Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters.
Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters.
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DOI：
10.1017/s1740925x10000219
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发表时间：
2010-08
影响因子：
--
通讯作者：
Khakh BS
中科院分区：
文献类型：
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作者：
Shigetomi E;Kracun S;Khakh BS
星形胶质细胞参与脑内突触和脑血管的调节。这些功能受细胞内钙信号的调节，该信号被认为反映了星形胶质细胞的一种兴奋性。在最近的一项研究中，我们报道了用膜系留结构域Lck修饰遗传编码的钙指示剂(Geci)GCaMP2，以产生Lck-GCaMP2。这种Geci使我们能够检测到新的微域钙信号。微域是随机的，本质上是“斑点”的。为了更可靠地检测这种信号，在本研究中，我们进一步修改了LCK-GCaMP2，使其携带GCaMP2部分的三个突变(EGFP中的M153K、T203V和CaM结构域的N60D)，以产生LCK-GCaMP3。我们通过评估LCK-GCaMP2和LCK-GCaMP3监测几种类型星形胶质细胞钙信号的能力来直接比较LCK-GCaMP2和LCK-GCaMP3。我们的数据显示，在基础荧光强度、信噪比和检测微域的能力方面，LCK-GCaMP3的性能比LCK-GCaMP2好两到四倍。因此，LCK-GCaMP3的使用代表了一种显著改进的监测星形胶质细胞钙信号的方法，包括微域，并将有助于详细探索其分子机制和生理作用。
Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.