First person – Makoto Nagano

Makoto Nagano

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

The endosome is a central membrane trafficking hub that sorts endocytic material to lysosomes for degradation or recycles them to the plasma membrane for reuse. This organelle plays a crucial role in multiple physiological processes, such as neurotransmission and hormone responses. Pathologically, endosomal trafficking is involved in cancer progression, viral infections and the entry of pathogenic bacteria into cells, making it an important target for disease therapies. Although endosomes are believed to be generated through the fusion of membrane vesicles, our knowledge about the molecular mechanisms that regulate endosome formation is still fragmented. The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying organelle dynamics and has also been utilized to investigate the molecular mechanisms of endosome formation and maturation. In this study, we aimed to clarify the molecular mechanism of endosome formation regulated at the trans-Golgi network (TGN), especially focusing on the functional relationships between clathrin adaptors at the TGN in the activation of Vps21p (the yeast Rab5 GTPase homolog). To do this, we used a novel biochemical method and live-cell imaging to detect Vps21p activity with high sensitivity and accuracy. We found a distinct role of several TGN-resident clathrin adaptors in the TGN–endosome trafficking pathway that mediate targeting, retention, and activation of Vps21p. Given that infectious viruses like SARS-CoV-2 enter the cytoplasm through an endosome-dependent trafficking process, this research could potentially lead to the development of new treatments for such diseases.

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

Small GTPases including Rab5, function as molecular switches by reversibly converting between GTP-bound active and GDP-bound inactive states in various cellular responses. In this study, we quantitatively analyzed the contributions of various TGN-resident proteins to Rab5 activation by comparing the levels of GTP-bound Rab5 in various yeast mutants. Because conventional methods for quantifying the active levels of small GTPase had several issues with accuracy, sensitivity or throughput, we needed a new method to address these issues. In this study, we successfully developed a new method to quantify intracellular active Rab5 levels with high sensitivity and accuracy using nanobodies. Dr Yohei Katoh and Prof. Kazuhisa Nakayama at Kyoto University helped us by sharing information and kindly providing us the plasmids for GFP-nanobody and ALFA-nanobody. If not for their help, the progression of our project would have been much more difficult. We really appreciate them!

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

At first, when observing the aberrant accumulation of GFP–Vps21p in a mutant yeast strain lacking ENT3, ENT5 and APL4 genes, I did not understand what was happening in the mutant cells. However, as I proceeded with the study, I found a significant reduction in the active state of Vps21p in the mutant strain. It was then that I realized the resemblance of this aberrant accumulation structure to that of GFP–Vps21p observed in the VPS9 mutant strain. This was such a ‘eureka’ moment for me.

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

JCS is a high-quality journal that covers a wide range of topics in cell biology, appealing to many readers with its extensive coverage. I myself am also a devoted reader of JCS.

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

I have met many researchers throughout my journey, and they have all been remarkable and passionate individuals, engaging in lively discussions about science. Among them, Dr Shuhei Ishikura, who was my first mentor, and Professors Jiro Toshima and Junko Y. Toshima, who are my current collaborators, have had a significant impact on me. Dr Ishikura's dedication to his research and logical thinking have become the base of my identity as a scientist. Professors J. Toshima and JY. Toshima are brilliant researchers filled with ideas and passion, and I draw much inspiration from them. I am grateful for these encounters.

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?

I enjoy designing new experimental setups or strategies. When the experimental setup I've created works well and leads to new discoveries, it brings me great pleasure.

What's next for you?

Currently, I am doing research with a focus on organelle dynamics and membrane trafficking mechanisms during viral infections. Specifically, I am interested in the role of endosomes, which serve as the site of virus entry into cells and play a key role in the release of infectious virus particles. The mechanisms of endosome formation that we have found in budding yeast intrigue me, and I am also interested in understanding their roles in mammalian cells. Therefore, I aim to further explore organelle dynamics using both budding yeast and mammalian cells to gain a deeper understanding of these processes.

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

I love hot springs and often go on hot spring trips with my family. I also enjoy bathing with bath salts at home and relaxing. Sometimes, I soak for as long as two hours.