Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
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.
Subach
Fedor V
FV
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.
Patterson
George H
GH
Renz
Malte
M
Lippincott-Schwartz
Jennifer
J
Verkhusha
Vladislav V
VV
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
D008164:Luminescent Proteins; D008565:Membrane Proteins; D000097573:Red Fluorescent Protein
Curr Biol. 2000 Nov 2;10(21):1395-8
11084343
Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21097-102
19934036
Biochemistry. 2008 Sep 23;47(38):10111-22
18759496
Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):15905-10
15505211
Nat Biotechnol. 2004 Nov;22(11):1435-9
15502815
Nat Biotechnol. 2006 May;24(5):577-81
16648840
Nat Methods. 2007 Sep;4(9):741-6
17721542
Nat Methods. 2009 Feb;6(2):153-9
19169259
J Am Chem Soc. 2007 Nov 14;129(45):13970-7
17956094
Biophys J. 2006 Dec 1;91(11):4258-72
16980368
Biophys J. 2008 Sep 15;95(6):2989-97
18658221
Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82
12060735
Nat Methods. 2006 Oct;3(10):793-5
16896339
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12651-6
12271129
J Biol Chem. 1993 Feb 15;268(5):3313-20
8381425
Photochem Photobiol. 2006 Mar-Apr;82(2):351-8
16613485
J Biol Chem. 2008 Oct 24;283(43):28980-7
18682399
Nat Chem Biol. 2009 Feb;5(2):118-26
19136976
J Am Chem Soc. 2007 Mar 21;129(11):3118-26
17326633
Science. 2004 Nov 19;306(5700):1370-3
15550670
Trends Cell Biol. 2009 Nov;19(11):555-65
19836954
Nat Methods. 2009 Feb;6(2):131-3
19169260
Biochim Biophys Acta. 1989 Apr 25;991(1):30-5
2713419
Biochemistry. 2005 Jul 26;44(29):9833-40
16026155
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):462-7
11209050
Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6672-7
17420458
Nat Methods. 2007 Jul;4(7):555-7
17572680
Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20308-13
18077327
Gene. 1989 Apr 15;77(1):51-9
2744487
Science. 2002 Sep 13;297(5588):1873-7
12228718
Nat Biotechnol. 2003 Feb;21(2):191-4
12524551
Curr Opin Chem Biol. 2010 Feb;14(1):23-9
19914857
Nat Biotechnol. 2006 Apr;24(4):461-5
16550175
J Virol. 1985 May;54(2):374-82
2985803
Chem Biol. 2008 Oct 20;15(10):1116-24
18940671
Nat Methods. 2008 Feb;5(2):155-7
18193054
Annu Rev Biochem. 2003;72:395-447
12651740
Nat Rev Mol Cell Biol. 2005 Nov;6(11):885-91
16167053
Biophys J. 2007 Nov 1;93(9):3285-90
17660318
Nat Biotechnol. 2004 Dec;22(12):1567-72
15558047
Structure. 2003 Mar;11(3):275-84
12623015
Nat Methods. 2004 Dec;1(3):209-17
15782196
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18343-8
19017808
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11984-9
11050229
Annu Rev Biochem. 2000;69:699-727
10966473
Science. 2006 Sep 15;313(5793):1642-5
16902090
Chem Biol. 2005 Mar;12(3):279-85
15797211
Biochemistry. 2006 Aug 15;45(32):9639-47
16893165