Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum

Overview

Journal Sensors (Basel)

Publisher MDPI

Specialty Biotechnology

Date 2015 Jun 9

PMID 26053751

Citations 12

Authors

Markus Waldeck-Weiermair

Helmut Bischof

Sandra Blass

Andras T Deak

Christiane Klec

Thomas Graier

Clara Roller

Rene Rost

Emrah Eroglu

Benjamin Gottschalk

Nicole A Hofmann

Wolfgang F Graier

Roland Malli

Affiliations

Soon will be listed here.

Abstract

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Citing Articles

Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease.

Liu L, Liu Y, Wu D, Cheng J, Li N, Zheng Y Neural Regen Res. 2023; 18(9):2019-2028.

PMID: 36926728 PMC: 10233771. DOI: 10.4103/1673-5374.366492.

Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting.

Erdogan Y, Altun H, Secilmis M, Ata B, Sevimli G, Cokluk Z Free Radic Biol Med. 2021; 177:132-142.

PMID: 34687864 PMC: 8639799. DOI: 10.1016/j.freeradbiomed.2021.10.023.

MICU1 controls cristae junction and spatially anchors mitochondrial Ca uniporter complex.

Gottschalk B, Klec C, Leitinger G, Bernhart E, Rost R, Bischof H Nat Commun. 2019; 10(1):3732.

PMID: 31427612 PMC: 6700202. DOI: 10.1038/s41467-019-11692-x.

Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes.

Bischof H, Burgstaller S, Waldeck-Weiermair M, Rauter T, Schinagl M, Ramadani-Muja J Cells. 2019; 8(5).

PMID: 31121936 PMC: 6562680. DOI: 10.3390/cells8050492.

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening.

Schaaf T, Li A, Grant B, Peterson K, Yuen S, Bawaskar P Biosensors (Basel). 2018; 8(4).

PMID: 30352972 PMC: 6315989. DOI: 10.3390/bios8040099.

References

Griesbeck O, Baird G, Campbell R, Zacharias D, Tsien R . Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001; 276(31):29188-94. DOI: 10.1074/jbc.M102815200. View

Knot H, Laher I, Sobie E, Guatimosim S, Gomez-Viquez L, Hartmann H . Twenty years of calcium imaging: cell physiology to dye for. Mol Interv. 2005; 5(2):112-27. PMC: 4861218. DOI: 10.1124/mi.5.2.8. View

Naghdi S, Waldeck-Weiermair M, Fertschai I, Poteser M, Graier W, Malli R . Mitochondrial Ca2+ uptake and not mitochondrial motility is required for STIM1-Orai1-dependent store-operated Ca2+ entry. J Cell Sci. 2010; 123(Pt 15):2553-64. DOI: 10.1242/jcs.070151. View

Lam A, St-Pierre F, Gong Y, Marshall J, Cranfill P, Baird M . Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods. 2012; 9(10):1005-12. PMC: 3461113. DOI: 10.1038/nmeth.2171. View

Gorlach A, Klappa P, Kietzmann T . The endoplasmic reticulum: folding, calcium homeostasis, signaling, and redox control. Antioxid Redox Signal. 2006; 8(9-10):1391-418. DOI: 10.1089/ars.2006.8.1391. View

Alam M, Groschner L, Parichatikanond W, Kuo L, Bondarenko A, Rost R . Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic β-cells. J Biol Chem. 2012; 287(41):34445-54. PMC: 3464549. DOI: 10.1074/jbc.M112.392084. View

Malli R, Naghdi S, Romanin C, Graier W . Cytosolic Ca2+ prevents the subplasmalemmal clustering of STIM1: an intrinsic mechanism to avoid Ca2+ overload. J Cell Sci. 2008; 121(Pt 19):3133-9. PMC: 4064434. DOI: 10.1242/jcs.034496. View

Samtleben S, Jaepel J, Fecher C, Andreska T, Rehberg M, Blum R . Direct imaging of ER calcium with targeted-esterase induced dye loading (TED). J Vis Exp. 2013; (75):e50317. PMC: 3679584. DOI: 10.3791/50317. View

Waldeck-Weiermair M, Alam M, Jadoon Khan M, Deak A, Vishnu N, Karsten F . Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon. PLoS One. 2012; 7(9):e45917. PMC: 3448721. DOI: 10.1371/journal.pone.0045917. View

10.

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

11.

Rudolf R, Magalhaes P, Pozzan T . Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle. J Cell Biol. 2006; 173(2):187-93. PMC: 2063810. DOI: 10.1083/jcb.200601160. View

12.

Palmer A, Giacomello M, Kortemme T, Hires S, Lev-Ram V, Baker D . Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol. 2006; 13(5):521-30. DOI: 10.1016/j.chembiol.2006.03.007. View

13.

Deak A, Blass S, Khan M, Groschner L, Waldeck-Weiermair M, Hallstrom S . IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake. J Cell Sci. 2014; 127(Pt 13):2944-55. PMC: 4077590. DOI: 10.1242/jcs.149807. View

14.

Fabiato A . Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245(1):C1-14. DOI: 10.1152/ajpcell.1983.245.1.C1. View

15.

Palmer A, Jin C, Reed J, Tsien R . Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A. 2004; 101(50):17404-9. PMC: 535104. DOI: 10.1073/pnas.0408030101. View

16.

Akerboom J, Carreras Calderon N, Tian L, Wabnig S, Prigge M, Tolo J . Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci. 2013; 6:2. PMC: 3586699. DOI: 10.3389/fnmol.2013.00002. View

17.

Jean-Quartier C, Bondarenko A, Alam M, Trenker M, Waldeck-Weiermair M, Malli R . Studying mitochondrial Ca(2+) uptake - a revisit. Mol Cell Endocrinol. 2011; 353(1-2):114-27. PMC: 3334272. DOI: 10.1016/j.mce.2011.10.033. View

18.

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

19.

Hofer A, Machen T . Technique for in situ measurement of calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2. Proc Natl Acad Sci U S A. 1993; 90(7):2598-602. PMC: 46142. DOI: 10.1073/pnas.90.7.2598. View

20.

Zhao Y, Araki S, Wu J, Teramoto T, Chang Y, Nakano M . An expanded palette of genetically encoded Ca²⁺ indicators. Science. 2011; 333(6051):1888-91. PMC: 3560286. DOI: 10.1126/science.1208592. View