Department of Biological Sciences, Graduate School of Science
JAPANESE

Research

Why focus on a single molecule or a single cell?

The term “average” is ubiquitous in our daily conversations, referring to various parameters such as annual income, temperature, stock price, height, sleep duration, and the list goes on. However, have you ever pondered why we rely so heavily on this term? The reason is that it provides a representative value that reflects the group in question, making it easier to discuss and understand. Nevertheless, this principle does not always apply universally, and I would like to provide two instances where it does not hold.

  • Disparity in average savings balance and life expectancy exist

For instance, on May 18, 2016, Japanese Ministry of Internal Affairs and Communications (MIC) released data stating an average savings balance of 18.5 million yen per household with two or more persons. However, this average is skewed by the top 10% of households with balances of over 40 million yen, while the remaining two-thirds of households fall below this average. Therefore, the average does not accurately represent the savings of most households, as there are two distinct groups: a small wealthy group and a larger group with lower savings.

Similarly, there is a discrepancy in average life expectancy across different regions and socioeconomic groups, which shows that the average does not necessarily reflect the experience of individuals in those groups.

  Moving on to life expectancy, as of 2014, the average life expectancy in Japan was 86.8 years for women and 80.5 years for men, marking an increase of approximately 30 years since the end of World War II. Interestingly, in 1878, the average life expectancy in Japan was just 38 years for women and 36 years for men. However, the same issue arises in this case as well.

According to some data, during the Edo period, deaths of infants aged 0-5 years accounted for 70-75% of all deaths. Nonetheless, it is known that a significant number of people in this period exceeded the average life expectancy. Thus, the fact that a large number of infant deaths was bringing down the average life expectancy was concealed. Therefore, the average life expectancy does not represent the population accurately. The problem lies in the fact that it is impossible to understand the distribution of the population by solely focusing on the average.

      The next crucial point to consider is the time fluctuation factor. The two average examples mentioned above do not account for changes over time. For instance, the average savings balance reflects data from 2016, and the average life expectancy is limited to a specific time frame. In other words, they depict a population distribution at a single moment in time (or a limited period). However, in reality, population distributions are subject to fluctuations over time.
      When understanding a particular population, two crucial factors to consider are:
  • Ⅰ.The value that represents the population, which is not always the average value.
  • Ⅱ.Populations are subject to dynamic changes due to time fluctuations.
      I would now like to broaden our discussion to the realm of life sciences.
      Life is a hierarchical structure, where an individual is made up of organs, which are comprised of cells, that in turn are composed of molecules, and finally, atoms. In essence, understanding life precisely requires understanding it in terms of these groups.
      If the aforementioned principles were not true of life, then the consequences could be significant. For example, without cells or molecules that deviate from the norm, there would be no cancer cells or evolutionary adaptations in response to the environment.
    Furthermore, without dynamic fluctuations in a population, it would not be considered a living system and would be akin to a static, lifeless state. Therefore, comprehending life accurately from the perspective of single cells and molecules, as per points I and II, is crucial, and is widely accepted at the forefront of contemporary life sciences.
      But how can we measure life at the single-cell/single-molecule level? This is not an easy task since cells and molecules are generally too small to be seen with the naked eye. Nonetheless, humans have developed various techniques to make them visible. For example, telescopes are used to observe celestial bodies, while microscopes can be used to view smaller objects, including single cells and molecules.
    However, visualizing a single cell or molecule with a microscope does not guarantee a full understanding of it. For instance, even if we could visualize a novel type of coronavirus, it would be impossible to differentiate it from a mutated or conventional type without analyzing its genetic sequence.
      Our aim is to precisely understand the nature of cellular and molecular mechanisms using various single-cell and single-molecule measurement techniques.
    The individuality of cells and molecules is a crucial characteristic, much like in human society where a diverse group is healthier than a uniform group lacking individuality. This is true at the cellular level as well.

Our objective is to uncover novel biological mechanisms through the utilization of our three original technologies, which are centered on the key concepts of single molecules and single cells.

Development of a method to explore and create novel functional molecules by the droplet technologies

Traditionally, liquid handling using micropipettes is restricted to the microliter (µL) scale, however, droplet handling allows for manipulation at the picoliter (pL) scale. By injecting an aqueous flow into an oil flow within a microfluidic channel, micrometer-sized droplets can be quickly and consistently generated (as depicted in the figure below). We are utilizing these droplets as individual test tubes to develop techniques for single molecule/single cell analysis and the discovery and synthesis of new functional molecules.

Development of unlabeled single-molecule measurement using novel nanopore technologies

Nanopore technology is a single-molecule measurement method that employs nano-scale pores to gather information about molecular structure. The primary advantage of this technology is the ability to obtain in-depth structural information, such as nucleotide information, of individual molecules without the use of labels. Our laboratory is focused on advancing this technology to better understand molecular functions and bring it to practical applications such as medical diagnostics.

Expansion of biological applications using ZMW

ZMW, or Zero-Mode Waveguide, is a cutting-edge technique that allows us to visualize single molecules at physiologically relevant concentrations of fluorescent dye, something that cannot be achieved through conventional total internal reflection fluorescence (TIRF) methods due to high background noise. Pacific Biosciences has developed a next-generation sequencing (NGS) system, known as “PacBio RS,” utilizing ZMW technology. PacBio RS is now readily available globally. A reference YouTube video.

Our group was the first to successfully utilize ZMW to observe the real-time transit of fluorescently labeled tRNAs to the ribosome during protein translation at codon-level resolution and at physiologically relevant concentrations. Through this, we were able to capture the process of tRNA incorporation into the ribosome during translation, as well as its subsequent dissociation. Additionally, we uncovered a heterogeneous pathway in which the translation initiation complex is formed, contradicting the previously accepted, simplistic single pathway outlined in traditional textbooks.

  • S. Uemura, et al., Real time tRNA transit on single translating ribosomes at codon resolution.
    Nature, 464, 1012-1017 (2010)
  • A. Tsai, A. Petrov, R. A. Marshall, J. Korlach, S. Uemura*, and J. D. Puglisi*,
    Heterogeneous pathways and timing of factor departure during translation initiation
    Nature, 487, 390-393 (2012) *corresponding author