| Project/Area Number |
18K19328
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| Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
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| Allocation Type | Multi-year Fund |
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| Review Section |
Medium-sized Section 44:Biology at cellular to organismal levels, and related fields
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
NAKATA Takao 東京医科歯科大学, 大学院医歯学総合研究科, 教授 (50218004)
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| Co-Investigator(Kenkyū-buntansha) |
稲葉 弘哲 東京医科歯科大学, 大学院医歯学総合研究科, 助教 (80791334)
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| Project Period (FY) |
2018-06-29 – 2021-03-31
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| Project Status |
Completed (Fiscal Year 2020)
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| Budget Amount *help |
¥6,370,000 (Direct Cost: ¥4,900,000、Indirect Cost: ¥1,470,000)
Fiscal Year 2019: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2018: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
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| Keywords | 光遺伝学 / クライオ電子線トモグラフィ / Rac1 / 葉状仮足 / RAC1 / アクチン細胞骨格 / クライオ電子線トモグラフィー |
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| Outline of Final Research Achievements |
This study aimed to analyze temporal changes of the F-actin cytoskeleton network in the lamellipodia formation with an ultra-spatiotemporal resolution by combining optogenetics and cryo-electron tomography. We induced lamellipodia formation with photoactivatable Rac1 (PA-Rac1) and analyzed its time-course with confocal microscopy. In addition, COS-7 cells were flash-frozen, and continuous tilt series images of the cell peripheral were acquired by cryo-electron microscopy, and 3D tomograms were reconstructed. We could manually segment the plasma membrane, microtubules, ribosomes, and actin filaments in the cell peripheral region. We also tried automatically segment the plasma membrane, microtubules, ribosomes, and actin filaments by deep learning.
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| Academic Significance and Societal Importance of the Research Achievements |
葉状仮足は枝分かれが豊富なアクチン線維のネットワークからなり、器官形成や創傷治癒、癌の浸潤転移などにおける細胞の移動において重要な役割を担うため、そのアクチンネットワークの再構成過程を詳細に観察することは、細胞生物学的にも病理学的にも意義がある。クライオ電子線トモグラフィによる細胞内構造の可視化は、まだ先行例が少ないが、今後益々重要な技術となることは間違いない。本研究期間中には到達できなかったが、光遺伝学によって電子顕微鏡の弱点である時間軸の導入を達成できれば、学術的に大きな意義がある。
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