| 研究課題/領域番号 |
22K15112
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| 研究種目 |
若手研究
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| 配分区分 | 基金 |
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| 審査区分 |
小区分44010:細胞生物学関連
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
Li Xue (馬場雪) 国立研究開発法人理化学研究所, 生命機能科学研究センター, 研究員 (50936507)
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| 研究期間 (年度) |
2022-04-01 – 2024-03-31
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| 研究課題ステータス |
交付 (2022年度)
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| 配分額 *注記 |
4,550千円 (直接経費: 3,500千円、間接経費: 1,050千円)
2023年度: 2,210千円 (直接経費: 1,700千円、間接経費: 510千円)
2022年度: 2,340千円 (直接経費: 1,800千円、間接経費: 540千円)
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| キーワード | KIF5C / Kinesin 1 / Microtubule binding / Cortical Neuron / TIRF / STED Microscopy / kinesin KIF5C / axon microtubule / TurboID / STED |
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| 研究開始時の研究の概要 |
1) optimizations of culture cortical neuron cells from mice Embryonic mice at E16 will be performed.
2) Several KIF5C-TurboID dimer constructs will be purified, and KIF5C binding activity to MT will be measured which is critical in our study.
3) Identify KIF5C partners using TurboID. The purified KIF5C-TurboID dimer will be delivered into semi-intact neurons, where KIF5C binds to MT and TurboID labels proteins in proximity of KIF5C with biotin. Then the biotinylated proteins will be identified by mass spectrometry, and we are going to verify the hits and uncover the binding mechanism behind.
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| 研究実績の概要 |
To identify the molecular machinery that controls the selective/preferential binding of KIF5C to axonal MTs, this project aims to identify the key factors that regulate or participate in this process through proximity-based labelling and mass spectrometry analyses in semi-intact cortical neuron cells.
In the past year, several efforts have been made to realize proximity-based labelling in semi-intact cortical neuron cells. First, different coating materials as well as combinations have been tested and optimized for cortical neuron cell growth in vitro. Second, the differentiation of cortical neuron cells after the isolation has been optimized to minimize the population of astrocytes and glia. Third, the purification of the KIF5C-TurboID construct has been performed using the MonoQ column on the Aekta system. In this way, several active, dimerized and highly concentrated KIF5C-TurboID constructs were prepared for the semi-intact labelling. Last but not least, the selective binding of KIF5C constructs on the MTs of the semi-intact cortical neurons has been optimized.
To summarize, the preparation work to conduct proximity-based labelling in semi-intact neuron cortical cells for mass spectrometry analysis has been completed. In the next step, the identification of key players controlling the selective/preferential binding of KIF5C to axonal MTs is going to be performed.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Different from normal cell lines in culture, the primary cortical neuron cells are very special and difficult to culture. They are isolated from the forebrain of mouse embryos at E16 so that the cell numbers are very limited. After isolation, the growth conditions such as plate materials, coating materials, and media conditions need to be established and optimized.
One tricky optimization is the AraC treatment. It is used to limit the growth of astrocytes and glia cells in the culture. Astrocytes and glia cells are important to keep cortical neuron cells alive and functionally, especially during the first several days after isolation. But as they can divide in vitro, the large number of those cells will then compete with cortical neuron cells for nutrition. To keep cortical neuron cells healthy, astrocytes and glial cells are required but the population need to be strictly controlled. Both the timing of the treatment and the concentration need to be carefully optimized.
Such optimizations for in vitro cortical neuron primary cell culture are time-consuming but very critical for this project. I believe that with the efforts on the optimization, a well-functional protocol is established for the smooth conduction of the following parts of this project.
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| 今後の研究の推進方策 |
Next, the identification of key players controlling the selective/preferential binding of KIF5C to axonal MTs is going to be performed in the semi-intact cortical neuron cell culture using mass spectrometry in collaboration with Tokushima University. Identification of different hits among different KIF5Cs samples by mass spectrometry is expected.
The following work will then focus on verifications and mechanism studies of KIF5C selective/preferential binding. Subsequently, I am going to study the alterations of KIF5C dynamic using TIRF and STED super-resolution microscopy.
This study aims to shed light on the molecular mechanism of controlling KIF5C affinity to MTs and the pathogenetic mechanism of MCD and microcephaly.
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