Calcium ions act as signals between cells, but their exact locations — at the nanometre scale — have been difficult to pinpoint. The latest biosensor promises to reveal these details in dynamic living systems.
Cell signalling is all about location. This concept is best illustrated with calcium signals — cells funnel bursts of calcium ions to specific locations, where the ions selectively activate a wide variety of physiological functions. Calcium signals ebb and flow to cellular hotspots that are confined to regions ranging in size from micrometres down to tens of nanometres. But despite the importance of localization for controlling the effects of calcium signals on cells, it has been a daunting task to study calcium and other transient cellular signals at the nanometre scale.
Reporting in Nature Chemical Biology, Tour et al.1 describe a promising approach to this long-standing problem. They have developed a calcium sensor that allows rapid, selective and sensitive tracking of localized calcium signals with high temporal and spatial resolution.
Fluorescence microscopy is a powerful technique for imaging, in real time, many aspects of communication within and between cells. The difficulty with this method for determining the movement of dynamic cell signals such as calcium is detecting the non-uniform variation in signal concentrations within highly localized regions. Being able to detect these signal fluctuations is essential, as they may lead to drastically different biological outcomes. Synthetic, small-molecule (that is, non-protein) fluorescent indicators — such as those in the Fura, Fluo and Calcium Green families of compounds — show very rapid and selective responses to calcium. But these indicators are distributed diffusely in cells and so are unable to provide resolutions using conventional light microscopy. Alternatively, protein-based biosensors can be introduced at specific subcellular locations using genetic engineering. This approach provides an easy way to place calcium probes into cells, but such sensors are limited by their slow responses, and their large sizes can perturb the system of interest.
The strategy now presented by Tour et al.1 combines the tunability and small size of synthetic chemical indicators with the spatial resolution and control of genetically targeted proteins. They have developed a prototype small-molecule sensor, known as Calcium Green FlAsH (CaGF; Fig. 1a). This molecule comprises a receptor that binds selectively to calcium, a fluorescent reporter that responds to calcium binding, and two arsenic groups that label proteins only at specially incorporated peptide sequences that consist of four cysteine amino acids. This study builds upon previous work from the same group2 that showed that small arsenic-containing dyes target tetracysteine peptide motifs. The addition of a calcium-reporting group to the dyes introduces an extra dimension that allows calcium's function in cellular systems to be studied using molecular imaging.
With the CaGF tool, Tour et al. explored the local dynamics of calcium signals in two cell systems. First, they studied gap junctions — the intersections that allow molecules and ions to pass freely between vertebrate cells. The authors used CaGF in cells expressing a connexin protein that had been tagged with a tetracysteine peptide motif. Connexins are major building-blocks of gap junctions, so the authors were able to use CaGF to monitor waves of calcium passing through the junctions in real time.
In a second set of experiments, Tour and colleagues targeted CaGF to the mouth of Cav 1.2, which is one of a family of channels that open and close to control the flow of calcium ions into cells (Fig. 1b). In this way, the authors could directly visualize calcium hotspots that colocalize with the clustered Cav 1.2 channels in the outer cell membrane. Although this technique does not yet allow calcium concentrations to be quantified or kinetic measurements to be made, the authors results do suggest that bursts of calcium signals exist that are only a few nanometres wide, and that calcium channels cluster together for signalling. More importantly, CaGF offers the tantalizing possibility of studying non-uniform calcium dynamics in more complex systems, such as brain neurons.
Tour and colleagues work provides a host of opportunities for the development of further chemical tools. A wish list for next-generation designs might include calcium-responsive groups with brighter fluorescence and more rapid turn-on responses; ratiometric readouts of fluorescence that allow the concentration of ions in nanometre-sized calcium bursts to be measured; and a choice of coloured reporters, so that different kinds of sensor can be used and identified in the same experiment. A range of sensors with different binding affinities for calcium would also be useful, as calcium-ion concentrations vary depending on the cell type.
Furthermore, the discovery and optimization of new peptide motifs that recognize small molecules should lead to sensors that bind with improved selectivity to proteins tagged with those peptides; this will improve the signal-to-noise ratios of sensors in localized cellular regions and allow multiple probes to be used that recognize different tags3,4,5,6,7. The authors general concept could also be exploited to visualize protein regions by using methods other than fluorescence, such as electron microscopy, magnetic resonance imaging, positron emission tomography and ultrasound. Different reporters could also be used, so that other metal ions, naturally occurring organic compounds and enzyme-reaction products can be detected. By opening the door to such possibilities, Tour et al.1 have taken a step towards the most ambitious goal of all–developing chemical probes that can visualize specific features of living systems with molecular resolution.