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Development of CFP-based calcium probes that emit blue fluorescence

开发基于 CFP 的发出蓝色荧光的钙探针

基本信息

批准号：
14570053
负责人：
NAKAI Junichi
金额：
$ 2.24万
依托单位：
RIKEN (The Institute of Physical and Chemical Research) Brain Science Institute (2003)Okazaki National Research Institutes (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570053/
关键词：
GFP CFP calcium myosin light chain kinase calmodulin バイオセンサー分子生物学蛍光

项目摘要

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.

钙离子作为肌肉收缩、神经元活动和激素分泌的细胞内介质发挥重要作用。因此，测定钙离子对研究细胞功能是有用的。本研究的目的是开发一种新型的基于绿色荧光蛋白（GFP）的钙离子传感器，该传感器能够发出蓝色荧光。这种类型的传感器是遗传编码的，因此，它可以很容易地在特定的细胞或组织中使用特定的启动子表达。该传感器可能成为体内钙测量的有用工具。为了制造这种传感器，我们将青色版本的GFP融合到钙调蛋白和来自肌球蛋白轻链激酶的M13片段。当DNA在HEK细胞或PC12细胞中表达时，我们可以用CCD摄像机或激光共聚焦显微镜监测细胞中的荧光变化。有趣的是，在静止状态下荧光更亮，而当细胞被激活时，荧光变得更暗。在钙传感器中有4个钙结合位点。为了降低钙缓冲效应并改变传感器的钙敏感性，我们接下来在钙结合位点中引入突变。当我们破坏超过3个钙结合位点时，传感器受到严重影响，不再作为钙传感器使用。另一方面，突变一个或两个钙结合位点是可以容忍的，并且这些突变体仍然可以用作钙传感器。令人惊讶的是，尽管他们的钙敏感性受到突变的影响，但影响并没有我们预期的那么大。这些在钙结合位点具有突变的传感器可能是很好的工具，因为它们具有降低的钙缓冲作用。