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

为了确定控制KIF5C与轴突mt选择性/优先结合的分子机制，本项目旨在通过基于接近度的标记和质谱分析，在半完整的皮质神经元细胞中确定调节或参与这一过程的关键因素。在过去的一年中，一些研究人员已经在半完整的皮质神经元细胞中实现了基于邻近度的标记。首先，不同的涂层材料以及组合已经测试和优化皮层神经元细胞的体外生长。其次，分离后皮质神经元细胞的分化得到优化，使星形胶质细胞和胶质细胞的数量最小化。第三，使用Aekta系统上的MonoQ柱对KIF5C-TurboID结构进行纯化。通过这种方法，制备了几种活性的、二聚的、高浓度的KIF5C-TurboID结构体，用于半完整的标记。最后，优化了KIF5C构建体在半完整皮质神经元mt上的选择性结合。总之，在半完整的神经元皮质细胞中进行基于邻近度的标记以进行质谱分析的准备工作已经完成。下一步，将确定控制KIF5C与轴突mt选择性/优先结合的关键参与者。

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.