GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca(2+) Dynamics in High-Ca(2+) Organelles.
Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe a ratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca(2+) oscillations in cultured astrocytes, (2) ex vivo functional mapping of cholinergic receptors triggering ER Ca(2+) release in acute hippocampal slices from transgenic mice, and (3) in vivo sarcoplasmic reticulum Ca(2+) dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca(2+) dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca(2+) signaling in animal models of health and disease.
Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), c/ Sanz y Forés 3, 47003 Valladolid, Spain.
Paloma
P
Navas-Navarro
Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), c/ Sanz y Forés 3, 47003 Valladolid, Spain.
Jonathan
J
Rojo-Ruiz
Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), c/ Sanz y Forés 3, 47003 Valladolid, Spain.
Macarena
M
Rodriguez-Prados
Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), c/ Sanz y Forés 3, 47003 Valladolid, Spain.
María Dolores
MD
Ganfornina
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA.
Loren L
LL
Looger
Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), c/ Sanz y Forés 3, 47003 Valladolid, Spain. Electronic address: talonso@ibgm.uva.es.
María Teresa
MT
Alonso
Instituto de Biología y Genética Molecular (IBGM), University of Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), c/ Sanz y Forés 3, 47003 Valladolid, Spain. Electronic address: jgsancho@ibgm.uva.es.
Javier
J
García-Sancho
D000331:Aequorin / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D004331:Drosophila melanogaster; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D057054:Molecular Imaging; D015388:Organelles / Q000737:chemistry / Q000378:metabolism*
D049452:Green Fluorescent Proteins; D000331:Aequorin; D002118:Calcium
GECI; GFP; Golgi apparatus; aequorin; biosensor; calcium; endoplasmic reticulum; organelles