Columns that reveal the world
- Getting up close and personal with the researchers -

This happened when I was still a student doing research at the undergraduate and graduate level. At the time, I was researching cellular slime molds, an amoeba cell, and my mentor, Professor Yasuo Maeda, always encouraged me by saying, "Create your own unique style from scratch," and "Think of something that no one else has done before." Of course, as a student, I couldn't do such groundbreaking research, but since I've always been the type of person who thought, "I don't want to do the same thing as other people," I was inspired and worked hard at my research. However, it was a series of hardships...I experienced the pain of giving birth. However, I feel that the hardships and experiences I experienced at that time are the foundation of who I am today.

In my third year of doctoral studies, I attended the Japan Society for Developmental Biology held in Fukuoka. On the way back on the bullet train, I was staring blankly at the scenery outside while thinking about my future. It suddenly occurred to me that deep-sea organisms are full of mysteries and I would be able to do something different from other people, so I thought I should give it a try. I returned to Sendai, where my graduate school was located, and immediately looked into it. I found out that there was a research institute called the Japan Marine Science and Technology Center (JAMSTEC/currently the Japan Agency for Marine-Earth Science and Technology), and when I called them, they happened to be recruiting several postdoctoral researchers. I was fortunate to have a connection with them, and in 1994 I was able to take my first steps as a researcher.

The leader of the DEEPSTAR group I belonged to at JAMSTEC was Professor Hiroki Horikoshi, a renowned microbiologist and world authority on alkaliphilic bacteria. However, my first work order was, "Try something interesting." I didn't really understand, but I had to think of something. At the time, microorganisms (called hyperthermophilic bacteria) that live in hot water wells on the deep sea floor were a hot topic around the world, and the field was booming. However, I thought again, "It's not interesting to follow what everyone else is doing," and "In that case, I'll try researching 'microorganisms that have adapted to high water pressure,' which no one else seems to be doing." However, since almost no one is doing it, there are very few examples to refer to. I didn't know how to proceed with the research, or even what kind of equipment I should use. Everything was a trial and error process, but after much trial and error with the manufacturer who developed the equipment, I was finally able to start my research.

However, a harsh reality awaited me. I have an extremely bad tendency to get seasick on ships. I had come here longing for the deep sea, but I couldn't get on a ship. I had dived to the bottom several thousand meters deep on the manned research submersibles "Shinkai 2000" and "Shinkai 6500" once each, but I couldn't do anything in the rocking laboratory on the mother ship. So after four years of research on board, I gave up and came ashore.

However, I wanted to do research related to high water pressure. I thought of "yeast". Yeast has been found in the deep sea, but I chose the common yeast that makes bread and alcohol. Yeast has been used in basic research for a long time, so there is a lot of genetic information, and whole genome analysis was also progressing at that time. A huge amount of biochemical knowledge has also been accumulated. So, rather than using a completely unknown organism as a research subject, I decided to use a common yeast with a known origin, and see how it would respond when the factor of "pressure" was added to it. It was about 4-5 years after I started working on it that I first noticed a "sign from yeast" that made me think "Oh?"

A differential interference contrast microscope image (left) and a transmission electron microscope image (right; taken at the Instrumental Analysis Center, Faculty College of Science and Engineering) of the budding yeast Saccharomyces cerevisiae. The diameter is approximately 5 μm.

Yeast bacteria stop growing when they sense high water pressure of hundreds of atmospheres (corresponding to a depth of several thousand meters). After pondering why this was, I thought that there might be a problem with the intake of nutrients, so I gave the culture medium an excess of sugar and amino acids and examined their growth. I found that only when a large amount of a single amino acid, tryptophan, was given, yeast bacteria grew well under a high pressure of 250 atmospheres, like the deep sea. This is the world of ultra-high pressure at a depth of 2,500 m. At first, I couldn't believe that such a thing could happen with just one amino acid. However, after further investigation, I found that the cell membrane transporter "Tat2", which is the tryptophan intake port, is very sensitive to pressure, and broken Tat2 is decomposed and removed by a mechanism called "ubiquitination". The large amount of tryptophan added from outside made up for the shortage. I submitted a paper on this, which was very well received, and the figure was used on the cover of the American journal Molecular and Cellular Biology. I still remember how happy I was.

Frankly speaking, I feel that it is thanks to the results I gained during that time that I am now able to have my own lab at this university and conduct research. I owe it all to my former professors and the leaders of my time at JAMSTEC, who allowed me to do everything the way I wanted.

Structural model of the ubiquitin ligase Rsp5, featured on the cover of the journal

I moved my research base to this university in 2010. Since then, I have been investigating various intracellular phenomena that occur under high pressure from a genetic perspective. Pressure is stress in English. Normally, it would be better to have no pressure, but deep-sea creatures cannot live in shallow waters. They like pressure. Astronauts staying on the space station and we who live on Earth also lose their bones and muscles if they do not exercise. In other words, living things should have a mechanism to sense "physical forces" such as gravity and pressure. How do living things recognize such invisible forces? I continue my research to find out the mechanism.

Yeast is one of the microorganisms whose entire genome sequence was revealed early on. However, of the more than 6,000 genes, there are nearly 700 whose functions are still unknown. The experiment we conducted was very mundane, and involved preparing about 5,000 strains of yeast with each gene deleted, placing them in a pressure vessel, and cultivating them under various conditions such as temperature and time. It is a very simple and painstaking task. It is difficult to say how long it will take, but in this experiment it took a year to find possible targets through screening, another year to narrow down the focus to a group of characteristic genes, and another year to conduct further experiments and compile the results as a paper. We found 84 interesting genes, and currently we are focusing on about 10 genes whose functions are almost unknown, and are conducting research with students. Of course, it is possible that we could have spent three years without any results, but no one would have known that until we tried. There is no guarantee of results.

A high-pressure vessel for culturing microorganisms under ultra-high pressures up to 1000 atmospheres. It was made with the cooperation of the Faculty College of Science and Engineering Workshop.

Genes are the blueprints for proteins. If genes are unknown, then the function of the resulting proteins is also unknown. If we compare "cells" to society, then humans are "proteins," and proteins are the executive forces that make cells work. Imagine that in the area where we live, there are dozens of mysterious people whose faces, names, and appearances are unknown. However, when a major disaster occurs and the city is destroyed, turning into an isolated island, those people suddenly appear and build a logistics network to save people. Such mysterious rescue team-like proteins are coded in the 10 or so unknown genes I mentioned earlier. The analogy to a major disaster is "high water pressure."

The "unknown genes" mentioned here mean that they certainly exist, but nothing came out of the investigation. They are a group of genes that have been reported in the past as causing no problems for yeast if they were not present. However, we discovered that they are essential for survival under high water pressure. It is believed that primitive life was born in extreme environments such as the deep sea, where hot water rises up under high water pressure. It is very mysterious that baker's yeast has "genes for high pressure adaptation" that such primitive life may have created. However, it was difficult from there. We made various inferences, put together experimental methods, investigated everything one by one, considered the results, inferred what was happening inside the cells, reviewed and carried out the experimental procedures, considered them again... and repeated this process, and we are still continuing the experiments endlessly.

Gene knockout library used to screen for high-pressure adaptable genes. Approximately 5,000 yeast strains are stored on 50 96-well plates.

I'll give you a few examples of unknown genes. One of the genes with unknown function that was found encoded a huge protein that binds to an organelle called the endoplasmic reticulum. If this gene is deleted, yeast cannot grow at high pressure or low temperature. Interestingly, lipid masses called lipid granules accumulated in the cells of the deleted strain in vast quantities beyond imagination. It seems that this protein is important for controlling the amount and distribution of lipids in yeast cells. It probably acts as a "tunnel" that transports lipids synthesized in the endoplasmic reticulum to each organelle. However, it is still a mystery why this is particularly important under high pressure and low temperature.

However, what is interesting is that we humans also have a protein in our cells that is very similar to this giant yeast protein. Of course, the function of this gene is unknown in humans, but children born with a mutation in this gene have been confirmed to have symptoms such as congenital brain malformations, heart abnormalities, and joint dysplasia. Furthermore, lipid-related diseases also include lifestyle-related diseases in modern society such as hyperlipidemia. In other words, if we can investigate the yeast gene we discovered more deeply and elucidate its role, it may provide a clue to the treatment and drug development of human diseases.

Another example is the protein complex TORC1, which has been studied very extensively in both yeast and humans. TORC1 is known as an intracellular nutrient sensor, and in humans it plays an important role in being activated by growth factors and insulin, and in detecting intracellular energy (ATP) levels. Abnormal activation of mTORC1 has also been confirmed in many cancers, including malignant brain tumors, and genetic diseases (congenital anomalies) of related proteins are known to cause various diseases, such as cerebral cortical abnormalities, epilepsy, autism, and tuberous sclerosis. Yeast TORC1 is bound to the surface of an organelle called the "vacuole." We found that if TORC1 or any of the components of the EGO complex that anchors it to the vacuole are missing, yeast cannot survive under high pressure. I noticed this and first presented it in a paper in 2008, before I was transferred to our university. Then, in 2011, the year after I came to our university, we began to elucidate the mechanism. However, this did not always go as expected, and meanwhile, the functions of TORC1 were being discovered one after another around the world in areas unrelated to high pressure, and there was a time when our research went unsolved.

Confocal laser scanning microscope for observing minute structures within cells at high resolution

The light finally shone after a long tunnel when we discovered that lack of TORC1 causes abnormal accumulation of an amino acid called "glutamine" in cells. Under high pressure, an imbalance occurs in the concentration of glutamine in cells, and survival is only possible when TORC1 restores this imbalance. It felt like the fog in front of my eyes suddenly cleared. This result was published in the British scientific journal "Journal of Cell Science" and selected as the "Research Highlight" by the editors' choice. Since pressure is also applied to the knees and hip joints during exercise, the main point of the evaluation was that "this research may be useful in improving "locomotor syndrome" (a condition in which mobility is reduced due to disorders of the musculoskeletal system) in an aging society." The research was supported by the education and research funds of our university's "AOYAMA VISION." Our project was adopted, and with that budget we were able to introduce a high-performance device called a confocal laser microscope. Using this, we were able to clearly observe structures inside cells that were previously invisible, which greatly advanced our research. AOYAMA VISION's budget is funded by valuable donations, so we are truly grateful to the many people who have donated.

Yeast belongs to the same eukaryotic organisms as humans, and the structure of its cells is very similar to that of humans. As a small microorganism, everything is compactly organized, making it easy to analyze. It is also said that about half of the genes found in yeast have similar counterparts in humans. For these reasons, various findings obtained from yeast are being applied to medical research. We hope that by observing what happens inside cells under high pressure and elucidating the mechanisms behind them, we can contribute to the treatment of diseases.

Research is empirical science. It is not enough to simply get the suggestion that "this gene seems to have some important function." The results have value only when we identify the actual operating principle and elucidate the mechanism. What changes, why, and how does that function occur? In order to propose a consistent model that everyone can agree with, we continue to conduct repeated experiments and continue to analyze and consider.

This article was selected as a "Research Highlight" by the Journal of Cell Science. The green fluorescence indicates the intracellular localization of TORC1.

Research is a repetition of mundane tasks such as conducting experiments and accumulating a lot of evidence. It is not something that will be immediately useful. However, in the process, we often have happy experiences. It goes back to when I was in my early 30s and gave a presentation at a conference in New York. At the time, I was researching transporters that transport amino acids into cells. I investigated the activity of the transporters under various pressure conditions and finally proposed a model in which "differences in the micro-regions (called lipid domains) of the cell membrane in which these transporters exist determine the transport properties." I still remember the roar of the audience at that time, as if to say, "Wow, that's a good idea!?" My English wasn't great, but perhaps there was something that resonated with the audience.

When we submit a paper to a major overseas journal, the reviewers also see the enormous amount of work that goes into the paper that is not reflected in the text. Three or even six months of repeated experiments may have gone into a single line of text. They can see that. In the course of our daily research, one day, we suddenly have a moment of inspiration that leads to that "one line." Our thinking suddenly expands, as if the fog has cleared, and we can see into the future. I think that moment comes because we have been gradually building up the things that need to be done.

Recently, retired professional tennis player Roger Federer said at a press conference, "I just enjoyed playing tennis, and before I knew it, I had come this far." Of course, the scale is different, but I feel similar. I guess it's like, "I liked doing things differently from other people, so I continued doing research, and before I knew it, I was here." When it comes to research results, impactful papers may look flashy in their output, but what you actually do is mundane and time-consuming. If you don't like it, you can't continue. In April of this year, the "Life Science Research Center" was newly established in College of Science and Engineering at our university, and I am serving as the first director of the center. Unlike national universities, our Faculty of Science College of Science and Engineering is small. That's why we are full of the spirit to work on originality and things that stand out, rather than the highly competitive fields where researchers from all over the world are competing. We also have an environment that supports this. I think it's a place where you can settle down and continue your steady research activities.

Preparation of reagents before PCR reaction

There are currently 20 students in my lab, including fourth-year undergraduates and graduate students who are doing their graduation research. What is important for them is to challenge difficult problems with curiosity, think for themselves, and take action. They plan their own experiments and try them, and if they don't work, they think about the cause and try again. If they do work, they think about what to do next. This training, which imprints the mind and body in tandem, overlaps with the daily training of athletes. Most athletes have a manager or coach. My role is similar to that. If there are 20 people, there are 20 different personalities. It is very difficult to guide them while identifying them, and I ask myself questions every day. The word "guidance" means that a teacher "instructs" students in a one-way manner. However, when I actually conduct research, there are many things that I did not know about the things that the students have researched and thought about, and I often learn from them. In that sense, I feel that "co-creation" with students is the source that makes research better and pushes it to a higher level.

Finding 1 from 0 is one of the major tasks that we basic researchers must accomplish. It is also important to turn that 1 into 99, or 99 into 100, but sometimes that can be done by someone else. It seems to me now that the "pain of birth" I experienced as a student was creating 1 from 0. What's important is to remain curious and to be able to immerse yourself in your research. I sincerely hope that many young people who aspire to be researchers will immerse themselves in research themes that only they can do, and send out original results to the world.

Discussion with students