| Project/Area Number |
07457010
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
KATAYAMA Yoshifumi Tokyo Med & Dent Univ, Med Res inst, Prof, 難治疾患研究所, 教授 (20014144)
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| Co-Investigator(Kenkyū-buntansha) |
TATSUMI Hitoshi Nagoya Univ, Sch Med, Assist Prof, 医学部, 助手 (20171720)
FURUKAWA Tetsushi Tokyo Med & Dent Univ, Med Res inst, Assist Prof, 難治疾患研究所, 助手 (80251552)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | Optical tweezers / Evanescent light / Membrane protein / Ion channel / Imaging / Cloning / Evanescent light microscopy / Optical technology / カルシウムチャネル / クロライドチャネル / メカノレセプター / 神経成長円錐 / トランスポータ / 微分干渉顕微鏡 / 近接場顕微鏡 |
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| Research Abstract |
It is necessary to apply recent advance in optical technology to studying the dynamics of ion channels and transporter of living cells. In the present study, we have attempted to employ laser optical tweezers and evanescent light in the field of cellular physiology. We improved the ability of the tweezers by using 100 mW Ar-Kr laser beam ; the tweezers could hold and move latex beads up to 1mum on the surface of growth cones of rat cultured neurons.
Evanescent light illumination was introduced into a high resolution video-enhanced differential interference contrast microscope. Growth cones and soma of neurons which were cultured on glass plates were stained with a fluorescent dye, DiI,and were observed with an internal total reflection evanescent light microscope. The evanescent images showed that growth cones and only limited areas of soma attached to the culture glass plate. At the same time, using an illumination mode evanescent light microscope developed by Ohtsu et al, we obtained
… More
images of bundles of tubulin or neurofilament in neurite or growth cone regions.
In the course of analyzing behavior of ion channels activated by extension, ClC-2 was cloned and ClC-2 currents expressed in Xenopus oocytes were characterized. And it is concluded that ClC-2 currents may regulate the oocyte volume. Furthermore an extension-actived ClC-2 channel-modulating protein was cloned and its structure was determined. We are trying to visualize this protein using evanescent light by preparing a fluorescence-conjugated antibody to the protein. At the same time, we could record inward currents in response to mechanical stimulation applied to growth cones of cultured DRG neurons.
Glutamate release from growth cones was detected by using an acutely dissociated hippocampal neuron as a biodetector from which whole cell patch clamp recordings were made. The release was dependent on Ca^<2+> and was abolished by omega-agatoxin. Cultured neurons were pretreated with the toxin, then incubated with the antibody to the toxin and further stained with gold-conjugated antibody. Because clusters of immuno-gold particles were observed over the membrane of growth cones by means of scanning and transmission electron microscope, it is suggested omega-agatoxin-sensitive Ca^<2+> channels were immunologically localized on growth cones. Less
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