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 - Institute for Frontier Medical Sciences
Nano Bioprocesses
Research Center for Nano Medical Engineering
Our laboratory is dedicated to methodology development for single-molecule observation and manipulation at nanometer precisions in living cells. The development is carried out simultaneously with the application for the studies of nano-bioprocesses occurring in living cells, in particular, signal transduction in the cell membrane and the formation and remodeling of the neuronal network. The smooth liaison between physics/engineering and biomedicine is a key for our methodology developments. On the basis of the knowledge of nano-bioprocesses learned in the cells (e.g., partitioning of the plasma membrane into submicron compartments and transient formation of signaling platforms in the cell membrane) and the single-molecule bionanotechnology developed here, we envisage the next-generation nanotechnology, regenerative medicine, and drug discovery protocols.

  Akihiro Kusumi, D. Sc.
Professor
Research and Education
The cell is surrounded by the cell membrane, and it is responsible for exchanging information, energy, and molecules with the outside world. Furthermore, 40% of cellular reactions take place in the membrane, and 30% of energy is stored in a form of the concentration gradient across the membrane. These functions are carried out by biomolecular nanosystems, but how exactly they are done is unknown. As described at the end of this part, these membranes have incredibly peculiar structures, and the cell appears to make the cell membrane work by taking advantage of the very peculiar structural features of the cell membrane.

We at the Kusumi laboratory are trying to understand the basic mechanisms for the function of the cell membrane at a very fundamental level. One of the most important features of our projects is to develop and apply single-molecule tracking/manipulation techniques (single-molecule nanobiotechnology) for the studies of living cells.

Our study is based on a hypothesis that “three fundamental physical properties of the cell membrane are essentially responsible for its function”. They are:
(1) its low (two) dimensionality greatly facilitates molecular collisions,
(2) molecular complexes and micro(nano)domains with various sizes and lifetimes continually form and disintegrate, and
(3) the cell membrane is partitioned into many small compartments due to the presence of the actin-based membrane skeleton fence and anchored transmembrane protein pickets aligned along the membrane skeleton.
In our laboratory, we hope to better define these three properties, and at the same time, to understand how these properties make possible the functions of the cell membrane, i.e., to reveal the “Membrane Mechanisms”.


Nano Bioprocesses
Research Center for Nano Medical Engineering
Institute for Frontier Medical Sciences
Professor Akihiro Kusumi
Associate
 Professor


Takahiro Fujiwara,
Kenichi Suzuki
TEL +81-75-751-4112
FAX +81-75-751-4113
e-mail akusumifrontier.kyoto-u.ac.jp
URL http://www.nanobio.frontier.kyoto-u.ac.jp
Figure 1. Left. Single-molecule tracking. A fluorescent or colloidal gold tag is attached to a membrane protein or a lipid molecule by way of a specific antibody’s Fab fragment or a ligand, and their movement in the cell membrane is visualized. Right. Using an optical trap (laser tweezers), a membrane molecule tagged by a gold particle is moved at will along the cell membrane, by moving the laser tweezers that trap the gold particle. The force exerted on this single molecule from the membrane skeleton or microdomains is registered at every pixel.
Figure 2. Inactive signaling protein Ras, located on the cytoplasmic surface of the plasma membrane (green), undergoes regulated Brownian diffusion (yellow trajectories). By the method we developed, the activation of this single Ras molecule was imaged (green color changes to red, due to FRET, at the center of this image), which entails the first successful observation of the activation of a single molecule. Many other cytoplasmic molecules are recruited to this activated Ras molecule, inducing immobilization of this activated Ras signaling complex. Surprisingly, such a molecular complex disintegrates within a second, suggesting a possibility that the basic unit of the cellular signal occurs like a digital pulse. Such observations are only possible with single-molecule imaging, rather than observing the average behavior of many molecules.
Figure 3. A discovery, which necessitated a paradigm shift of the concept of the plasma membrane structure and function, has been made. The entire plasma membrane is partitioned into many small compartments of 30 – 200 nm (depending on the cell type) due to the actin-based membrane skeleton (fence, left) and various transmembrane proteins anchored to the membrane skeleton (pickets, right).
Recent Publications
1. N. Morone et al. Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. J. Cell Biol. 174, 851-62 (2006).
2. K. G. N. Suzuki et al. GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J. Cell Biol. 177, 717-730 (2007).
3. K. G. N. Suzuki et al. Dynamic recruitment of phospholipase Cγ at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2. J. Cell Biol. 177, 731-742 (2007).
4. K. A. K. Tanaka et al. Membrane molecules mobile even after chemical fixation. Nature Methods 7, 865-866 (2010).
5. R. S. Kasai et al. Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. J. Cell Biol. 192, 463-480 (2011).