The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

PLoS Computational Biology
Shaoying LuYingxiao Wang

Abstract

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2)/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2)/sec and outside: 0.18+/-0.02 microm(2)/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained rela...Continue Reading

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Nov 14, 2013·Proceedings of the National Academy of Sciences of the United States of America·Jihye SeongYingxiao Wang
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Methods Mentioned

BETA
biosensors
FRET
biosensor
fluorescence recovery after photobleaching
myristoylation
PCR

Software Mentioned

MetaFluor
MATLAB

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