A Deep Dive into Neuronal Nuclear Transport

PhD student at Graduate School of Biostudies, Kyoto University (Kengaku Lab)

Zhou Chuying

Zhou Chuying

Dr Zhou is a PhD student working at Kengaku Lab to study the molecular mechanisms governing neuronal migration during brain development. She has recently uncovered the critical role of a nuclear membrane molecule, Nesprin-2, in mediating the dynamic movements of the neuronal nucleus.

Please share with us the significance of your manuscript in terms of its research achievements, impact, or uniqueness.

During brain development, the nucleus of migrating neurons must translocate forward to position the cell body correctly, a crucial step in establishing the layered structure of the cortex. While it has long been thought that the microtubule-minus-end-directed motor protein dynein is primarily responsible for driving forward nuclear movements along the peri-nuclear microtubules, our group’s previous study suggest that the microtubule-plus-end-directed motor kinesin-1 is also involved, though its role remains unclear. My research revealed that dynein and kinesin-1 indeed work together to facilitate nuclear movement through Nesprin-2, a nuclear membrane protein that recruits and coordinates the activities of both motors. Interestingly, rather than simply driving dynein-dominated forward motion, Nesprin-2 promotes persistent bidirectional cargo movements driven coordinately by opposing motors. When the peri-nuclear microtubules shift forward, Nesprin-2 links the nucleus to these advancing microtubule tracks, generating forward jumps of the nucleus. This discovery provides new insights into the mechanisms of intracellular cargo transport and offers fresh perspectives on diseases such as brain malformations and cancer cell migration.

Please tell us what was the most gratifying or inspiring moment for you during this research project.

Failure has been a constant companion throughout my PhD journey, which makes me cherish every small success even more. One of my most unforgettable moments came when I successfully observed granule neuron migration for the first time using organotypic cultures from mouse cerebellum lobes. Before this, I had struggled with the experiment for over four months, as the preparation of organotypic cultures demands meticulous skill. Neurons are incredibly sensitive, and even the slightest damage to the sample would halt migration. After numerous rounds of troubleshooting, discussions with colleagues, and countless hours of practice, I finally observed active cell migration under the microscope. The excitement kept me awake all night, and I spent those hours imaging my sample. Though this wasn't a major scientific breakthrough, it marked the first time I truly felt a sense of achievement in my research. This experience taught me that every small piece of data in a figure represents immense effort and perseverance.

Please tell us about the biggest challenge or problem you experienced while conducting your research. How did you overcome it?

One of the biggest challenges in this study was analyzing the movement of peroxisomes. To characterize the cargo transport activity mediated by Nesprin-2, I used an intracellular peroxisome trafficking assay. This analysis posed several difficulties, such as distinguishing between active transport and random Brownian motion, ensuring a fast imaging rate, and maintaining cell health for fluorescence observation while preserving proper cell morphology and cytoskeleton structure.
To address these issues, I implemented dual-color imaging to visualize both the cargo and microtubules, selectively analyzing episodes where the cargo moved along microtubules over a defined distance. For high-speed imaging, I tested various microscopes and ultimately chose the Dragonfly fast-imaging system at the iCeMS Analysis Center, optimizing its settings for rapid acquisition. Additionally, I refined cell plating techniques and carefully selected healthy cells suitable for imaging.
Through expert consultations and trial-and-error, I successfully collected data and learned to develop computer programs to analyze the complex, dynamic behavior of peroxisomes. While there were many frustrating moments requiring repeated experiments with updated microscope setups and refined analysis criteria, this process taught me a great deal and boosted my confidence in tackling unknown challenges.

Would you consider this work to be a turning point in your overall research direction? If so, how has your research direction changed as a result of this work?

As a PhD student, this study has offered me comprehensive training and laid a solid foundation for entering the research world. After graduation, I plan to explore new topics in neuroscience and refine my research interests by gaining broader experience. Although my postdoctoral work will likely focus on a different subject, the knowledge and skills I've gained through this project will undoubtedly serve as valuable assets for my future career.

Please describe the current situation of your career. What is your current position? How has the knowledge and experience gained at iCeMS influencing your career?

I recently defended my PhD thesis and expect to graduate next month. Over the past six years at iCeMS, I have been fortunate to experience both scientific exploration and significant personal growth. When I first arrived in Japan as an international student with no prior knowledge of Japanese, the welcoming and international environment at iCeMS made it easy for me to adapt. I built many lasting friendship here, whom I hope to stay connected with throughout my life. I have thoroughly enjoyed the seminars and the social events, learning a great deal from interacting with other research groups.
In particular, the iCeMS Analysis Center has been invaluable to my research, and without their professional support, this study would not have been possible. I am deeply grateful to be part of iCeMS, and its spirit of bridging disciplines and bringing together researchers from diverse backgrounds will continue to inspire my future work.

Paper information

Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons

Chuying Zhou, You Kure Wu, Fumiyoshi Ishidate, Takahiro K. Fujiwara, Mineko Kengaku

Journal of Cell Biology

Published: August 2024

DOI: 10.1083/jcb.202405032