Article

Differential Ras activation between caveolae/raft and non-raft microdomains.

Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN.
Cell Structure and Function (impact factor: 2.29). 02/2007; 32(1):9-15. pp.9-15
Source: PubMed

ABSTRACT Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu-Ras, was fused to the C-terminal hypervariable regions of H-Ras and K-Ras to create indicators for Ras activation within caveolae/rafts (Raichu-tH) and non-raft domains (Raichu-tK) of the plasma membrane, respectively. Raichu-tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device (DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer (FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.

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Keywords

C-terminal hypervariable regions
 
digital micromirror device
 
DMD
 
extensively
 
fluorescence resonance energy transfer
 
fluorescent indicator
 
fringe projection
 
fused
 
growth factors
 
indicators
 
K-Ras
 
non-raft domains
 
physiological modes
 
Ras activation
 
reliable signals
 
signal transduction
 
temporally unique patterns
 
two indicators
 
wide-field fluorescence microscope
 
yellow mutants