Graduate School of Frontier Biosciences, Osaka University

Japanese

Laboratory of Single Molecule Biology

  Name Email TEL
Professor UEDA, Masahiro
Assistant Prof. MIYANAGA, Yukihiro
FAX +81-6-6879-4613
Postal Mail Address Laboratory of Single Molecule Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 Japan
QBiC labo HP http://www.qbic.riken.jp/csd/en/index.html

Cells are complex but well-organized systems comprising various kinds of biomolecules. Because biomolecules operate stochastically under the strong influence of thermal fluctuations, living cells can be referred to as stochastically-operating biomolecular computation systems. Through the dynamic processes in reaction networks of biomolecules, cells can respond flexibly and adaptively to environmental changes. Recent progress in single molecule imaging techniques has made it possible to monitor directly the stochastic behaviors of biomolecules in living cells, in which the locations, movements, turnovers, and complex formations of biomolecules can be detected quantitatively at the single molecule level, providing powerful tools to elucidate molecular mechanisms of intracellular signaling processes. Our laboratory develops quantitative single-molecule imaging methods, computational modeling methods and biochemical synthetic methods to reveal the molecular mechanisms of cellular chemotaxis with single-molecule resolution.

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1. Development of automated in-cell single-molecule imaging system (AiSIS)

Single-molecule imaging, which measures the spatiotemporal dynamics of individual molecules and also their statistical distribution, is a popular method for investigating the molecular mechanisms of biological phenomena. However, the technical expertise required for single molecule imaging prevents the efficient acquisition of massive amounts of data. We have overcome this issue by developing an automated single-molecule imaging system using artificial intelligence. Our developed technology can be applied to genome-wide and drug screening to discover novel and physiologically active substances.

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2. Single-molecule biology of chemotactic signaling system

Living cells can sense and respond to environmental signals through dynamic signaling processes in the reaction networks of biomolecules. Because biomolecules operate stochastically under the strong influence of thermal fluctuations, living cells can be referred to as stochastically-operating biomolecular computation systems. Recent progress in single molecule detection techniques has identified the stochastic nature of biomolecules in vitro and in living cells. On-off fluctuations in individual molecules inevitably cause the number fluctuations in the ensemble of the molecules, and thus making intracellular signaling processes inherently noisy. This leads to a fundamental question on intracellular signaling processes in general: how does the signaling system operate reliably under thermal and stochastic fluctuations? To gain insights into how signals are received, processed and transduced by stochastically-operating molecules, we study the chemotactic signaling system of eukaryotic cells as a typical example of a stochastic computation system.

Chemotaxis is a fascinating phenomenon in which cells sense chemical gradients and move with directional preference toward or away from the source of the chemical cues. Eukaryotic cells can sense the differences in chemoattractant concentration across the cell body and respond by extending pseudopod directed up the chemical gradient. In Dictyostelium cells, extracellular cAMP functions as a chemoattractant. Only 2% gradients can induce a biased movement of the cells toward the source of cAMP in a wide range from 10 pM~10 μM. Ligand binding to the receptors is a stochastic process, so that receptor occupancy should fluctuate with time and space. The input signals for chemotaxis become noisy due to the fluctuations in ligand-binding to the receptors. Such fluctuations in signal inputs have been observed directly by single molecule imaging of the attractant bound to living Dictyostelium cells. Chemotactic signaling systems should amplify small changes in input signals. But by the same systems, small random changes (noise) in the input signals would be also amplified, resulting in the propagation of noise as well as signal. Thus, how chemotactic cells reliably obtain information regarding the gradient from such noisy inputs is a critical question for directional sensing in chemotaxis. To adderss this question, we apply single-molecule imaging analysis to chemotactic signaling of Dictyostelium cells.

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3. Single-molecule biology of ErbB signaling

ErbB membrane receptors, which consist of four families designated ErbB1 - ErbB4, transmit extra-cellular signals into the cytoplasm. Because each ErbB protein interacts with family-specific ligands and activates a receptor-specific signaling pathway, a cell reaction depends on the extracellular ligand-receptor combination. For example, ErbB1 which binds the epidermal growth factor (EGF) ligand is also called the EGF receptor (EGFR) and a principal receptor controlling cell proliferation. If the regulation of ErbB1 expression or receptor function is lost, extraordinary proliferation is induced that leads to serious diseases such as cancer.

Single-molecule imaging of ErbB proteins conjugated with a fluorescent probe enables a direct observation of the individual receptors on the plasma membrane. By analyzing motility or brightness of fluorescent spots in the acquired images, we obtain detailed information about ErbB including its molecular behaviors, interactions with ligands, and reactions with other signaling proteins. Elucidating the regulatory processes in cellular signaling based on this information, we aim to understand the mechanism of cellular reactions and initiation / progression of diseases.

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In addition, we are also studying the following research items.

4. Synthetic biology of chemotactic signaling system

5. Single-molecule imaging analysis in immune system

6. Imaging analysis of multicellular morphogenesis in the developmental process of the cellular slime mold Dictyostelium


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