| Budget Amount *help |
¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000)
Fiscal Year 2009: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2008: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Research Abstract |
In this project, I have set two goals : i) identification of substrate proteins of the redox-sensitive dynein components in Chlamydomonas flagella ; ii) elucidation of functions of redox-signaling pathway(s) in Chlamydomonas flagella including proteins identified in i).
With regard to i), mutagenized and HA-tagged LC3/LC5/DC3 were successfully expressed in Chlamydomonas cells and incorporated to flagella as the dynein subunits. The mutations were designed to trap substrate proteins in vivo via intermolecular disulfide bonds. However, because of low expression of those mutagenized proteins, I have not purified sufficient amounts of substrate proteins for identification via mass spec. This experiment is still underway.
With regard to ii), because the experiments for i) are still underway, I have started a new experiment to quantify the redox state of flagellar cytoplasm. From our previous study, it has been shown that the flagellar cytoplasmic redox state changes depending on the photosynthetic activity. The redox-signaling pathways must sense the flagellar redox state.
Using a newly generated transformant expressing redox-sensitive GFP, currently I am trying to quantify the redox state of the Chlamydomonas flagellar redox state. Two novel regulation mechanisms for flagellar motility have been discovered in this project other than the results above. First, the flagellar beat frequency elevates upon mechanical stimuli in a calcium-dependent manner. Second, the sign (or direction) of phototaxis in Chlamydomonas is redox-regulated. The first study is published as a paper in Cell Motility and the Cytoskeleton, and the second study has been submitted and is under review.
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