Measurement of cytoplasmic and cilioplasmic calcium in a single living cell

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.