title: Measuring EGFR separations on cells with ~10 nm resolution via fluorophore localization imaging with photobleaching.
abstract: Detecting receptor dimerisation and other forms of clustering on the cell surface depends on methods capable of determining protein-protein separations with high resolution in the ~10-50 nm range. However, this distance range poses a significant challenge because it is too large for fluorescence resonance energy transfer and contains distances too small for all other techniques capable of high-resolution in cells. Here we have adapted the technique of fluorophore localisation imaging with photobleaching to measure inter-receptor separations in the cellular environment. Using the epidermal growth factor receptor, a key cancer target molecule, we demonstrate ~10 nm resolution while continuously covering the range of ~10-80 nm. By labelling the receptor on cells expressing low receptor numbers with a fluorescent antagonist we have found inter-receptor separations all the way up from 8 nm to 59 nm. Our data are consistent with epidermal growth factor receptors being able to form homo-polymers of at least 10 receptors in the absence of activating ligands.
authors: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Oxford, Didcot, Oxfordshire, United Kingdom.
authors: Sarah R
authors: SR
authors: Needham
authors: Michael
authors: M
authors: Hirsch
authors: Daniel J
authors: DJ
authors: Rolfe
authors: David T
authors: DT
authors: Clarke
authors: Laura C
authors: LC
authors: Zanetti-Domingues
authors: Richard
authors: R
authors: Wareham
authors: Marisa L
authors: ML
authors: Martin-Fernandez
mesh_terms: D000465:Algorithms; D045744:Cell Line, Tumor; D003198:Computer Simulation; D066246:ErbB Receptors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D055503:Protein Multimerization; D021381:Protein Transport; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; C512478:EGFR protein, human; D066246:ErbB Receptors
references: Nat Struct Mol Biol. 2011 Oct 23;18(11):1244-9
references: 22020299
references: J Cell Physiol. 1988 Mar;134(3):413-20
references: 3258312
references: J Biol Chem. 2010 Jan 29;285(5):3145-56
references: 19959837
references: Cell. 2006 Jun 16;125(6):1137-49
references: 16777603
references: Cell. 2002 Sep 20;110(6):763-73
references: 12297049
references: Mol Cell Biol. 1995 Dec;15(12):6496-505
references: 8524214
references: Biochemistry. 2007 Apr 17;46(15):4589-97
references: 17381163
references: Biochemistry. 1993 Aug 24;32(33):8602-7
references: 8395211
references: Mol Cell. 2003 Feb;11(2):507-17
references: 12620237
references: Mol Cell Biol. 2010 Aug;30(15):3795-804
references: 20516214
references: Annu Rev Biochem. 2011;80:247-71
references: 21548785
references: Biophys J. 2005 Aug;89(2):1362-73
references: 15908575
references: PLoS One. 2012;7(9):e45655
references: 23049831
references: Nat Struct Biol. 2000 Sep;7(9):735-9
references: 10966640
references: Biophys J. 2002 May;82(5):2775-83
references: 11964263
references: J Cell Biol. 1992 Oct;119(2):349-55
references: 1383230
references: J Am Chem Soc. 2011 Aug 24;133(33):12902-5
references: 21793568
references: Exp Cell Res. 1992 Mar;199(1):90-7
references: 1735465
references: Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3477-81
references: 8622961
references: Cell. 2010 Jun 25;141(7):1117-34
references: 20602996
references: Opt Lett. 1994 Jun 1;19(11):780-2
references: 19844443
references: Cell. 2002 Sep 20;110(6):775-87
references: 12297050
references: EMBO J. 1984 Aug;3(8):1843-7
references: 6090128
references: Biophys J. 2006 Jan 15;90(2):668-71
references: 16258038
references: Biochem Biophys Res Commun. 2004 Nov 19;324(3):1143-9
references: 15485674
references: J Cell Biol. 1995 Jun;129(6):1543-58
references: 7790353
references: Annu Rev Biophys. 2011;40:169-86
references: 21314430
references: Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6462-5
references: 15096603
references: Biophys J. 2007 Aug 1;93(3):1021-31
references: 17496034
references: Science. 2000 Nov 24;290(5496):1567-70
references: 11090353
references: Biophys J. 2002 May;82(5):2415-27
references: 11964230
references: J Chromatogr. 1991 Dec 20;587(2):129-36
references: 1783666
references: Trends Biochem Sci. 2011 Nov;36(11):604-15
references: 21917465
references: J Cell Biol. 1981 Apr;89(1):141-5
references: 7014571
references: Eur Biophys J. 2011 Oct;40(10):1167-86
references: 21928120
references: Science. 2003 Jun 27;300(5628):2061-5
references: 12791999
references: Biochem Biophys Res Commun. 2009 Jan 16;378(3):376-82
references: 19014905
references: J Cell Biol. 2007 Oct 22;179(2):341-56
references: 17938246
references: Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16524-9
references: 20813958
references: J Biol Chem. 1989 Sep 15;264(26):15501-7
references: 2549066
references: Science. 2006 Sep 15;313(5793):1642-5
references: 16902090
references: Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11298-303
references: 15277661
references: Nat Methods. 2006 Oct;3(10):793-5
references: 16896339
references: Biochem Soc Trans. 2001 Aug;29(Pt 4):480-4
references: 11498013
references: Biophys J. 2007 Jul 15;93(2):684-98
references: 17468161
references: J Biol Chem. 2005 Aug 26;280(34):30392-9
references: 15994331
references: Proc Natl Acad Sci U S A. 1967 Aug;58(2):719-26
references: 5233469