Sherpa Rinzhin T, Pala Rajasekharreddy, Mohieldin Ashraf M, Nauli Surya M
Department of Biomedical & Pharmaceutical Sciences, Chapman University School of Pharmacy (CUSP), Chapman University, Irvine, CA, United States.
Department of Biomedical & Pharmaceutical Sciences, Chapman University School of Pharmacy (CUSP), Chapman University, Irvine, CA, United States; Department of Medicine, University of California Irvine, Irvine, CA, United States.
Methods Cell Biol. 2019;153:25-42. doi: 10.1016/bs.mcb.2019.03.015. Epub 2019 Apr 17.
Cellular signaling represents an evolution of biological systems to sense external stimuli and communicate extracellular microenvironment to the intracellular compartments. The processes underlying molecular signaling have been widely studied due to their important cellular functions. There are numerous techniques available to quantitate the different molecules involved in cellular processes. Among them, calcium is a ubiquitous signaling molecule involved in many biological pathways. Over time the methods to measure intracellular calcium have advanced to better understand its role as a second messenger. In this chapter, we introduce a method to study a single cilium, a mechanosensor that elicits a calcium signaling cascade. To successfully observe the calcium changes in this thin cylindrical-like projection from the cell surface, we utilize a genetically encoded sensor with a high spatial and temporal resolution. In addition, the probe must be localized to the ciliary compartment in order to observe the intraciliary calcium signaling dynamics. To this end, a cilium targeting genetically encoded indicator is used to observe calcium fluxes in both cytoplasm and cilioplasm.
细胞信号传导代表了生物系统的一种进化，用于感知外部刺激并将细胞外微环境传递到细胞内区室。由于分子信号传导的重要细胞功能，其潜在过程已得到广泛研究。有许多技术可用于定量细胞过程中涉及的不同分子。其中，钙是一种参与许多生物途径的普遍存在的信号分子。随着时间的推移，测量细胞内钙的方法不断进步，以便更好地理解其作为第二信使的作用。在本章中，我们介绍一种研究单个纤毛的方法，纤毛是一种引发钙信号级联反应的机械传感器。为了成功观察从细胞表面伸出的这种细圆柱状突起中的钙变化，我们使用具有高空间和时间分辨率的基因编码传感器。此外，为了观察纤毛内的钙信号动态，探针必须定位于纤毛区室。为此，使用一种靶向纤毛的基因编码指示剂来观察细胞质和纤毛质中的钙通量。
