Imaging and analysis of subplasmalemmal Ca^<2+> dynamics by FRET
| Project/Area Number |
15590724
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ISSHIKI Masashi The University of Tokyo, Faculty of Medicine, Assistant, 医学部附属病院, 助手 (70302734)
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| Co-Investigator(Kenkyū-buntansha) |
IIRI Taro The University of Tokyo, Faculty of Medicine, Assistant, 医学部附属病院, 助手 (90313022)
YAMAMOTO Kimiko The University of Tokyo, Graduate School of Medicine, Lecturer, 大学院・医学系研究科, 講師 (00323618)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | endothelium / calcium signal / cameleon / FRET / nitric oxide (NO) / imaging / PLC / カベオラ |
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| Research Abstract |
Subplasmalemmal Ca^<2+>, dynamically equilibrated with extracellular Ca^<2+>, affects numerous signaling molecules, effectors, and events within this restricted space. We demonstrated the presence of a novel Ca^<2+> wave propagating beneath the plasma membrane in response to acute elevation of extracellular[Ca^<2+>], by targeting a Ca^<2+> sensor, cameleon, to the endothelial plasmalemma. These subcortical waves, spatially distinct from classical cytosolic Ca^<2+> waves, originated in localized regions and propagated throughout the subplasmalemma. Translocation of an expressed GFP fused with a PH domain of PLCδ from the plasma membrane to the cytosol accompanied these subcortical waves, and U73122 attenuated not only the GFP-PHδ translocation, but also the peak amplitude of the subcortical Ca^<2+> waves ; this finding suggests the involvement of local IP_3 production through PLC-mediated PIP_2 hydrolysis in the initiation of these waves. Changes in NO production as well as PKCβ-GFP translocation from the cytosol to the plasma membrane, but not of GFP-PLA_2 to perinuclear endomembranes, were associated with the subplasmalernrnal Ca^<2+> changes. Thus, extracellular Ca^<2+> maintains the basal PLC activity of the plasma membrane, is involved in the initiation of compartmentalized subcortical Ca^<2+> waves, and regulates CA^<2+>-dependent signaling molecules residing in or translocated to the plasma membrane.
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Report
(3 results)
Research Products
(3 results)
