| Co-Investigator(Kenkyū-buntansha) |
ITO Hiroyasu Hamamatsu Photonics Researcher, 筑波研究所, 研究員
MORIKAWA Kaoru Kousyu Eiseiin Chif, 衛生薬学部, 室長 (40147020)
FUJITA Masahiko Kousyu Eiseiin Manager, 衛生薬学部, 部長 (00083741)
SUZUKI Kazuo NIH of Japan Chief, 抗生物質部, 室長 (20192130)
ARATA Yoji Univ. of Tokyo Fac. Pharm. Sci. Professor, 薬学部, 教授 (40011499)
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| Research Abstract |
There is a revolution in the use of light microscopy in basic biological research. Several new technologies, used in conjunction with the light microscope, give us a new possibility of seeing and measuring events in the living cell. Thus, in this research project, we have tried to prepare a new system of light microscopy to study signal transduction in immunological cells. By our new confocal fluorescence microscope with an argon-ion laser ( 488 nm) and a He-Cd laser (325 nm) we were able to study spatial heterogeneity of the calcium signals in rat basophilic leukemia cells (RBL-2H3), B lymphocytes and T lymphocytes. Confocal fluorescence images of these cells showed that th e receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus. This confocal fluorescence microscope was used to the exocytotic secretory process of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its qantum yield with decreasing intramolecular rotation. Then, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramaticaly in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D. However, fluo-3 fluorescence intensities still increased after addition of compound 48/80. Thus, this imaging system became a powerful tool to study the signal transduction in t he immune responses.
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