| Budget Amount *help |
¥44,850,000 (Direct Cost: ¥34,500,000、Indirect Cost: ¥10,350,000)
Fiscal Year 2005: ¥8,450,000 (Direct Cost: ¥6,500,000、Indirect Cost: ¥1,950,000)
Fiscal Year 2004: ¥8,450,000 (Direct Cost: ¥6,500,000、Indirect Cost: ¥1,950,000)
Fiscal Year 2003: ¥27,950,000 (Direct Cost: ¥21,500,000、Indirect Cost: ¥6,450,000)
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| Research Abstract |
We have tried to construct a foundation of single molecular biochemistry of G protein mediated signal transduction system in vertebrate rod photoreceptor cells. By using a single fluorescent molecule imaging technique, we have succeeded to observe single molecular behavior of three key components of vertebrate phototransduction, i.e. a typical GPCR rhodopsin, G protein transducin, and its target enzyme cGMP-PDE. We have employed near far-red fluorescent-labeled antibody Fab' fragment or direct conjugation of these dyes for labeling these proteins. Rhodopsin was proved with Cy7-labeled Fab' fragment of monoclonal antibody (1D4) against the C-terminus of rhodopsin. The single molecular behavior of rhodopsin on the disc membrane was observed at video rate and 70 nm/pixel in spatial resolution. It was found that the rhodopsin does not form static paracrystalline as reported recently on the basis of AFM observation. Rhodopsin undergoes anomalous diffusion changing diffusion rate frequently. FRET experiments revealed that rhodopsins form dimer or a cluster of short lifetime. Artificially dimerized rhodopsin by the use of IgG also shows slow diffusion rate. The formation of cluster of low diffusion rate is apparently disadvantageous for rapid amplification in the phototransduction. Thus, it was suggested paradoxically that the cluster formation has an important meaning for photo-transduction. In addition, I succeeded in labeling transducin by Cy7 maintaining its activity to bind rhodopsin and PDE. Single molecular behavior of transducin was totally different from that of rhodopsin. Single molecular biochemistry of phototransduction has started to evolve.
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