Differential Ras activation between caveolae/raft and non-raft microdomains.
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.
Fukano
Takashi
T
Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN.
Sawano
Asako
A
Ohba
Yusuke
Y
Matsuda
Michiyuki
M
Miyawaki
Atsushi
A
D000818:Animals; D019556:COS Cells; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D004815:Epidermal Growth Factor; D018631:ras Proteins