title: Identification of plasma membrane macro- and microdomains from wavelet analysis of FRET microscopy.
abstract: In this study, we sought to characterize functional signaling domains by applying the multiresolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes. A genetically encoded FRET reporter of protein kinase C (PKC)-dependent phosphorylation was expressed in COS1 cells. Differences between wavelet coefficient matrices revealed several heterogeneous domains (typically ranging from 1 to 5 microm), reflecting the dynamic balance between PKC and phosphatase activity during stimulation with phorbol-12,13-dibutyrate or acetylcholine. The balance in these domains was not necessarily reflected in the overall plasma membrane changes, and observed heterogeneity was absent when cells were exposed to a phosphatase or PKC inhibitor. Prolonged exposure to phorbol-12,13-dibutyrate and acetylcholine yielded more homogeneous FRET distribution in plasma membranes. The proposed wavelet-based image analysis provides, for the first time, a basis and a means of detecting and quantifying dynamic changes in functional signaling domains, and may find broader application in studying fine aspects of cellular signaling by various imaging reporters.
authors.affiliation: National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Kobrinsky
authors.forename: Donald E
authors.initials: DE
authors.lastname: Mager
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Bentil
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Murata
authors.forename: Darrell R
authors.initials: DR
authors.lastname: Abernethy
authors.forename: Nikolai M
authors.initials: NM
authors.lastname: Soldatov
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000737:chemistry; D015233:Models, Statistical; D015240:Phorbol 12,13-Dibutyrate / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases
references.citation: J Cell Biol. 2003 Jun 9;161(5):899-909
references.pmid: 12782683
references.citation: J Cell Sci. 2004 Nov 1;117(Pt 23):5521-34
references.pmid: 15479718
references.citation: Circ Res. 2004 Apr 16;94(7):866-73
references.pmid: 15087426
references.citation: J Histochem Cytochem. 2003 Jul;51(7):951-8
references.pmid: 12810845
references.citation: Biophys J. 1993 Sep;65(3):1135-46
references.pmid: 8241393
references.citation: J Microsc. 2004 May;214(Pt 2):190-200
references.pmid: 15102066
references.citation: Biophys J. 2003 Oct;85(4):2170-85
references.pmid: 14507683
references.citation: J Microsc. 2004 Nov;216(Pt 2):110-22
references.pmid: 15516222
references.citation: Curr Opin Cell Biol. 2002 Aug;14(4):483-7
references.pmid: 12383800
references.citation: Cell. 2004 Feb 20;116(4):577-89
references.pmid: 14980224
references.citation: Science. 2002 May 3;296(5569):913-6
references.pmid: 11988576
references.citation: Nat Med. 2004 Mar;10(3):248-54
references.pmid: 14966518
references.citation: Phys Rev Lett. 1995 Apr 17;74(16):3293-3296
references.pmid: 10058160
references.citation: J Biol Chem. 2003 Feb 14;278(7):5021-8
references.pmid: 12473653
references.citation: Biophys J. 2001 Oct;81(4):2395-402
references.pmid: 11566809
references.citation: Cytometry. 2001 Feb 1;43(2):94-100
references.pmid: 11169573
references.citation: J Biochem. 2002 Nov;132(5):663-8
references.pmid: 12417013
references.citation: Methods. 2001 Jul;24(3):289-96
references.pmid: 11403577
references.citation: Annu Rev Biomed Eng. 2000;2:511-50
references.pmid: 11701522
references.citation: Biophys J. 2004 Aug;87(2):844-57
references.pmid: 15298893
references.citation: Biophys J. 2003 Jul;85(1):559-71
references.pmid: 12829510