FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca2+dynamics
FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca2+dynamics
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DOI：
10.1111/j.1365-313x.2011.04780.x
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发表时间：
2012-01-01
期刊：
影响因子：
7.2
通讯作者：
Schumacher, Karin
中科院分区：
文献类型：
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作者：
Krebs, Melanie;Held, Katrin;Schumacher, Karin
时间和空间定义的钙信号是许多信号传导途径的组成部分。因此，以高空间和时间分辨率监测钙动力学对于了解这种无处不在的第二信使如何控制不同的细胞反应至关重要。黄驼龙（YCs）是一种基于荧光共振能量转移（FRET）的基因编码的Ca 2+传感器，它为监测Ca 2+通量的时空动态提供了一个强有力的工具。在这里，我们提出了一套先进的载体和转基因植物活细胞钙离子成像线。由花椰菜花叶病毒（CaMV）35 S启动子介导的转基因沉默严重限制了离子和代谢物纳米传感器的应用，因此我们使用了来自拟南芥的UBQ 10启动子，并在此表明这导致了YC在转基因植物中的组成型和稳定表达。为了提高空间分辨率，我们的载体库包括可以针对定义位置的YC版本。使用这个工具包，我们确定了时间上不同的反应，外部ATP在质膜，在细胞质和相邻的根细胞的细胞核。此外，对百脉根中Ca ~（2+）动态的分析显示，结瘤因子诱导的细胞核和胞质中Ca ~（2+）峰化模式不同。因此，这里介绍的构建体和转基因株系能够详细分析不同细胞室和不同植物物种中的Ca 2+动态，并将促进新的方法来破译钙信号的时间和空间特征。
Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca2+ -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca2+ fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca2+ imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca2+ dynamics in Lotus japonicus revealed distinct Nod factor induced Ca2+ spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca2+ dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.