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Proceedings Paper

Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation
Author(s): Horst Wallrabe; Yuansheng Sun; Xiaolan Fang; Ammasi Periasamy; George Bloom
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Paper 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 TFPNWASP/ 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.

Paper Details

Date Published: 15 February 2012
PDF: 10 pages
Proc. SPIE 8226, Multiphoton Microscopy in the Biomedical Sciences XII, 82260J (15 February 2012); doi: 10.1117/12.906432
Show Author Affiliations
Horst Wallrabe, Univ. of Virginia (United States)
Keck Ctr. for Cellular Imaging, Univ. of Virginia (United States)
Yuansheng Sun, Univ. of Virginia (United States)
Xiaolan Fang, Keck Ctr. for Cellular Imaging, Univ. of Virginia (United States)
Ammasi Periasamy, Keck Ctr. for Cellular Imaging, Univ. of Virginia (United States)
George Bloom, Keck Ctr. for Cellular Imaging, Univ. of Virginia (United States)

Published in SPIE Proceedings Vol. 8226:
Multiphoton Microscopy in the Biomedical Sciences XII
Karsten König, Editor(s)

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