Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology
Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology
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DOI：
10.1111/nph.12652
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发表时间：
2014-04-01
期刊：
影响因子：
9.4
通讯作者：
Frommer, Wolf B.
中科院分区：
文献类型：
--
作者：
Lanquar, Viviane;Grossmann, Guido;Frommer, Wolf B.
锌作为酶的辅因子和蛋白质充分折叠的结构元素，在所有活细胞中发挥着核心作用。在真核细胞中，金属是高度区域化和螯合的。我们的工作结合了遗传编码的Forster共振能量转移(FRET)传感器和一种新的微流控技术RootChip，来监测拟南芥根细胞胞液中锌离子浓度的动态变化。我们的实验提供了在充足(0.4 NM)和过量(2 NM)锌离子供应下生长的拟南芥根细胞胞质游离锌离子浓度的估计。此外，监测细胞内[Zn2+]对外界供应的响应表明，高亲和力和低亲和力的摄取系统以及从内部存储器释放的参与。在这项研究中，我们证明了遗传编码的FRET传感器和微流控技术的结合为监测根细胞中广泛浓度范围内细胞金属离子浓度的动态提供了一个有吸引力的工具。
Zinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn2+ homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine.Our work combines the use of genetically encoded Forster resonance energy transfer (FRET) sensors and a novel microfluidic technology, the RootChip, to monitor the dynamics of cytosolic Zn2+ concentrations in Arabidopsis root cells.Our experiments provide estimates of cytosolic free Zn2+ concentrations in Arabidopsis root cells grown under sufficient (0.4 nM) and excess (2 nM) Zn2+ supply. In addition, monitoring the dynamics of cytosolic [Zn2+] in response to external supply suggests the involvement of high- and low-affinity uptake systems as well as release from internal stores.In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells.