Research

Molecules in cells do not work in synchronization (work in essentially heterogenous!), but do randomly.
The interaction periods of individual molecules are often less than a second. In addition, the fraction of interacting molecules is frequently less than 10%. If we measure averaged behavior of many molecules in cells, we easily miss such interaction, and it is very hard to unravel molecular mechanisms in cells.
Therefore, we try to unravel the molecular mechanisms in cells by observing proteins and lipids at the level of single molecules (sometimes, at 10,000 frames/sec which is world’s fastest rate!), and by evaluating periods and frequencies of individual events.
Then, we perform statistical analysis by observing many events at the level of single molecules. (Of course, the results obtained by single-molecule imaging should be consistent with those by observation of multiple molecules.)

1. Unraveling of cell membrane structure, especially lipid raft structure and signal transduction

Lipid rafts have been drawing extensive attention as a signaling platform in cell membranes for about 30 years (Simons and Ikonen, Nature 1997). However, since rafts are too small to be observed by light microscopy, and molecules enter and go out of rafts very rapidly, rafts are still enigmatic. In early studies, nonionic mild detergents were used to extract “raft fraction”, or immunostaining methods were employed to detect rafts in cell membranes. However, it turned out that both methods tend to induce artifacts (Tanaka and Suzuki, Nature Methods, 2011). Therefore, to reduce the perturbation as small as possible, we observe the dynamic behavior of single molecules of representative raft molecules, GPI-anchored proteins (CD59, DAF, etc.) in living cell plasma membranes. We found that GPI-anchored proteins transiently form homodimers via protein-protein interactions, and them the homodimers are stabilized by cooperative raft-lipid interactions (Fig. 1, left). In other words, protein-protein interactions enhance raft-lipid interactions. We propose that the homodimers are one of the basic units to form greater rafts. At present, we are examining this model.

Furthermore, we are investigating how GPI-anchored proteins which lack transmembrane domain interact with signaling molecules in inner leaflet of cell membranes

2. Unraveling of dynamic behaviors of glycans in cell membranes

Glycans play very important roles in a variety of biological events. However, due to difficulty of synthesizing fluorescent probes, dynamic behaviors of glycans in living cell membranes have hardly been investigated. We tried to solve the issue and elucidate how glycans work in living cell membrane. Among glycans, we focus on “ganglioside” which is one of glycosphingolipid because it is a representative raft marker.

First, in collaboration with a group of Prof. Ando and Emeritus Prof. Kiso (G-CHAIN/Gifu University) we have recently developed four new fluorescent ganglioside probes (GM1, GM2, GM3, GD1b, Fig. 2) by using an entirely chemical method. These ganglioside probes act similarly to their parental molecules in terms of raft partitioning and binding affinity.

Since there have been no ganglioside fluorescent probes so far, dynamic behaviors of gangliosides have hardly been investigated in living cell plasma membranes. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave very tiny rafts at steady-state cells. Meanwhile, gangliosides also enter and leave rafts featuring CD59, a GPI-anchored protein. As the cluster size of CD59 increase (monomer→dimer→tetramer), the residency time of ganglioside probes in CD59 cluster was elongated (Fig. 3). In other words, we found that GPI-anchored proteins recruit other raftophilic lipids upon clustering, and stabilize the cluster rafts. This is also endorsed by another collaborative work with Prof. Murata (Osaka Univ.) and Prof Matsumori (Kyushu Univ.) in which new sphingomyelin probe we developed was used (Kinoshita and Suzuki et al., JCB, 2017).

Previous studies suggest that gangliosides regulate receptor activation in cell membranes, but the mechanisms in living cells have never known yet. At present, we are investigating how gangliosides regulate receptor clustering and activation, especially focusing on “glycan interactions”.

3．Unraveling of digital-like signaling system

Our results obtained by dual-color single-molecule imaging or single-molecule FRET of receptors and signaling molecules in living cell membranes indicate that signaling molecules are recruited to receptors for only less than a second, and suggest that signaling molecules are activated for very short terms (Fig. 4. Suzuki et al., 2007ab, J. Cell Biol.). On the other hand, cell bulk signals observed by western blotting of phosphorylation of signaling molecules continued for a few minutes to 20 minutes. Based on these results, we proposed a hypothesis of “digital-like signaling system” in which bulk signals continue over several minutes, but such bulk signals must be generated by the superposition of pulse-like individual events. In other words, the activation level of the bulk signal is determined by the superposition (integration) of the recruitment/activation of thousands of copies of the signaling molecule. The digital-like signaling system does not need complicated regulation and would be robust against noise. At present, we are testing this hypothesis.

Press release 2007　[Suzuki et al., 2007a, J. Cell Biol.]　[Suzuki et al. 2007b, J. Cell Biol.]

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