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2015 ; 87
(6
): 580-8
Nephropedia Template TP
gab.com Text
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Three-color confocal Förster (or fluorescence) resonance energy transfer
microscopy: Quantitative analysis of protein interactions in the nucleation of
actin filaments in live cells
#MMPMID25755111
Wallrabe H
; Sun Y
; Fang X
; Periasamy A
; Bloom GS
Cytometry A
2015[Jun]; 87
(6
): 580-8
PMID25755111
show ga
Experiments using live cell 3-color Förster (or fluorescence) resonance energy
transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution
of the same proteins were conducted to evaluate actin filament nucleation. A
novel application of 3-color FRET data is demonstrated, extending the analysis
beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells
were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1,
Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or
CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for
spectral bleedthrough correction. Using confirmed E% as an entry point,
fluorescence levels and related ratios were correlated at discrete accumulating
levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were
correlated with lower overall actin fluorescence, whereas the
CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low
ratios, but was neutral at higher ratios. The new FRET analyses also indicated
that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or
suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET
assays further support our in vitro results about the role of IQGAP1, Rac1, and
Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the
association of N-WASP with IQGAP1. In addition, this study emphasizes the power
of 3-color FRET as a systems biology strategy for simultaneous evaluation of
multiple interacting proteins in individual live cells.
|Actin Cytoskeleton/physiology
[MESH]
|Animals
[MESH]
|Cell Line
[MESH]
|Dogs
[MESH]
|Fluorescence Resonance Energy Transfer/*methods
[MESH]