title: Visualization of peroxynitrite-induced changes of labile Zn2+ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes.
abstract: Zn(2+) plays essential roles in biology, and the homeostasis of Zn(2+) is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn(2+), and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn(2+), ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N3O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable Kd values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn(2+) binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn(2+) thus providing evidence for a direct connection between ER stress and ER Zn(2+) homeostasis.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.forename: Daniela
authors.initials: D
authors.lastname: Buccella
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D048588:Benzoxazines / Q000737:chemistry*; D002470:Cell Survival; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D058953:Neural Stem Cells / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000138:chemical synthesis / Q000737:chemistry*; D015032:Zinc / Q000032:analysis* / Q000737:chemistry
chemical_list: D048588:Benzoxazines; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D030421:Peroxynitrous Acid; D015032:Zinc
references.citation: Brain Res Brain Res Rev. 2000 Dec;34(3):137-48
references.pmid: 11113504
references.citation: J Neurosci. 2012 Oct 31;32(44):15521-32
references.pmid: 23115189
references.citation: J Proteome Res. 2006 Jan;5(1):196-201
references.pmid: 16396512
references.citation: Neuron. 2011 Sep 22;71(6):1116-26
references.pmid: 21943607
references.citation: Nature. 2006 May 25;441(7092):513-7
references.pmid: 16724068
references.citation: Annu Rev Nutr. 2009;29:133-52
references.pmid: 19400701
references.citation: Proc Natl Acad Sci U S A. 2012 Dec 18;109(51):E3530-8
references.pmid: 23213233
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17822-7
references.pmid: 17098870
references.citation: J Fluoresc. 2004 Jan;14(1):17-23
references.pmid: 15622856
references.citation: Free Radic Biol Med. 2011 Jun 15;50(12):1717-25
references.pmid: 21463677
references.citation: J Am Chem Soc. 2011 Mar 23;133(11):4101-14
references.pmid: 21351756
references.citation: J Biol Chem. 2006 Apr 7;281(14):9460-70
references.pmid: 16431921
references.citation: Protein Cell. 2012 Apr;3(4):251-61
references.pmid: 22549585
references.citation: Stem Cells. 2011 Dec;29(12):1915-22
references.pmid: 22009633
references.citation: J Neurosci. 2004 Nov 24;24(47):10616-27
references.pmid: 15564577
references.citation: J Neurosci. 2011 Nov 9;31(45):16076-85
references.pmid: 22072659
references.citation: Nat Methods. 2009 Oct;6(10):737-40
references.pmid: 19718032
references.citation: Biochim Biophys Acta. 2006 Jul;1763(7):711-22
references.pmid: 16675045
references.citation: J Phys Chem A. 2010 Sep 30;114(38):10427-34
references.pmid: 20809640
references.citation: J Am Chem Soc. 2008 Nov 26;130(47):15788-9
references.pmid: 18975868
references.citation: J Org Chem. 2008 Mar 21;73(6):2424-7
references.pmid: 18302409
references.citation: Nat Neurosci. 2002 May;5(5):438-45
references.pmid: 11953752
references.citation: Biochemistry. 1990 Jun 19;29(24):5647-59
references.pmid: 2200508
references.citation: Int J Biochem Cell Biol. 2012 Aug;44(8):1295-8
references.pmid: 22564438
references.citation: Chem Biol. 2004 Feb;11(2):203-10
references.pmid: 15123282
references.citation: Mol Cell Neurosci. 1997;8(6):389-404
references.pmid: 9143557
references.citation: J Am Chem Soc. 2009 May 27;131(20):7142-52
references.pmid: 19405465
references.citation: J Am Chem Soc. 2006 Dec 6;128(48):15517-28
references.pmid: 17132019
references.citation: Cancer Res. 2010 Aug 1;70(15):6119-27
references.pmid: 20610630
references.citation: Inorg Chem. 2013 Mar 18;52(6):3285-94
references.pmid: 23461436
references.citation: Biometals. 2007 Dec;20(6):891-901
references.pmid: 17279325
references.citation: Neuron. 2004 Feb 5;41(3):351-65
references.pmid: 14766175
references.citation: J Cardiovasc Pharmacol. 1989;14 Suppl 11:S13-22
references.pmid: 2484692
references.citation: J Am Chem Soc. 2001 Aug 15;123(32):7831-41
references.pmid: 11493056
references.citation: J Neurobiol. 1999 Jan;38(1):65-81
references.pmid: 10027563
references.citation: Metallomics. 2012 Jul;4(7):614-8
references.pmid: 22610492
references.citation: Biometals. 2001 Sep-Dec;14(3-4):331-41
references.pmid: 11831463
references.citation: Photochem Photobiol. 2002 Oct;76(4):385-90
references.pmid: 12405144
references.citation: J Biol Chem. 2005 Aug 5;280(31):28811-8
references.pmid: 15961382
references.citation: Plant Signal Behav. 2011 Jan;6(1):77-9
references.pmid: 21242724
references.citation: Mol Med. 2007 Jul-Aug;13(7-8):371-5
references.pmid: 17622324
references.citation: J Biol Inorg Chem. 2011 Oct;16(7):1123-34
references.pmid: 21660546
references.citation: Acc Chem Res. 2009 Jan 20;42(1):193-203
references.pmid: 18989940