Annual Report 2024
Division of Advanced Bioimaging
Kenichi Suzuki, Rinshi Kasai, Ryo Yoshizawa
Introduction
To understand how anticancer drugs work, clarifying how cancer-related gene products behave in cells is mandatory. Molecules in cells do not work synchronously but essentially work in a stochastic process theory (randomly). Individual molecules engage in interactions lasting less than a second, with the proportion of interacting molecules rarely exceeding 10%. Single-molecule imaging is a powerful tool to unravel such stochastic molecular events occurring in cells.
The Team and What We Do
We investigate signaling mechanisms of membrane proteins and cytosolic proteins that play critical roles in oncogenic transformation, using high-resolution single-molecule imaging techniques. We have established a total internal reflection fluorescence microscope system capable of simultaneous three-color single-molecule observation with a temporal resolution of 1 millisecond, along with image-analysis programs. Based on these techniques, we perform three-dimensional super-resolution movie observations with a spatial resolution of 20 nm. In addition, we engaged in various collaborative studies within the institute.
Research Activities
1) Elucidation of the mechanism of extracellular vesicle uptake by target cells
In recent years, extracellular vesicles (EVs) have attracted considerable attention as mediators of intercellular communication. However, the mechanisms by which EVs bind to and are internalized by target cells have remained largely unclear. We hypothesized that this is due to the highly heterogeneous nature of EV populations, and therefore performed single-particle tracking of EVs. Furthermore, by employing a super-resolution live-cell imaging method developed on the basis of our ultra single-molecule imaging technology, we succeeded in visualizing the entire process of EV uptake through specific structures on the target cell membrane, thereby revealing the following findings:
a) EVs contain subtypes that differ in their levels of tetraspanin proteins.
b) The membrane fluidity of EVs containing only CD63 was lower than that of other subtypes.
c) All subtypes of EVs were primarily internalized into target cells via clathrin-independent endocytosis. Only the CD63-containing EVs were additionally internalized through caveolae-mediated pathways.
d) All subtypes of EVs, upon binding to target cells, recruited integrin b1 beneath the binding sites, thereby initiating adhesion signaling and triggering intracellular calcium responses. The elevation of intracellular calcium levels was found to enhance dynamin activity through calcineurin, thereby promoting the internalization of EVs.
2) Development of a non-toxic sphingomyelin marker protein
So far, no sphingomyelin marker protein applicable to living cell membranes has been available, and thus the localization of sphingomyelin on the plasma membrane of living cells remained unclear. By introducing mutations into Equinatoxin II isolated from sea anemone, we developed a sphingomyelin marker protein suitable for use in living cells. Super-resolution microscopy revealed that sphingomyelin is present in the inner leaflet of many plasma membranes, where it forms raft domains approximately 180 nm in size.
Education
We accepted one graduate student from the United Graduate School of Agricultural Science of Gifu University, one graduate student from the Graduate School of Natural Science and Technology of Gifu University, and four undergraduate students from Hoshi University as observership visitors.
Future Prospects
We aim to elucidate how signaling molecules, receptors, and transcription factors exploit membrane structures such as lipid rafts and nuclear structures such as super-enhancers to efficiently promote signal transduction and transcription, and how cancer-related mutants alter these processes, thereby contributing to cancer research.
List of papers published in 2024
Journal
1. Ren WW, Kawahara R, Suzuki KGN, Dipta P, Yang G, Thaysen-Andersen M, Fujita M. MYO18B promotes lysosomal exocytosis by facilitating focal adhesion maturation. The Journal of cell biology, 224:e202407068, 2025
2. Hirosawa KM, Sato Y, Kasai RS, Yamaguchi E, Komura N, Ando H, Hoshino A, Yokota Y, Suzuki KGN. Uptake of small extracellular vesicles by recipient cells is facilitated by paracrine adhesion signaling. Nature communications, 16:2419, 2025
3. Isogai T, Hirosawa KM, Kanno M, Sho A, Kasai RS, Komura N, Ando H, Furukawa K, Ohmi Y, Furukawa K, Yokota Y, Suzuki KGN. Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin. The Journal of cell biology, 224:e202404064, 2025
4. Shintani Y, Higashi SL, Shibata A, Hirosawa KM, Suzuki KGN, Kawano S, Katagiri H, Ikeda M. Modulable supramolecular hydrogels via coassembly using cyclic dipeptides: influence of one methyl group. Chem Mater, 37:2241-2250, 2025
5. Harada A, Kunii M, Kurokawa K, Sumi T, Kanda S, Zhang Y, Nadanaka S, Hirosawa KM, Tokunaga K, Tojima T, Taniguchi M, Moriwaki K, Yoshimura SI, Yamamoto-Hino M, Goto S, Katagiri T, Kume S, Hayashi-Nishino M, Nakano M, Miyoshi E, Suzuki KGN, Kitagawa H, Nakano A. Dynamic movement of the Golgi unit and its glycosylation enzyme zones. Nature communications, 15:4514, 2024
6. Miura A, Manabe Y, Suzuki KGN, Shomura H, Okamura S, Shirakawa A, Yano K, Miyake S, Mayusumi K, Lin CC, Morimoto K, Ishitobi J, Nakase I, Arai K, Kobayashi S, Ishikawa U, Kanoh H, Miyoshi E, Yamaji T, Kabayama K, Fukase K. De Novo Glycan Display on Cell Surfaces Using HaloTag: Visualizing the Effect of the Galectin Lattice on the Lateral Diffusion and Extracellular Vesicle Loading of Glycosylated Membrane Proteins. Journal of the American Chemical Society, 146:22193-22207, 2024
7. Kusumi A, Tsunoyama TA, Suzuki KGN, Fujiwara TK, Aladag A. Transient, nano-scale, liquid-like molecular assemblies coming of age. Current opinion in cell biology, 89:102394, 2024
8. Mori T, Niki T, Uchida Y, Mukai K, Kuchitsu Y, Kishimoto T, Sakai S, Makino A, Kobayashi T, Arai H, Yokota Y, Taguchi T, Suzuki KGN. A non-toxic equinatoxin-II reveals the dynamics and distribution of sphingomyelin in the cytosolic leaflet of the plasma membrane. Scientific reports, 14:16872, 2024
9. Isogai T, Hirosawa KM, Suzuki KGN. Recent Advancements in Imaging Techniques for Individual Extracellular Vesicles. Molecules (Basel, Switzerland), 29:5828, 2024
10. Sugiura S, Higashi SL, Shintani Y, ShibataA, Hirosawa KM, Suzuki KGN, Ikeda M. 9-fluorenylmethoxycarbonyl (Fmoc)-modified taurine as a hydrogelator bearing sulfonate group. Chem Lett, 53:upae189, 2024
Book
1. Suzuki KGN, Hirosawa KM, Isogai T, Yasuda T, Hanashima S. Membrane dynamics of exosomes as revealed by single-molecule imaging. In: Baba Y, Hanayama R, Akita H, Yasui T (ed), Extracellular Fine Particles, Singapore, Springer Singapore, pp 69-80, 2025