ABSTRACT
First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Shingo Koinuma and Misa Miyaji are co-first authors on ‘TC10 on endosomes regulates the local balance between microtubule stability and dynamics through the PAK2-JNK pathway and promotes axon outgrowth’, published in JCS. Shingo is a research assistant in the lab of Takeshi Nakamura at Tokyo University of Science, investigating axon biology. Misa is a Master's student in the same lab investigating axon growth and regeneration.
Shingo Koinuma
How would you explain the main findings of your paper in lay terms?
S.K. and M.M.: Microtubules maintain neuronal structure, act as rails for material transport, and play an important role in axon formation, elongation and maintenance. In our study, we find that the Rho family G-protein TC10 efficiently stabilizes microtubules on endosomes and suppresses axon retraction. Downstream of active TC10 on recycling endosomes, local activation of PAK and JNK occurs. JNK phosphorylates the neuron-specific microtubule regulators SCG10 and MAP1B, stabilizing microtubules and counteracting axon retraction.
Were there any specific challenges associated with this project? If so, how did you overcome them?
S.K. and M.M.: The inevitable damage caused by electroporation in mouse hippocampal neurons made long-term live imaging very difficult. By improving the preparation process, using conditioned medium, and keeping appropriate temperature and humidity, we achieved a survival rate of 90%. In addition, we were eager to obtain a TC10 mutant that localized exclusively to recycling endosomes to directly test our hypothesis. However, we found no suitable tools available. By fusing the N-terminus of SCG10 to TC10, we created the TC10 mutant that suited our needs.
When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?
M.M.: I was surprised that the TC10 mutant localized only to endosomes, and not the plasma membrane, and yet it rescued microtubule stability and prevented axon retraction caused by TC10 ablation.
Misa Miyaji
TC10 on endosomes counteracts excessive MT destabilization. Hippocampal neurons from WT (left) or TC10 KO (middle and right) mice were electroporated with pCAGGS-EGFP or pCAGGS-SN-TC10ΔCT-EGFP. In the SN-TC10ΔCT-EGFP mutant, the N-terminal lipid-modified region of SCG10 was added to the N-terminus of TC10; therefore, SN-TC10ΔCT was localized only at Rab11-positive endosomes. At 2.5 days in vitro, neurons were immunostained with an anti-acetylated-α-tubulin antibody. White arrows indicate distal axons. Scale bar: 50 μm.
TC10 on endosomes counteracts excessive MT destabilization. Hippocampal neurons from WT (left) or TC10 KO (middle and right) mice were electroporated with pCAGGS-EGFP or pCAGGS-SN-TC10ΔCT-EGFP. In the SN-TC10ΔCT-EGFP mutant, the N-terminal lipid-modified region of SCG10 was added to the N-terminus of TC10; therefore, SN-TC10ΔCT was localized only at Rab11-positive endosomes. At 2.5 days in vitro, neurons were immunostained with an anti-acetylated-α-tubulin antibody. White arrows indicate distal axons. Scale bar: 50 μm.
Why did you choose Journal of Cell Science for your paper?
S.K.: Our study focused on the cellular and molecular mechanisms by which TC10 promotes axonal elongation. Journal of Cell Science is a high-quality, well-established journal highly regarded in this field and an ideal platform for this work.
Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?
M.M.: I vividly remember the day I first learned about the research in Professor Nakamura's lab and e-mailed him. He guided me on experimental techniques, advised me on my research, and taught me how to tackle challenging problems.
What's next for you? (If you are planning on leaving academia, please tell us why!)
S.K.: I am deeply committed to continuing my career in academia. I hope to further investigate the molecular mechanisms underlying various events in the axon regeneration process, with a particular focus on TC10.
Tell us something interesting about yourself that wouldn't be on your CV
S.K.: As you know, Japan is home to many creative and fantastic game companies, such as Nintendo and SONY. I regularly participate in online gaming parties with people I met at my former workplace. Playing new games is refreshing and fun, as it engages my brain in a different way than usual, and the social connection helps me maintain a balance in life.
Shingo Koinuma's and Misa Miyaji's contact details: Division of Biosignaling, Research Institute for Biomedical Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan.
E-mails: [email protected]; [email protected]
