Cellular Informatics Laboratory

Outline

Our aim is to understand principles of signal processing carried out by biological systems in proteins, protein networks, cells and cell communities. We are studying how bio-molecules assemble to process intra- and extra-cellular information and express flexible higher-order cellular responses by developing and applying techniques of single-molecule measurements, optical microscopy, cell engineering and reconstruction of biosignal systems, as well as mathematical analysis and computer simulations of reaction networks. Our recent main targets are intracellular protein reaction networks called RTK-Ras-MAPK systems. These systems are responsible for cell fate decisions, including cell proliferation, differentiation and apoptosis. We are studying the functions and dynamics of proteins involved in these systems. We are also analyzing how various dynamics of reaction systems emerge from the accumulations of elemental protein reactions.

Recent Research Topic

Single-molecule analysis of information processing in living cells

Fig. 1 Associations between extracellular ligands and ErbB

Upper: Single-molecule imaging of fluorescently labeled EGF or HRG on the surface of a living cell.

Lower: Associations between EGF and ErbB1 are accelerated by predimerization and allosteric formation of the association intermediate. Associations between HRG and ErbB3/4 take place basically in the same manner.

Decision-making in biological cells is carried out by an intracellular reaction network of proteins. To understand this process, quantitative measurements of intracellular reactions followed by theoretical and computational analysis are indispensable. We are analyzing intracellular reaction networks called RTK-Ras-MAPK systems, which are responsible for cell fate decision into proliferation, differentiation, apoptosis and even carcinogenesis. Quantitative single-molecule measurement in living cells in combination with mathematical analysis is our main technology. Recently, we have been studying the ErbB-Ras-MAPK system, a member of the RTK-Ras-MAPK system. By stimulating this system, EGF induces cell proliferation, but using the same system, HRG induces cell differentiation. The membrane receptor ErbB family consists of four members, of which EGF and HRG associate to ErbB1 and B3/4, respectively. We have measured association between EGF and HRG to their receptors that induces dimerization of receptors and found that for both receptor systems, predimerization and allosteric conformational change of receptors regulate dimerization of the ligand/receptor complex, which is required for cell signaling (Fig. 1). We have suggested that after ligand association, dynamic clustering of ErbB is important for signal amplification and propagation. Applying quantitative PALM (photoactivation localization microscopy) that allows super resolution imaging of fluorescent molecules, we analyzed the cluster size distribution of ErbB1 molecules on the cell surface (Fig. 2). At the same time, we measured lateral diffusion movements of ErbB1 clusters. These measurements provided basic information for the modeling of receptor dynamics, which is in progress. We also measured dynamics of various cytoplasmic proteins that recognize activation of ErbB receptors using FCS (fluorescence correlation spectroscopy) and FCCS (fluorescence cross correlation spectroscopy). We have detected differences in the regulation of protein dynamics after cell stimulation depending on the species of ligands (EGF or HRG) in cases of some cytoplasmic proteins (Fig. 3). By integrating the results of these measurements with the aid of computational biology, we expect to better understand the mechanism of cell signaling.

Fig. 2 Super localization imaging of ErbB clusters

Even though the spatial resolution of single-molecule images in conventional fluorescence microscopy (left) is less than 250 nm, the center of mass of each image can be determined with <20 nm accuracy (right). By determining the location of single molecules one-by-one, we can construct super resolution images of ErbB clusters on the cell surface.

Fig. 3 FCS measurements of PI3K

FCS is a technique to measure lateral diffusion of fluorescent molecules in living cells. In cells before stimulation (no ligand), PI3K-GFP diffuses rapidly in the cytoplasm. After stimulation of cells with EGF or HRG, a slow diffusion component representing the movements of PI3K complexed with ErbB's has been observed. The diffusion coefficients and percent fractions of the slow diffusion component were different for EGF and HRG.

Fig. 1

Reproduced, with permission, from Y. Teramura, et al. Single-molecule analysis of epidermal growth factor binding on the surface of living cells, EMBO J. 2006, 25, 4215. © (2012) Macmillan Publishers Ltd: The EMBO Journal