Intraorganellar calcium imaging in Arabidopsis seedling roots using the GCaMP variants GCaMP6m and R-CEPIA1er
Intraorganellar calcium imaging in Arabidopsis seedling roots using the GCaMP variants GCaMP6m and R-CEPIA1er
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DOI：
10.1016/j.jplph.2020.153127
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发表时间：
2020-03-01
影响因子：
4.3
通讯作者：
Han, Shengcheng
中科院分区：
文献类型：
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作者：
Luo, Jin;Chen, Lvli;Han, Shengcheng
Ca2+在真核生物中作为普遍的第二信使。在动物中，各种各样的环境和发育刺激触发细胞器中的Ca2+动力学，如细胞质、细胞核和内质网（ER）。然而，内质网Ca2+ ([Ca2+](ER))稳态及其在植物胞质和/或核质Ca2+动力学中的作用仍然是未知的。gcamp由圆形排列形式的增强绿色荧光蛋白与钙调蛋白和肌球蛋白轻链激酶M13融合组成，用于监测哺乳动物细胞中的Ca2+动态。在这里，我们针对具有细胞质核输出信号的GCaMP高亲和力变体（ns - gcamp6m），具有核定位信号的核定位信号（nns - gcamp6m），以及具有内质网腔内质网定位蛋白Calreticulin la信号肽序列（CRT1a-R-CEPIA1er）的GCaMP低亲和力变体（也称为钙测量细胞器捕获蛋白指标（CEPIA）），用于拟南芥细胞器内ca2 +成像。我们发现250 mM山梨糖醇作为渗透胁迫刺激，50 μ M脱落酸（ABA）或1 mM碳糖醇（CCh）诱导的细胞质Ca2+ ([Ca2+](cyt))增加主要是由于细胞外Ca2+内流，而渗透胁迫，ABA或CCh触发的核质Ca2+ ([Ca2+](nuc))增加是由[Ca2+]（er）释放引起的。此外，[Ca2+]（er）动态在不同的刺激（如渗透胁迫、ABA或CCh）下呈现出特定的模式，表明Ca2+信号在植物内质网中发生。这些结果为拟南芥根细胞对不同胁迫的亚细胞Ca2+动力学响应提供了有价值的见解，并证明GCaMP成像是进一步了解植物细胞器功能的有用工具。
Ca2+ acts as a universal second messenger in eukaryotes. In animals, a wide variety of environmental and developmental stimuli trigger Ca2+ dynamics in organelles, such as the cytoplasm, nucleus, and endoplasmic reticulum (ER). However, ER Ca2+ ([Ca2+](er)) homeostasis and its contributions in cytosolic and/or nucleosolic Ca2+ dynamics in plants remain elusive. GCaMPs are comprised of a circularly permutated form of enhanced green fluorescent protein fused to calmodulin and myosin light-chain kinase M13 and used for monitoring Ca2+ dynamics in mammalian cells. Here, we targeted a high-affinity variant of GCaMP with nuclear export signal in the cytoplasm (NES-GCaMP6m), with a nuclear-localised signal in the nucleus (NLS-GCaMP6m), and a low-affinity variant of GCaMP, also known as calcium-measuring organelle-entrapped protein indicators (CEPIA), with a signal peptide sequence of the ER-localised protein Calreticulin la in the ER lumen (CRT1a-R-CEPIA1er) for intraorganellar Ca 2+ imaging in Arabidopsis. We found that cytosolic Ca2+ ([Ca2+ ](cyt)) increases induced by 250 mM sorbitol as an osmotic stress stimulus, 50 mu M abscisic acid (ABA), or 1 mM carbachol (CCh) were mainly due to extracellular Ca 2+ influx, whereas nucleosolic Ca2+ ([Ca2+](nuc)) increases triggered by osmotic stress, ABA, or CCh were contributed by [Ca2+](er) release. In addition, [Ca2+](er) dynamics presented specific patterns in response to different stimuli such as osmotic stress, ABA, or CCh, indicating that Ca2+ signalling occurs in the ER in plants. These results provide valuable insights into subcellular Ca2+ dynamics in response to different stresses in Arabidopsis root cells and prove that GCaMP imaging is a useful tool for furthering our understanding of plant organelle functions.