title: APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.
abstract: BACKGROUND
  Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.
  
  
  METHODS
  Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.
  
  
  CONCLUSIONS
  All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.
authors.affiliation: Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America. chial.heidi@gmail.com
authors.forename: Heidi J
authors.initials: HJ
authors.lastname: Chial
authors.forename: Peter
authors.initials: P
authors.lastname: Lenart
authors.forename: Yong Q
authors.initials: YQ
authors.lastname: Chen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: C464801:APPL1 protein, human; C528555:APPL2 protein, human; D048868:Adaptor Proteins, Signal Transducing
references.citation: J Biol Chem. 2009 Aug 14;284(33):22426-22435
references.pmid: 19520843
references.citation: Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8407-12
references.pmid: 8710884
references.citation: Biophys J. 2004 Jun;86(6):3923-39
references.pmid: 15189889
references.citation: Nat Methods. 2007 Oct;4(10):767-8
references.pmid: 17901864
references.citation: EMBO Rep. 2000 Sep;1(3):287-92
references.pmid: 11256614
references.citation: Biochem J. 2003 May 15;372(Pt 1):33-40
references.pmid: 12662152
references.citation: Mol Cell Endocrinol. 2007 Jan 2;260-262:93-9
references.pmid: 17030088
references.citation: Cell. 2004 Feb 6;116(3):445-56
references.pmid: 15016378
references.citation: Biophys J. 2003 Jun;84(6):3992-4010
references.pmid: 12770904
references.citation: EMBO J. 2006 Jun 21;25(12):2889-97
references.pmid: 16763557
references.citation: Mol Cell Biol. 2006 Dec;26(23):8942-52
references.pmid: 17015470
references.citation: Structure. 2007 May;15(5):525-33
references.pmid: 17502098
references.citation: EMBO J. 2007 Jul 25;26(14):3484-93
references.pmid: 17581628
references.citation: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7461-6
references.pmid: 12032305
references.citation: Biol Reprod. 2004 Aug;71(2):629-36
references.pmid: 15070827
references.citation: J Microsc. 2005 Jun;218(Pt 3):253-62
references.pmid: 15958019
references.citation: Biochem Biophys Res Commun. 2008 May 2;369(2):493-9
references.pmid: 18307981
references.citation: EMBO J. 2006 Jun 21;25(12):2898-910
references.pmid: 16763559
references.citation: J Cardiovasc Pharmacol. 2004 Nov;44 Suppl 1:S30-3
references.pmid: 15838305
references.citation: J Biol Chem. 2007 Nov 2;282(44):32280-7
references.pmid: 17848569
references.citation: EMBO J. 1996 Aug 15;15(16):4246-53
references.pmid: 8861953
references.citation: Mol Cell Biol. 2006 Dec;26(23):8928-41
references.pmid: 17000777
references.citation: Nat Cell Biol. 2006 May;8(5):516-23
references.pmid: 16622416
references.citation: J Biol Chem. 2009 Jul 3;284(27):18115-28
references.pmid: 19433865
references.citation: Biophys J. 1998 May;74(5):2702-13
references.pmid: 9591694
references.citation: Biophys J. 2001 Oct;81(4):2395-402
references.pmid: 11566809
references.citation: Oncogene. 1999 Sep 2;18(35):4891-8
references.pmid: 10490823
references.citation: Dev Cell. 2007 Sep;13(3):377-90
references.pmid: 17765681
references.citation: Biochem J. 2009 Oct 12;423(3):389-400
references.pmid: 19686092
references.citation: Diabetologia. 2009 Oct;52(10):2190-200
references.pmid: 19636528
references.citation: Nat Methods. 2006 Jul;3(7):491-2; author reply 492-3
references.pmid: 16791203
references.citation: Sci STKE. 2000 Jun 27;2000(38):pl1
references.pmid: 11752595
references.citation: J Biol Chem. 2009 Nov 13;284(46):31608-15
references.pmid: 19661063
references.citation: Traffic. 2008 Feb;9(2):215-29
references.pmid: 18034774
references.citation: Diabetes. 2007 May;56(5):1387-94
references.pmid: 17287464
references.citation: Nat Methods. 2005 Nov;2(11):801
references.pmid: 16278647
references.citation: J Biol Chem. 2002 Jul 19;277(29):26281-5
references.pmid: 12011067