Here we investigate FSH-and LH-induced Ca 2+ signaling in intact follicles of mice expressing genetically encoded Ca 2+ sensors, Twitch-2B and 5 GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca 2+ within the granulosa cells of preantral and antral follicles. The Ca 2+ rise begins several minutes after FSH application, peaks at ~10 minutes, remains above baseline for another ~10 minutes, and depends on extracellular Ca 2+ . However, suppression of the FSH-induced Ca 2+ increase by reducing extracellular Ca 2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol 10 production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca 2+ , when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca 2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 hours and depend on the activity of G q family G-proteins. Suppression of the oscillations by G q inhibition does not inhibit meiotic resumption, but does delay the time to 50% 15 ovulation by about 3 hours. In summary, both FSH and LH increase Ca 2+ in the granulosa cells of intact follicles, but the functions of these Ca 2+ rises are only starting to be identified. 2 1

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Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

Cited by 44 publications

References 47 publications

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

Follicle-stimulating hormone and luteinizing hormone increase Ca²⁺ in the granulosa cells of mouse ovarian follicles¹

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Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

Cited by 44 publications

References 47 publications

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles1

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Follicle-stimulating hormone and luteinizing hormone increase Ca²⁺ in the granulosa cells of mouse ovarian follicles¹