| 研究実績の概要 |
The kinesin KIF5C (Kinesin 1) transports various cargos from the neuronal cell body towards axon terminal along microtubules (MTs) in motor neuron. The importance of KIF5C affinity to MTs has been highlighted after the analysis of KIF5C mutants found in patients with severe malformations of cortical development and microcephaly. Our in vitro and in vivo studies of KIF5C binding to MTs reveled the existence of different binding affinity levels, indicating that there is molecular machinery in cells regulating or adjusting the binding behaviors of KIF5C to MTs.
In this study, isolated and differentiated cortical neurons from mice embryos were used. To identify the key protein players, we used proximity-based labelling method KIF5C-TurboID in semi-intact cortical neuron cells. We made several KIF5C binding constructs to mimic selective binding and established the protocol to let in vitro purified dimerized active KIF5C-TurboID proteins bind to MTs of the semi-intact cortical neurons and label the proteins in close proximity.
However, due to the highly active labelling efficiency and long required labeling time (~18h) of TurboID, it is prone to have many false-positive hits identified and is difficult to control the labelling process. Therefore, we decided to switch our system to the newly introduced proximity labeling methods APEX2. APEX2 has a lower labeling activity and a rapid labeling time (~10 min). Currently, we have optimized the labeling of KIF5C-APEX2 in semi-intact cortical neuron cells and are proceeding to hits identification using mass spectrometry analyses.
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