First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Keiichiro Sakai is first author on ‘ Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system’, published in JCS. Keiichiro is a PhD student in the lab of Kazuhiro Aoki at the Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Aichi, Japan, investigating how phycocyanobilin, a linear tetrapyrrole, brightens near-infrared fluorescent proteins, including iRFP, as a chromophore more efficiently than biliverdin in fission yeast.

Keiichiro Sakai

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

Fluorescent proteins (FPs) are widely used for visualization of biological processes in living cells and tissues. A near-infrared fluorescent protein, iRFP, is known as a FP that emits the longest wavelength fluorescence among FPs. Unlike other conventional FPs, such as GFP, iRFP requires biliverdin (BV) as a chromophore, which is responsible for absorbing and emitting photons. To our surprise, we found that iRFP does not fluoresce in a model organism, the fission yeast Schizosaccharomyces pombe, due to the lack of its ability to produce BV. That is, fission yeast cannot make the ‘material’ for the iRFP fluorescence. Accidentally, we found that a similar compound phycocyanobilin (PCB) functions as a ‘better material’ for iRFP fluorescence than the BV. We introduced genes for PCB biosynthesis in fission yeast cells, resulting in brighter iRFP fluorescence than in cells synthesizing BV. Our study provides the potential application of PCB to near-infrared fluorescence imaging in a broader range of living organisms, as well as a near-infrared imaging platform for fission yeast.

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

Until my Master's degree program, I was at a different university (Tohoku University) and was involved in a different field of research on bacterial gene expression systems. In my PhD program, I started to work with fission yeast using cutting-edge microscopes and therefore had to acquire a lot of knowledge and experimental techniques, such as on yeast genetics, live-cell imaging, biochemistry, among others. Sometimes it was hard, but thanks to my lab members, especially the obliging help of Prof. Kazuhiro Aoki and Dr Yuhei Goto, I was able to complete this research project.

One of the specific challenges raised by reviewers during the revision was to estimate the percentage of holo-iRFP that binds to chromophores. To address this issue, I adopted fluorescence correlation spectroscopy (FCS). By properly preparing the samples and optimizing the observation conditions, I could finally obtain the percentage of holo-iRFP.

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

I have experienced two ‘eureka’ moments during a series of experiments. One was when we found that iRFP is not fluorescent in fission yeast. iRFP has been used in various organisms, such as mammalian cells, mice and budding yeast, so I totally did not expect that it could not be used in fission yeast. Another was when we found that PCB enhanced iRFP fluorescence more efficiently than BV in fission yeast. It was generally known that BV is the authentic chromophore for iRFP, and therefore I was surprised to find that PCB is a better chromophore for iRFP than BV.

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

Journal of Cell Science publishes research articles including new tools and resources, which are highly practical, well-characterized and widely used by researchers in cell biology. So, we decided to submit our manuscript to JCS, and we hope that our experimental tools and methods developed here will be known and used by a lot of cell biology researchers.

Multiplexed imaging of fission yeast expressing NLS-mTagBFP2-NLS (nucleus), Turquoise2-GL-ras1ΔN200 (plasma membrane), Mis12-mNeonGreen (kinetochore), mCherry-Atb2 (tubulin), and Lifeact-iRFP (F-actin).

Multiplexed imaging of fission yeast expressing NLS-mTagBFP2-NLS (nucleus), Turquoise2-GL-ras1ΔN200 (plasma membrane), Mis12-mNeonGreen (kinetochore), mCherry-Atb2 (tubulin), and Lifeact-iRFP (F-actin).

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

In my lab, I have been fortunate to have kind mentors and focused on my research freely. Prof. Kazuhiro Aoki has keen insight and gave me good advice, which helped me move my research in the right direction. Dr Yuhei Goto always discussed the results of experiments and experimental ideas with me, and I learned how to logically consider scientific data, helping me get closer to becoming an independent scientist. Dr Yohei Kondo revealed to me the interest and importance of scientific discussion, and the delicious tastes of beers worldwide.

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 want to pursue a career in science as I am fascinated by the clever survival strategies of microorganisms. Most unicellular organisms show an excellent ability to adapt to even the harshest environments where there is no nutrition at all. It is an exciting moment for me to imagine how they live in a natural environment and how I can understand their adaptation strategies through experiments using a microscope. Although this paper focuses on a fluorescent protein, iRFP, I originally discovered the main findings of this work while studying fission yeast spore formation, which is a typical survival strategy. I hope to study the life cycle of microorganisms using the methods described in this paper and others.

What's next for you?

Currently, I am a PhD student and will be enrolled for another one and a half years. I am planning a new research topic about the germination of fission yeast spores in relation to the cytoplasmic physical properties. After graduation, I would like to stay in academia and further enjoy studying microorganisms.

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

I love drinking beer and running. When I'm not in the laboratory, I refresh myself with these hobbies.

Keiichiro Sakai's contact details: Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.

E-mail: k-sakai@nibb.ac.jp

Sakai
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Kondo
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Fujioka
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Kamiya
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2021
). Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system.
J. Cell Sci.
134
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