Department of Biological Sciences, Graduate School of Science
JAPANESE

Research

We aim to elucidate novel biological mechanisms by applying the following three original technologies, whose key words are single molecules and single cells.

Expansion of biological applications using ZMW

ZMW is an abbreviation for Zero-Mode Waveguide, a new technique which enables us to visualize single molecules at physiologically relevant concentrations of fluorescent dye. This is impossible to achieve with conventional total internal reflection fluorescence (TIRF) methods due to high background. Pacific Biosciences has succeeded in releasing a next generation sequencing (NGS) system named “PacBio RS” based on ZMW technology. PacBio RS is now commercially available worldwide. A reference YouTube video is below.

Using ZMW, our group was the first to successfully visualize the real-time transit of fluorescently labeled tRNAs to the ribosome during protein translation at codon-level resolution and at physiologically relevant concentrations. As a result, we were able to capture the incorporation of tRNA into the ribosome during translation and its subsequent dissociation. We also succeeded in discovering the heterogeneous pathway in which the translation initiation complex is formed, overturning the simple single pathway classically described in 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

We are further expanding this technology to target novel biological phenomena, not only translation but including “Genome editing”, “RNA silencing”, “Membrane proteins” and “Molecular motors”.
These projects are mainly sponsored by the JST CREST “Biodynamics” grant.

Development of an amplification-free single cell sequencing system

In order to study the characteristics of a single cell in detail, specially developed technologies are required. While understanding the genome and translated proteins offers a glimpse of a cell’s characteristics, it is thought that analysis of total RNA in a single cell, in addition to selected parts of the genome, is required to characterize and reflect the dynamic changes that occur. In doing so, rather than measuring a mixed population of cells, we can achieve true “single cell biology”. Special instrumentation is required to analyze single cells since it is not easy to handle them. To analyze whole RNA, we use next generation sequencing. Conventional methods require an inescapable amplification step during sample preparation to obtain the necessary levels of material for analysis. However, amplification brings about strong bias and sample loss. As much as possible, this should be eliminated to approach a real single cell measurement. We are taking advantage of the single molecule paradigm and developing amplification-free single cell sequencing in collaboration with the Preventive Medicine and Diagnosis Innovation Program at RIKEN.

Expansion of real-time single cell secretion imaging technologies

Life is maintained at the organism level and at the organ level by the skillful orchestration of cell to cell communication and regulation. Cells can communicate with surrounding cells and even over long distances mainly through soluble factors such as hormones or cytokines. Traditionally, these secreted factors have been purified from the supernatant of a large number of cultured cells and analyzed by gel electrophoresis or  immunoassays. However, it is impossible to understand when and how secretion occurs on a single cell level with this conventional approach. Y. Shirasaki et al have developed new technology to monitor single cell secretion dynamics on nanofabricated chips by merging conventional sandwich immunoassays with TIRF (Total Internal Reflection Fluorescence). Together with this advancement in real-time single cell secretion imaging, we are also upgrading pre-existing techniques such as live cell imaging and microfluidics to elucidate cell to cell communication and secretion mechanisms. We hope to apply these techniques to personalized medicine and development of drugs targeting allergies or inflammatory autoimmune diseases. This project is sponsored by the ImPACT (Impulsing Paradigm Change through Disruptive Technologies, Cabinet Office Japan) program.

Secretion of IL-1β by macrophages (blue fluorescence)
Despite all cells being macrophages, this image demonstrates the individualistic nature of protein secretion.
Y. Shirasaki et al, Scientific Reports 4, #: 4736 (2014)