title: Specific in vivo labeling of tyrosinated α-tubulin and measurement of microtubule dynamics using a GFP tagged, cytoplasmically expressed recombinant antibody.
abstract: GFP-tagged proteins are used extensively as biosensors for protein localization and function, but the GFP moiety can interfere with protein properties. An alternative is to indirectly label proteins using intracellular recombinant antibodies (scFvs), but most antibody fragments are insoluble in the reducing environment of the cytosol. From a synthetic hyperstable human scFv library we isolated an anti-tubulin scFv, 2G4, which is soluble in mammalian cells when expressed as a GFP-fusion protein. Here we report the use of this GFP-tagged scFv to label microtubules in fixed and living cells. We found that 2G4-GFP localized uniformly along microtubules and did not disrupt binding of EB1, a protein that binds microtubule ends and serves as a platform for binding by a complex of proteins regulating MT polymerization. TOGp and CLIP-170 also bound microtubule ends in cells expressing 2G4-GFP. Microtubule dynamic instability, measured by tracking 2G4-GFP labeled microtubules, was nearly identical to that measured in cells expressing GFP-α-tubulin. Fluorescence recovery after photobleaching demonstrated that 2G4-GFP turns over rapidly on microtubules, similar to the turnover rates of fluorescently tagged microtubule-associated proteins. These data indicate that 2G4-GFP binds relatively weakly to microtubules, and this conclusion was confirmed in vitro. Purified 2G4 partially co-pelleted with microtubules, but a significant fraction remained in the soluble fraction, while a second anti-tubulin scFv, 2F12, was almost completely co-pelleted with microtubules. In cells, 2G4-GFP localized to most microtubules, but did not co-localize with those composed of detyrosinated α-tubulin, a post-translational modification associated with non-dynamic, more stable microtubules. Immunoblots probing bacterially expressed tubulins confirmed that 2G4 recognized α-tubulin and required tubulin's C-terminal tyrosine residue for binding. Thus, a recombinant antibody with weak affinity for its substrate can be used as a specific intracellular biosensor that can differentiate between unmodified and post-translationally modified forms of a protein.
authors.affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, United States of America. lc07@lehigh.edu
authors.forename: Lynne
authors.initials: L
authors.lastname: Cassimeris
authors.forename: Laurence
authors.initials: L
authors.lastname: Guglielmi
authors.forename: Vincent
authors.initials: V
authors.lastname: Denis
authors.forename: Christian
authors.initials: C
authors.lastname: Larroque
authors.forename: Pierre
authors.initials: P
authors.lastname: Martineau
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D001921:Brain / Q000473:pathology; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008870:Microtubules / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D057127:Single-Chain Antibodies / Q000737:chemistry*; D013552:Swine; D014404:Tubulin / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014404:Tubulin; D049452:Green Fluorescent Proteins; D014443:Tyrosine
references.citation: Protein Eng Des Sel. 2011 Dec;24(12):873-81
references.pmid: 21997307
references.citation: BMC Biotechnol. 2007 Nov 22;7:81
references.pmid: 18034894
references.citation: Mol Biol Cell. 2002 Oct;13(10):3614-26
references.pmid: 12388761
references.citation: Science. 2008 Nov 28;322(5906):1353-6
references.pmid: 18927356
references.citation: Curr Opin Cell Biol. 1999 Feb;11(1):81-94
references.pmid: 10047525
references.citation: Cell. 2010 Nov 12;143(4):564-78
references.pmid: 21074048
references.citation: Traffic. 2006 May;7(5):499-507
references.pmid: 16643273
references.citation: Nat Rev Mol Cell Biol. 2008 Apr;9(4):309-22
references.pmid: 18322465
references.citation: J Cell Sci. 2004 Dec 1;117(Pt 25):6129-41
references.pmid: 15564376
references.citation: J Cell Biol. 2008 Feb 25;180(4):729-37
references.pmid: 18283108
references.citation: EMBO J. 1987 Sep;6(9):2597-606
references.pmid: 3315650
references.citation: J Cell Sci. 2001 Nov;114(Pt 21):3885-97
references.pmid: 11719555
references.citation: Mol Biol Cell. 2006 Aug;17(8):3557-68
references.pmid: 16775012
references.citation: Curr Biol. 2006 Jul 25;16(14):R516-7
references.pmid: 16860721
references.citation: Nat Rev Mol Cell Biol. 2011 Nov 16;12(12):773-86
references.pmid: 22086369
references.citation: EMBO J. 2012 Feb 15;31(4):856-69
references.pmid: 22193721
references.citation: J Mol Biol. 2002 Aug 2;321(1):49-56
references.pmid: 12139932
references.citation: Science. 2004 Feb 6;303(5659):836-9
references.pmid: 14764879
references.citation: J Biol Chem. 2000 Jan 28;275(4):2795-803
references.pmid: 10644744
references.citation: Biochemistry. 1992 Feb 11;31(5):1270-9
references.pmid: 1736986
references.citation: Methods Enzymol. 1982;85 Pt B:376-85
references.pmid: 7121276
references.citation: Mol Biol Cell. 2003 Mar;14(3):916-25
references.pmid: 12631713
references.citation: Curr Biol. 2006 Nov 7;16(21):2166-72
references.pmid: 17084703
references.citation: J Biol Chem. 2007 Feb 23;282(8):5570-81
references.pmid: 17178729
references.citation: Science. 2003 May 9;300(5621):984-7
references.pmid: 12738866
references.citation: Mol Biol Cell. 2011 Apr;22(7):1045-57
references.pmid: 21307336
references.citation: Curr Opin Cell Biol. 1998 Feb;10(1):16-22
references.pmid: 9484591
references.citation: Mol Biol Cell. 2001 Apr;12(4):971-80
references.pmid: 11294900
references.citation: Cell Motil Cytoskeleton. 2009 Aug;66(8):535-45
references.pmid: 19373773
references.citation: Nat Rev Mol Cell Biol. 2009 Nov;10(11):765-77
references.pmid: 19851335
references.citation: Oncogene. 2010 Jun 3;29(22):3217-27
references.pmid: 20228842
references.citation: Mol Biol Cell. 2009 Aug;20(15):3451-8
references.pmid: 19515833
references.citation: Curr Biol. 2012 Jun 19;22(12):1066-74
references.pmid: 22658592
references.citation: PLoS One. 2012;7(12):e51442
references.pmid: 23251535
references.citation: Biochem Soc Trans. 2009 Oct;37(Pt 5):1007-13
references.pmid: 19754441
references.citation: J Biol Chem. 1991 Sep 15;266(26):17597-605
references.pmid: 1680130
references.citation: J Cell Biol. 2006 Sep 11;174(6):839-49
references.pmid: 16954346
references.citation: Nat Methods. 2008 Jul;5(7):605-7
references.pmid: 18536722
references.citation: J Cell Sci. 2001 Aug;114(Pt 16):3025-33
references.pmid: 11686305
references.citation: J Cell Biol. 1984 Dec;99(6):2175-86
references.pmid: 6501419
references.citation: Mol Biol Cell. 2005 Dec;16(12):5686-98
references.pmid: 16195341