2003 Fiscal Year Final Research Report Summary
Development of CFP-based calcium probes that emit blue fluorescence
| Project/Area Number |
14570053
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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 |
General physiology
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| Research Institution | RIKEN (The Institute of Physical and Chemical Research) Brain Science Institute (2003)
Okazaki National Research Institutes (2002) |
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Principal Investigator |
NAKAI Junichi RIKEN (The Institute of Physical and Chemical Research) Brain Science Institute, Laboratory for Neuronal Circuit Dynamics, Deputy teamleader, 神経回路ダイナミクス研究チーム, 副チームリーダー (80237198)
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| Project Period (FY) |
2002 – 2003
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| Keywords | GFP / CFP / calcium / myosin light chain kinase / calmodulin |
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| Research Abstract |
Calcium ions play important roles as an intracellular mediator of muscle contraction, neuronal activity and hormone secretion. Therefore, it is useful to measure calcium ion to study cellular function. The purpose of this research is to develop new Green fluorescent protein (GFP) based calcium sensors that emit blue fluorescence. This type of sensors are genetically-encoded, therefore, it can be easily expressed in specific cells or tissues using a specific promoter. This sensor may become a useful tool for in vivo calcium measurements. To make such kind of sensors we fused a cyan version of GFP to calmodulin and the M13 fragment from myosin light chain kinase. When the DNA ~vas expressed HEK cells or PC12 cells. we could monitor fluorescence changes in the cells with a CCD camera or a laser confocal microscope. Interestingly, the fluorescence was brighter at the resting condition and it became dimmer when the cells were activated. There are 4 calcium binding sites in a calcium sensor. To reduce the calcium buffering effect and to change the calcium sensitivity of the sensor, we next introduced mutations in there calcium binding sites. When we destroyed more than 3 calcium binding sites, the sensor was severely affected and it was not useful as a calcium sensor any more. On the other hand, mutating one or two calcium binding sites is tolerable and those mutants were still useful as calcium sensors. To be surprised, although their calcium sensitivities were affected by the mutations, the effects were not as large as we expected. These sensors which have mutations in calcium binding sites may be good tools because they have reduced calcium buffering effects.
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