Identification of molecular machinery to control the selective/preferential binding of kinesin KIF5C to axonal microtubules by proximity labelling

通过邻近标记鉴定控制驱动蛋白 KIF5C 与轴突微管选择性/优先结合的分子机制

• 批准号: 22K15112
• 负责人: Li Xue
• 金额: $ 2.91万
• 依托单位: Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22K15112/
• 关键词: KIF5C; Kinesin 1; Microtubule binding; Cortical Neuron; TIRF; STED Microscopy; kinesin KIF5C; axon microtubule; TurboID; STED

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.

本项目旨在通过对半完整皮层神经元细胞的近端标记和质谱分析，确定控制KIF 5C与轴突MT选择性/优先结合的分子机制。在过去的一年中，我们进行了几项努力，以实现半完整皮层神经元细胞的近端标记。首先，不同的涂层材料以及组合已被测试和优化的皮层神经元细胞的体外生长。第二，分离后皮层神经元细胞的分化已被优化，以最大限度地减少星形胶质细胞和神经胶质细胞的数量。第三，使用Aekta系统上的MonoQ柱进行KIF 5C-TurboID构建体的纯化。以这种方式，制备了几种活性、二聚化和高度浓缩的KIF 5C-TurboID构建体用于半完整标记。最后但并非最不重要的是，KIF 5C构建体在半完整皮层神经元的MT上的选择性结合已经被优化。综上所述，在半完整神经元皮层细胞中进行基于邻近的标记以用于质谱分析的准备工作已经完成。在下一步中，将进行控制KIF 5C与轴突MT选择性/优先结合的关键参与者的鉴定。