title: Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
abstract: Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.
authors.affiliation: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Fedor V
authors.initials: FV
authors.lastname: Subach
authors.forename: George H
authors.initials: GH
authors.lastname: Patterson
authors.forename: Malte
authors.initials: M
authors.lastname: Renz
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D003116:Color; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D016296:Mutagenesis; D055668:Photochemical Processes; D013696:Temperature; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D008565:Membrane Proteins; D000097573:Red Fluorescent Protein
references.citation: Curr Biol. 2000 Nov 2;10(21):1395-8
references.pmid: 11084343
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21097-102
references.pmid: 19934036
references.citation: Biochemistry. 2008 Sep 23;47(38):10111-22
references.pmid: 18759496
references.citation: Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):15905-10
references.pmid: 15505211
references.citation: Nat Biotechnol. 2004 Nov;22(11):1435-9
references.pmid: 15502815
references.citation: Nat Biotechnol. 2006 May;24(5):577-81
references.pmid: 16648840
references.citation: Nat Methods. 2007 Sep;4(9):741-6
references.pmid: 17721542
references.citation: Nat Methods. 2009 Feb;6(2):153-9
references.pmid: 19169259
references.citation: J Am Chem Soc. 2007 Nov 14;129(45):13970-7
references.pmid: 17956094
references.citation: Biophys J. 2006 Dec 1;91(11):4258-72
references.pmid: 16980368
references.citation: Biophys J. 2008 Sep 15;95(6):2989-97
references.pmid: 18658221
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82
references.pmid: 12060735
references.citation: Nat Methods. 2006 Oct;3(10):793-5
references.pmid: 16896339
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12651-6
references.pmid: 12271129
references.citation: J Biol Chem. 1993 Feb 15;268(5):3313-20
references.pmid: 8381425
references.citation: Photochem Photobiol. 2006 Mar-Apr;82(2):351-8
references.pmid: 16613485
references.citation: J Biol Chem. 2008 Oct 24;283(43):28980-7
references.pmid: 18682399
references.citation: Nat Chem Biol. 2009 Feb;5(2):118-26
references.pmid: 19136976
references.citation: J Am Chem Soc. 2007 Mar 21;129(11):3118-26
references.pmid: 17326633
references.citation: Science. 2004 Nov 19;306(5700):1370-3
references.pmid: 15550670
references.citation: Trends Cell Biol. 2009 Nov;19(11):555-65
references.pmid: 19836954
references.citation: Nat Methods. 2009 Feb;6(2):131-3
references.pmid: 19169260
references.citation: Biochim Biophys Acta. 1989 Apr 25;991(1):30-5
references.pmid: 2713419
references.citation: Biochemistry. 2005 Jul 26;44(29):9833-40
references.pmid: 16026155
references.citation: Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):462-7
references.pmid: 11209050
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6672-7
references.pmid: 17420458
references.citation: Nat Methods. 2007 Jul;4(7):555-7
references.pmid: 17572680
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20308-13
references.pmid: 18077327
references.citation: Gene. 1989 Apr 15;77(1):51-9
references.pmid: 2744487
references.citation: Science. 2002 Sep 13;297(5588):1873-7
references.pmid: 12228718
references.citation: Nat Biotechnol. 2003 Feb;21(2):191-4
references.pmid: 12524551
references.citation: Curr Opin Chem Biol. 2010 Feb;14(1):23-9
references.pmid: 19914857
references.citation: Nat Biotechnol. 2006 Apr;24(4):461-5
references.pmid: 16550175
references.citation: J Virol. 1985 May;54(2):374-82
references.pmid: 2985803
references.citation: Chem Biol. 2008 Oct 20;15(10):1116-24
references.pmid: 18940671
references.citation: Nat Methods. 2008 Feb;5(2):155-7
references.pmid: 18193054
references.citation: Annu Rev Biochem. 2003;72:395-447
references.pmid: 12651740
references.citation: Nat Rev Mol Cell Biol. 2005 Nov;6(11):885-91
references.pmid: 16167053
references.citation: Biophys J. 2007 Nov 1;93(9):3285-90
references.pmid: 17660318
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: Structure. 2003 Mar;11(3):275-84
references.pmid: 12623015
references.citation: Nat Methods. 2004 Dec;1(3):209-17
references.pmid: 15782196
references.citation: Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18343-8
references.pmid: 19017808
references.citation: Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11984-9
references.pmid: 11050229
references.citation: Annu Rev Biochem. 2000;69:699-727
references.pmid: 10966473
references.citation: Science. 2006 Sep 15;313(5793):1642-5
references.pmid: 16902090
references.citation: Chem Biol. 2005 Mar;12(3):279-85
references.pmid: 15797211
references.citation: Biochemistry. 2006 Aug 15;45(32):9639-47
references.pmid: 16893165