| Project/Area Number |
17370071
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | Okayama University |
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Principal Investigator |
TAKEI Kohji Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Professor (40322226)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Hiroshi Okayama University, Graduate School of Medicine and Pharmaceutical Sciences, Lecturer (80325092)
LI Shun-ai Okayama University, Graduate School of Medicine and Pharmaceutical Sciences, Assistant Professor (30403497)
TANAE Kenji Okayama University, Graduate School of Medicine and Pharmaceutical Sciences, Assistant Professor (80423341)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,280,000 (Direct Cost: ¥14,800,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥10,000,000 (Direct Cost: ¥10,000,000)
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| Keywords | endocvtosis / phagocytosis / vesicular traffic / dvnamin / amphiphysin / actin / live imaging / riffle / 無細胞系 / アンフィファシジン / リポソーム |
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| Research Abstract |
In order to elucidate the role of endocytic proteins in membrane dynamics, localization and intracellular dynamics of dynamin and amphiphysin during phagocytosis were examined. For this purpose, testicular Sertoli cell phagocytosis was stimulated by phosphatidylserine containing liposomes, and intracellular localization and dynamics of the endocytic proteins were examined. By immunofluorescence and by live cell imaging, both dynamin2 and amphiphysin were concentrated at the leading edge of lamellipodia and ruffles. Ruffle formation, actin formation, and phagocytosis were markedly inhibited in Amphiphysin siRNA treated cells indicating that amphiphysin is essential for these processes. Furthermore, these effects in the amphiphysin 1-knocked down cells were rescued by co-overexpression of constitutive active Rac 1, suggesting that Rac 1 is involved in the process at the downstream of amphiphysin.
Next, effect of amphiphysin 1 on actin polymerization activity was examined in vitro. For this purpose, mouse testis cytosol supplemented with pyrene-conjugated actin, were subjected to quantitative actin in vitro polymerization assay. Actin polymerization was also observed under fluorescent microscopy using cytosol supplemented with rhodamine-conjugated actin. Actin assembly activity was considerably reduced in cytosol from amphiphysin 1 knock out mice, which can be recovered by adding back recombinant proteins.
Thus, amphiphysin 1, an endocytic protein, play a role in the regulation of actin dynamics, by which it stimulate in membrane dynamics and phagocytosis.
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