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In vivo Ca2+ imaging in the endoplasmic reticulum with GAP sensors

AutorAlonso, María Teresa
Fecha de publicación2016
Citación14th International Meeting of the European Calcium Society (2016)
ResumenProper functioning of organelles such as the endoplasmic reticulum (ER) or the Golgi apparatus requires luminal accumulation of Ca2+ at high concentrations. Direct intra-organellar measurement of Ca2+ concentrations in intact preparations is essential to address complex physiological questions. Few genetically encoded Ca2+ indicators have been developed for high-Ca2+ organelles, and none of these have been used in vivo in the context of transgenic expression. We have recently developed a new family of fluorescent Ca2+ sensors based on the fusion of two Aequorea victoria proteins, GFP and apoaequorin, named GAP. Here we report the generation of a novel low affinity Ca2+ indicator, optimized for measurements in high Ca2+ concentration environments. Transgenic animals (mice and flies) expressing the ER-targeted sensor enabled monitoring fast physiological Ca2+ responses under minimally disturbing conditions, both ex vivo and in vivo.The applicability of the sensor was demonstrated under three experimental paradigms: i) ER Ca2+ oscillations in cultured astrocytes, ii) ex vivo functional mapping of cholinergic receptors triggering ER Ca2+ release in acute hippocampal slices from transgenic mice, and iii) in vivo sarcoplasmic reticulum Ca2+ dynamics in the muscle of transgenic flies. Our results provide proof of concept for the suitability of the new biosensors to monitor Ca2+ dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca2+ signalling in animal models of health and disease.
DescripciónResumen del trabajo presentado al 14th International Meeting of the European Calcium Society, celebrado en Valladolid (España) del 25 al 29 de septiembre de 2016.
URIhttp://hdl.handle.net/10261/158198
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