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

2022 Fiscal Year Research-status Report

• Project/Area Number: 22K15112
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: 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)
• Project Period (FY): 2022-04-01 – 2024-03-31
• Keywords: KIF5C / Kinesin 1 / Microtubule binding / Cortical Neuron / TIRF / STED Microscopy

Outline of Annual Research Achievements

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.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

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.

Strategy for Future Research Activity

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.

Causes of Carryover

In the first fiscal year, as the primary cortical neuron cells used in this project are very special and difficult to culture, many optimizations and preparations have been done to establish the working protocol to conduct proximity-based labelling in semi-intact neuron cortical cells for mass spectrometry analysis.
In the next step, both the identification of key players controlling the selective/preferential binding of KIF5C to axonal MTs using Mass spectrometry and the following study of alterations of KIF5C dynamic using TIRF and STED super-resolution microscopy will require a lot of expenses.
Consequently, the consumables that are available in the lab are first used in the preparation work and the incurring amount is going to be used in the next fiscal year for a large amount of culture of primary cortical neuron cells for mass spectrometry, TIRF and STED super-resolution microscopy analysis.
I believe that this plan will help us achieve our objectives effectively and efficiently, and I am committed to ensuring that the funds are used in a responsible and transparent manner.