Properties and use of genetically encoded FRET sensors for cytosolic and organellar Ca2+ measurements

Abstract

In the last 15 years, there has been an explosion in the development of genetically encoded biosensors that report enzyme activity, chemical transformation, or concentration of ions and molecules in living cells. Currently, there are well over 120 biosensors of different cellular targets. As a general design principle, these sensors convert a molecular event, such as the binding of a molecule to a sensing domain or a signal-induced change in protein conformation, into a change in the sensor's fluorescence properties. In contrast to small-molecule sensors, genetically encoded sensors are generated when sensor-encoding nucleic acid sequences, which have been introduced by transgenic technologies, are translated in cells, tissues, or organisms. One of the best developed classes of biosensors is the genetically encoded Ca(2+) indicators (GECIs). Here, we briefly summarize the properties of ratiometric GECIs and describe how they are used to quantify Ca(2+) in specific cellular locations, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria.

FIGURE 1

Design of genetically-encoded, ratiometric, FRET-based Ca²⁺ sensors. (A) A Ca²⁺ binding domain is sandwiched between a donor FP (usually cyan FP) and an acceptor FP (usually yellow FP). When Ca²⁺ ions reversibly bind to the sensor’s binding domain, the sensor’s conformation changes, which leads to a change in FRET efficiency. (B) Emission spectrum of a sensor in the presence and absence of Ca²⁺. The FRET ratio of the unbound sensor (R_free) is distinct from that of the bound sensor (R_bound).