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Cas9-mediated Genome Editing Reveals a Significant Contribution of Calcium Signaling Pathways to Anhydrobiosis in Pv11
bioRxiv - Molecular Biology Pub Date : 2020-10-15 , DOI: 10.1101/2020.10.15.340281
Yugo Miyata , Hiroto Fuse , Shoko Tokumoto , Yusuke Hiki , Ruslan Deviatiiarov , Yuki Yoshida , Takahiro G. Yamada , Richard Cornette , Oleg Gusev , Elena Shagimardanova , Akira Funahashi , Takahiro Kikawada

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin - calcineurin - NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin - calmodulin Kinase - CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose-induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway.

中文翻译:

Cas9介导的基因组编辑揭示了钙信号通路对Pv11中脱水生物的重要贡献。

Pv11是一种从the蚊(Polypedilum vanderplanki)建立的昆虫细胞系,具有极强的脱水耐受性,被称为无水生物。Pv11还具有海藻糖处理诱导的抗生水能力。在这里,我们报告成功构建了Pv11细胞的基因组编辑系统,并将其用于确定脱水生物中的信号通路。使用Cas9介导的基因敲入系统,我们建立了稳定表达GCaMP3的Pv11细胞,以监测细胞内Ca2 +的动员。有趣的是,海藻糖处理引起细胞内Ca2 +浓度的瞬时增加,进一步的实验表明钙调蛋白-钙调神经磷酸酶-NFAT途径对海藻糖处理的耐受性以及干燥耐受性的影响,而钙调蛋白-钙调蛋白激酶-CREB途径仅赋予Pv11细胞干燥耐性。因此,我们的结果显示了海藻糖诱导的Ca2 +激增对脱水生物的关键作用以及每个信号通路在时间上的不同作用。
更新日期:2020-10-17
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