title: Three-Color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation.
abstract: With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFP-NWASP/Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1-F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.
authors.affiliation: Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA ; Department of Biology, University of Virginia, Charlottesville, VA.
authors.forename: Horst
authors.initials: H
authors.lastname: Wallrabe
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Sun
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Fang
authors.forename: Ammasi
authors.initials: A
authors.lastname: Periasamy
authors.forename: George
authors.initials: G
authors.lastname: Bloom
keywords: E%; IQGAP1; N-WASP; Teal; Three-color FRET; Venus; actin; mCherry; quantitative analysis
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