Imaging Intracellular Free Ca²⁺ Concentration Using Yellow Cameleons

Abstract

Green fluorescent protein (GFP)-based fluorescent indicators for Ca 2+ offer significant promise for monitoring Ca 2+ in previously unexplored organisms, tissues, and submicroscopic environments because they are genetically encoded, function without cofactors, can be targeted to any intracellular location, and are bright enough for single-cell imaging. These probes use simple GFP variants, circularly permuted GFP (cpGFP), in which the amino and carboxyl portions have been interchanged and reconnected by short spacers between the original termini, or pairs of GFP variants that permit fluorescence resonance energy transfer (FRET). Yellow cameleons (YCs) use FRET between cyanand yellow-emitting variants of Aequorea GFP (cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP], respectively). YCs are composed of a linear combination of CFP, calmodulin (CaM), a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and YFP. Binding of Ca 2+ to the CaM moiety of the YC initiates an intramolecular interaction between the CaM and the M13 domains, causing the chimeric protein to shift from an extended conformation to a more compact one, thereby increasing the efficiency of FRET from CFP to YFP. This technique is amenable to emission ratioing, which is more quantitative than single-wavelength monitoring. YC3.60 was engineered to enhance its performance as a fluorescent Ca 2+ indicator and here we describe the use of this cameleon to image rapid changes in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) within HeLa cells. FRET imaging is performed using a laser-scanning confocal microscope. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. ReagentsCa 2+ -free medium For fast and simultaneous acquisition of cpVenus173 and CFP images from HeLa cells expressing YC3.60, this camera, which comprises three charge-coupled device (CCD) chips (RGB: red, green, and blue) and a prism, can be used. The cpVenus173 and CFP images are captured by the G and B chips, respectively. In addition, to improve spatial resolution along the z-axis, a spinning-disk unit (e.g., CSU21 from Yokogawa Electric Corporation) can be placed in front of the camera.Diode-pumped solid-state laser (430 nm) (Melles Griot) Glass-bottomed dishes (35 mm) (Matsunami Glass Ind., Ltd., D111300) Image acquisition software to control the Ashura 3CCD camera (e.g., . These include intensity of the laser, scanning speed, binning, and sensitivity of the camera.In our experience, the intensity of the laser should be attenuated greatly using neutral-density filters for Ca 2+ imaging on a timescale of subseconds. Photobleaching does not happen with a laser that is sufficiently attenuated.6. Observe fluorescence signals from CFP and cp173Venus by the blue and green channels of the 3CCD camera, respectively. 7. Set 4 × 4 binning for high-sensitive fluorescence ...

The Development of Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) During Gastric Repair Is Absent in the Aged Stomach

“…Gastric organoids derived from whole stomach glands were transplanted into acetic acid–induced ulcerated gastric tissue of young (age, 2–3 mo) and aged (age, 18–24 mo) mice ( Figure 7 A ). To visualize engraftment directly within the gastric epithelium, gastric organoids were derived from the whole stomachs of yellow chameleon 3.0 mice 28 ; therefore, the organoids expressed YFP ( Figure 7 B ). When compared with injection into the lumen, organoids transplanted into the submucosa of the stomach efficiently promoted repair ( Figure 7 C ).…”

Intravital Two-photon Imaging of Ca2+ signaling in Secretory Organs of Yellow Cameleon Transgenic Mice

“…The other well-recognized GECI is GCaMPs, a single fluorophore probe based on a circularly permuted green fluorescent protein, their monitoring of Ca 2+ signaling is depending on fluorescence intensity. Compared to GCaMPs, YCs have less dynamics range and photostability but the advantages of them are less sensitive to motion artifact and expression level differences because of rationing of two fluorescent proteins 13 , 14 .…”

Receptomics, design of a microfluidic receptor screening technology

“…When signals are low, it is important to use a probe that is the most sensitive in the resting range of cytosolic calcium levels. Previously, in reverse transfected arrays with bitter receptors, calcium assays were performed using the Cameleon YC3.6 sensor protein, because of its wide dynamic range (5-6 fold change in the ratio of YFP/CFP upon Ca 2+ binding in vitro) and brightness [131,200]. The arrays were typically printed using two plasmids at identical concentrations; one encoding the G protein coupled receptor (GPCR) and one the calcium sensing protein.…”

“…In this study we aimed to evaluate ratiometric probes with a calcium affinity that would ensure sufficient sensitivity for relatively minor calcium transients of bitter taste receptors above the resting levels of ~100 nM in HEK293 cells [206]. Cameleon YC3.6 [131,200,204], Nano140 [204] and Twitch2B [205] have K d values of ~140-250nM that fit this criterion in principle, but these probes are distinctly different: YC3.6 and Nano140 both include a modified calmodulin domain, but with different cooperative binding and dissociation constants, and Twitch2B contains a troponin domain with only one calcium binding pocket and a more linear calcium binding curve. An added advantage of the use of troponin over calmodulin is that it does not potentially interact with host cell calmodulin binding proteins [202].…”

“…An added advantage of the use of troponin over calmodulin is that it does not potentially interact with host cell calmodulin binding proteins [202]. Next to sensor type, we also aimed to evaluate the effect of the cytosolic sensor concentration, because a high sensor concentration may increase signal-to-noise levels, but may also result in a lower signal intensity because of calcium buffering [200,207].…”