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. Tadayuki Komori is first author on ‘ Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2’, published in JCS. Tadayuki conducted the research described in this article while a PhD student in Takeshi Iwatsubo's lab at University of Tokyo, Japan. He is now a postdoc in the lab of Mitsunori Fukuda at Tohoku University, Sendai, Japan, investigating Rab GTPases and membrane trafficking in general.

Tadayuki Komori

How would you explain the main findings of your paper in lay terms?

Lysosomes are the centers of digestion for intracellular components. Stressing lysosomes causes swelling, and ultimately failures in important cell functions. Cells have mechanisms to cope with this stress. The protein I investigated, Rab29, is known to move to the surface of lysosomes to regulate their size, but how this happens was not clear. In this report, we found that phosphorylation, the addition of a negatively charged phosphate group, of Rab29 happens when lysosomes are stressed, and this phosphorylation makes Rab29 stay on the surface of stressed lysosomes, ultimately decreasing their size. We also found the putative kinase enzyme that catalyzes this phosphorylation.

Were there any specific challenges associated with this project? If so, how did you overcome them?

The main cell types used in this study are RAW264.7 and HEK293, which are mouse macrophage and human embryonic kidney cell lines, respectively. The phenomenon we found, chloroquine-induced enlargement of lysosomes, was so prominent in RAW cells compared to HEK cells that we tried to do all the experiments with RAW cells. Unfortunately, the RAW cells we had were unexpectedly resistant to DNA transfection. I spent a whole year just trying to transfect these cells, using various reagents and viruses, and the only method I found to accomplish GFP expression was using lentivirus. I gave up on RAW cells after a year of practically achieving nothing and switched to transfection of HEK cells, which was far easier. I was lucky that HEK cells were responsive enough so that I could observe changes in lysosomes elicited by chloroquine.

Another challenge in this project was my unusual career path. I became a PhD student through the University of Tokyo's Ph.D.-M.D. program, in which you could enter a PhD course after the 4th year of the total six years of medical school, postponing the last 2 years of medical school until completing the PhD. Owing to this, combined with the effects of COVID-19, I had to complete my research project during my medical training. Even though I managed to work through both, it was surely not the best choice to sacrifice all my free time.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

The best moment in this project was when the Phos-tag SDS-PAGE for the alanine mutants of putative phosphorylation sites showed a clear and major decrease in the phosphorylation of Rab29. When I obtained this result, I had a gut feeling that this was worth digging into.

The ‘eureka’ moment in this project was when I dug out a long-forgotten preliminary result lying around our lab that showed PMA-induced phosphorylation in Rab29. As seen in Table S1 of the report, I had trouble identifying the kinase responsible for the phosphorylation at S185. One day I was looking through some of the progress reports of previous lab members and the letters Rab7L1 (our lab's old name for Rab29), PKC and Phos-tag jumped into my eyes. This particular result was obtained just before a report of LRRK2-mediated phosphorylation of Rabs came out, and our objectives turned towards LRRK2, leaving this PKC-phosphorylation untouched and forgotten.

Why did you choose Journal of Cell Science for your paper?

Journal of Cell Science publishes specialized and scientifically interesting articles. Some that I personally really like are Balderhaar et al., 2010 and Yasuda et al., 2016. Additionally, I have always liked to read (or to gaze at) the epic series of ‘Cell Science at a Glance’ posters. These aspects of the Journal of Cell Science helped us decide to submit to this journal.

Rab29 S185 phosphomimetics cope well with lysosomal stress. Chloroquine (CQ) causes lysosomal stress, swollen lysosomes (lysosomal marker: LAMP1) and Rab29 localization to lysosomes in normal cells. Cells expressing Rab29 that mimic lysosomal stress-induced phosphorylation (S185D or S185E) have smaller lysosomes upon lysosomal stress induction.

Rab29 S185 phosphomimetics cope well with lysosomal stress. Chloroquine (CQ) causes lysosomal stress, swollen lysosomes (lysosomal marker: LAMP1) and Rab29 localization to lysosomes in normal cells. Cells expressing Rab29 that mimic lysosomal stress-induced phosphorylation (S185D or S185E) have smaller lysosomes upon lysosomal stress induction.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

The co-first author, lecturer Dr T. Kuwahara, was especially compassionate towards Master and PhD students, resulting in an effective increase in their motivations. He has the magic ability to encourage and empower everyone he talks with, and I couldn't have finished my PhD or completed this project without him.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

My motivations are broad interests in molecular biology that originated in high school biology classes, combined with a personal grudge against neurodegenerative disorders (specifically MSA), which took my grandfather's and my father's lives.

A very important event for me in choosing to go to academia was a brief internship at Professor Christian Ungermann's laboratory at Osnabrück University. There I took part in research on endosomal Rab proteins, and through discussions and lab meetings, greatly opened my interests in membrane trafficking.

Who are your role models in science? Why?

Although I don't know him personally, I would say Dr Rei Narikawa, a PI at the Tokyo Metropolitan University working on cyanobacteria and photobiology. He shares his thoughts as well as his enthusiasm for his work (for example, videos of protein solutions changing color) and also some of his funding application documents on the internet, which I think is a really good way to reach out to the public and at the same time inform or educate the next generation of scientists.

What's next for you?

My interests expanded from Rab29 to Rab proteins in general, and now I am a postdoc in one of the leading laboratories in Rab GTPase research. I will be looking for a permanent position that would fulfill my interests, anywhere in the world, after finishing this postdoc.

Tell us something interesting about yourself that wouldn't be on your CV

I am a really big fan of logic puzzles like sudoku, not only solving them but also making them on my own. I sometimes host online puzzle solving competitions. I love elegant techniques and logic; using them to both solve and make puzzles are joyous moments of my life.

Tadayuki Komori's contact details: Tohoku University, Building H02, Room 915, Aoba 6-3, Aoba-ku, Sendai, Miyagi 980-8578, Japan.

E-mail: tadayuki74659576@gmail.com

Komori
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T.
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Kuwahara
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Fujimoto
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T.
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Sakurai
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M.
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Koyama-Honda
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Fukuda
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Iwatsubo
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2023
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Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2
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J. Cell Sci
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136
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jcs261003
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