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

点击翻译按钮获取中文摘要

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.