Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii.
Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii.
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DOI：
10.1007/978-1-4939-9857-9_11
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发表时间：
2019-11
影响因子：
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通讯作者：
S. Vella;Abigail Calixto;Beejan Asady;Zhu-Hong Li;S. Moreno
中科院分区：
文献类型：
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作者：
S. Vella;Abigail Calixto;Beejan Asady;Zhu-Hong Li;S. Moreno
胞质钙离子（Ca2+）浓度的波动调节了所有真核生物的多种细胞功能。细胞表达一套复杂的机制来平衡胞质Ca2+水平，并且胞质中升高Ca2+的信号被降低Ca2+的机制所补偿。Ca2+依赖性内平衡机制的改变是人类许多突出疾病的原因，如心力衰竭或神经元死亡。弓形虫的遗传易感性和遗传工具的可用性使细胞质中表达的遗传编码钙指示剂（GECIs）的使用成为可能，这开启了弓形虫钙信号研究的新时代。最终有可能在寄生虫从宿主细胞中退出之前看到Ca2+振荡。在Endo等人证明离子载体触发细胞输出数年后，振荡先于宿主细胞输出的假设已经通过使用geci的实验得到验证。GECIs允许可视化活的细胞内寄生虫中的特定Ca2+信号，并将这些信号与宿主细胞钙波动区分开来。在本章中，我们概述了我们实验室首次使用GECI中毒体的“可靠”方法，包括GECI的选择，转染方法和理想克隆的选择，它们的特性，以及使用表达GECI的寄生虫进行荧光和显微镜分析。
Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+levels and the signals that elevate Ca2+in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.The genetic tractability ofToxoplasma gondiiand the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies ofToxoplasmacalcium signaling. It was finally possible to see Ca2+oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s inToxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.