GAP, an Aequorin-based Fluorescent Indicator for Imaging Ca2+ in Organelles

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2014 Feb 7

PMID 24501126

Citations 25

Authors

Arancha Rodriguez-Garcia

Jonathan Rojo-Ruiz

Paloma Navas-Navarro

Francisco Javier Aulestia

Sonia Gallego-Sandin

Javier Garcia-Sancho

Maria Teresa Alonso

Affiliations

Soon will be listed here.

Abstract

Genetically encoded calcium indicators allow monitoring subcellular Ca(2+) signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca(2+) sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg(2+), tunable Ca(2+) affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca(2+) indicators for organelles and becomes a valuable tool for in vivo Ca(2+) imaging applications.

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References

Chamero P, Manjarres I, Garcia-Verdugo J, Villalobos C, Alonso M, Garcia-Sancho J . Nuclear calcium signaling by inositol trisphosphate in GH3 pituitary cells. Cell Calcium. 2007; 43(2):205-14. DOI: 10.1016/j.ceca.2007.05.005. View

Alonso M, Garcia-Sancho J . Nuclear Ca(2+) signalling. Cell Calcium. 2010; 49(5):280-9. DOI: 10.1016/j.ceca.2010.11.004. View

Kendall J, Dormer R, Campbell A . Targeting aequorin to the endoplasmic reticulum of living cells. Biochem Biophys Res Commun. 1992; 189(2):1008-16. DOI: 10.1016/0006-291x(92)92304-g. View

Crameri A, Whitehorn E, Tate E, Stemmer W . Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol. 1996; 14(3):315-9. DOI: 10.1038/nbt0396-315. View

Tian L, Hires S, Mao T, Huber D, Chiappe M, Chalasani S . Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods. 2009; 6(12):875-81. PMC: 2858873. DOI: 10.1038/nmeth.1398. View

Palmer A, Tsien R . Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc. 2007; 1(3):1057-65. DOI: 10.1038/nprot.2006.172. View

Mitchell K, Pinton P, Varadi A, Tacchetti C, Ainscow E, Pozzan T . Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera. J Cell Biol. 2001; 155(1):41-51. PMC: 2150797. DOI: 10.1083/jcb.200103145. View

Whitaker M . Genetically encoded probes for measurement of intracellular calcium. Methods Cell Biol. 2010; 99:153-82. PMC: 3292878. DOI: 10.1016/B978-0-12-374841-6.00006-2. View

Rizzuto R, Pozzan T . Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol Rev. 2005; 86(1):369-408. DOI: 10.1152/physrev.00004.2005. View

10.

Yamano K, Mori K, Nakano R, Kusunoki M, Inoue M, Satoh M . Identification of the functional expression of adenosine A3 receptor in pancreas using transgenic mice expressing jellyfish apoaequorin. Transgenic Res. 2007; 16(4):429-35. DOI: 10.1007/s11248-007-9084-0. View

11.

Miyawaki A, Llopis J, HEIM R, McCaffery J, Adams J, Ikura M . Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997; 388(6645):882-7. DOI: 10.1038/42264. View

12.

Hara M, Bindokas V, Lopez J, Kaihara K, Landa Jr L, Harbeck M . Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice. Am J Physiol Cell Physiol. 2004; 287(4):C932-8. DOI: 10.1152/ajpcell.00151.2004. View

13.

LaFerla F . Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease. Nat Rev Neurosci. 2002; 3(11):862-72. DOI: 10.1038/nrn960. View

14.

Brini M, Murgia M, Pasti L, Picard D, Pozzan T, Rizzuto R . Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin. EMBO J. 1993; 12(12):4813-9. PMC: 413931. DOI: 10.1002/j.1460-2075.1993.tb06170.x. View

15.

Rudolf R, Mongillo M, Rizzuto R, Pozzan T . Looking forward to seeing calcium. Nat Rev Mol Cell Biol. 2003; 4(7):579-86. DOI: 10.1038/nrm1153. View

16.

Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T . Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J. 1995; 14(22):5467-75. PMC: 394660. DOI: 10.1002/j.1460-2075.1995.tb00233.x. View

17.

Rogers K, Picaud S, Roncali E, Boisgard R, Colasante C, Stinnakre J . Non-invasive in vivo imaging of calcium signaling in mice. PLoS One. 2007; 2(10):e974. PMC: 1991622. DOI: 10.1371/journal.pone.0000974. View

18.

Palmer A, Qin Y, Park J, McCombs J . Design and application of genetically encoded biosensors. Trends Biotechnol. 2011; 29(3):144-52. PMC: 3433949. DOI: 10.1016/j.tibtech.2010.12.004. View

19.

Clapham D . Calcium signaling. Cell. 2007; 131(6):1047-58. DOI: 10.1016/j.cell.2007.11.028. View

20.

Jiang L, Collins J, Davis R, Lin K, Decamp D, Roach T . Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway. J Biol Chem. 2007; 282(14):10576-84. PMC: 2526465. DOI: 10.1074/jbc.M609695200. View