Properties and Use of Genetically Encoded FRET Sensors for Cytosolic and Organellar Ca2+ Measurements

Changes in intracellular Ca2+ concentrations are integral to important cellular processes, including cell division, growth, signaling, and death. GECIs have been optimized during the past few decades to monitor changes in Ca2+ in living cells. In contrast to small-molecule Ca2+ probes, GECIs can be targeted to subcellular organelles and to specific cell types, and they can be expressed for days, or longer, in cultured cells and transgenic organisms. Fluorescent GECIs have been engineered to respond to Ca2+ in two different ways: the fluorescence intensity of intensiometric GECIs, or the absorption or emission spectrum of ratiometric GECIs change with fluctuations in Ca2+ concentration. Intensiometric GECIs, such as the GCaMP sensors, can be monitored using one fluorescence channel, and ratiometric GECIs are monitored by calculating the ratio of two different fluorescence emission intensities. Several excellent reviews outline the evolution, advantages, and disadvantages of the major GECI families, discuss important considerations when choosing a GECI for a specific application, and detail primary differences between GECIs and small molecule probes (Dean et al. 2012; Tian et al. 2012; Whitaker 2010). Most ratiometric GECIs are designed in the following way1 (see Figure 1): a donor fluorescent protein (FP) is attached to an acceptor FP by a linker containing a Ca2+-binding domain. The GECI undergoes a conformational change upon Ca2+ binding, which alters the distance between the two FPs, the orientation of the FPs relative to each other, and, consequently, the efficiency of Forster resonance energy transfer (FRET) from the donor to the acceptor FP (reviewed in (Lakowicz 2006; Zhang et al. 2002)). The FRET ratio, which is the ratio of the acceptor FP emission intensity (also called the FRET intensity) to the donor FP emission intensity, is the metric used to monitor the change in FRET. For example, two images – a FRET image and a donor FP image – of a cell expressing a GECI can be used to calculate the FRET ratio, which is the cell’s mean intensity in the FRET image divided by its mean intensity in the donor FP image. The FRET ratio can be converted to a Ca2+ concentration when the following are available: (1) the sensor’s affinity, in terms of its dissociation constant (Kd’), (2) its empirical Hill coefficient (n) if cooperativity is involved, (3) the FRET ratio in the absence of Ca2+ (Rfree), and (4) the FRET ratio when the sensor is saturated with Ca2+ (Rbound). The sensor’s affinity and Hill coefficient can be measured in vitro or in situ (i.e. in cells) and is usually published in the literature, while Rfree and Rbound are measured during a calibration procedure at the end of each experiment. Over the last decade, sensor calibrations have been optimized in several different cell types and organelles (Mccombs and Palmer 2008; Palmer and Tsien 2006; Pozzan and Rudolf 2009; Rudolf et al. 2006). 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 protocols in this chapter focus on using ratiometric FRET-based GECIs for quantitative measurement of cytosolic, nuclear, mitochondrial, or ER Ca2+. Table 1 highlights current, state-of-the-art, ratiometric GECIs. These GECIs are preferred over intensiometric GECIs for quantitative measurement of Ca2+ because their FRET ratios are independent of their concentration in each cell and the path length (i.e. the thickness of a cell), and hence Ca2+ can be estimated with greater confidence. TABLE 1 Selected ratiometric GECIs Perhaps one of the single greatest advantages of using ratiometric GECIs is that it is possible to rigorously define the intracellular concentration of the sensor and assess whether the sensor itself perturbs cellular Ca2+. This can be done because the fluorescence intensity of the acceptor FP, upon direct excitation, is independent of the level of Ca2+ within the cell. For example, the fluorescence intensity of a yellow cameleon sensor, using 495/10 nm excitation and 535/25 nm emission filters, is proportional to its concentration. Thus, a standard curve relating the acceptor FP intensity to the sensor concentration can be made by measuring the intensity of purified sensor protein, at a range of known concentrations, using a fluorescence microscope under conditions identical to a live cell imaging experiment. The standard curve can then be used to calculate the sensor concentration from a fluorescence image of an unknown sample (i.e. a cell). This method necessitates creating micro-cuvettes with volumes similar to that of cell, as has been done using the wedge method (Chen et al. 2005; Miyawaki et al. 1999), glass capillaries (Dittmer et al. 2009), and microwells fabricated in polydimethylsiloxane (PDMS) (our work, unpublished). Having a means to quantify the sensor concentration in cells allows researchers to assess whether the measured Ca2+ concentration is independent of the sensor’s concentration. We have found that for most sensors under control of a standard CMV promoter there is a wide range of sensor expression levels, typically varying from low μM to tens of μM (Dittmer et al. 2009; Qin et al. 2011), and hence there is natural variation of sensor concentration that enables researchers to examine estimated Ca2+ concentration as a function of sensor concentration. However, it is also possible to put the sensor under control of an inducible promoter to tightly control expression levels. For sensor concentrations ranging from low μM to tens of μM, we have observed little perturbation of resting Ca2+ measurement. In contrast, small molecule probes with acetoxymethyl (AM) esters are known to concentrate in cells at levels close to hundreds of μM (Dineley et al. 2002; Kao et al. 2010). Ratiometric GECIs can be used to compare Ca2+ concentrations at subcellular resolution among different cell types, in cells with genetic or chemical changes, or using different instruments. For example, the ER-targeted D1ER probe enabled researchers to compare the resting levels of Ca2+ in the ER in cells expressing different mutant forms of the protein presenilin-1, a protein that is dysfunctional in inherited forms of Alzheimer’s disease (Mccombs et al. 2010). D1ER also revealed altered ER Ca2+ related to Gaucher’s disease and demonstrated that small molecule drugs that modulate ER Ca2+ partially relieve the disease associated proteostasis defect (Mu et al. 2008). The mitochondrial GECI 4mt-D3cpV allowed investigators to observe that knockdown of a putative regulator of mitochondrial Ca2+ uptake, MICU1, abolished mitochondrial Ca2+ uptake in single HeLa cells in response to thapsigargin or histamine (Perocchi et al. 2010). Lastly, in a particularly elegant example of the capabilities of ratiometric GECIs, Tay et al. fused TN-XL to the N-type Ca2+ channel (CaV2.2) and quantified Ca2+ in a nanodomain surrounding the channel (Tay et al. 2012). The protocols that follow detail the experimental steps of using ratiometric GECIs to quantify Ca2+ in the cytosol, nucleus, mitochondria, or ER (Protocol 1). In addition, a protocol is presented for carrying out in situ titrations to determine the binding parameters (Kd and n) of sensors in particular cells and organelles (Protocol 2).