Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

Horst Wallrabe; Yuansheng Sun; Xiaolan Fang; Ammasi Periasamy; George Bloom

doi:10.1117/12.906432

15 February 2012 Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

Horst Wallrabe, Yuansheng Sun, Xiaolan Fang, Ammasi Periasamy, George Bloom

Author Affiliations +

Proceedings Volume 8226, Multiphoton Microscopy in the Biomedical Sciences XII; 82260J (2012) https://doi.org/10.1117/12.906432
Event: SPIE BiOS, 2012, San Francisco, California, United States

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.

Citation Download Citation

Horst Wallrabe, Yuansheng Sun, Xiaolan Fang, Ammasi Periasamy, and George Bloom "Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation", Proc. SPIE 8226, Multiphoton Microscopy in the Biomedical Sciences XII, 82260J (15 February 2012); https://doi.org/10.1117/12.906432

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