Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca2+) detection have found diverse applications in vitro and in vivo across a series of model animal systems. Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.

Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

The present work describes the engineering and characterization of a new Ca2+-activated photoprotein (Photina®) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein—coupled receptors (GPCRs), targeting Photina® to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina.® The mitochondrial-targeted Photina® also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina® provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR3, FLIPRTETRA, CyBi®-®Lumax flash HT, Lumilux®, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS. ( Journal of Biomolecular Screening 2007:694-704)

Coupling of Calcium Homeostasis to Axonal Sodium in Axons of Mouse Optic Nerve

Axonal populations in neonatal and mature optic nerves were selectively stained with calcium dyes for analysis of calcium homeostasis and its possible coupling to axonal Na. Repetitive nerve stimulation causes a rise in axonal [Ca2+]i the posttetanus recovery of which is impeded by increasing the number of action potentials in the tetanus. This effect is augmented in 4-aminopyridine (4-AP; 1 mM), which dramatically increases the calcium and presumably sodium load during the tetanus. Increasing axonal [Na]i with the Na-ionophore monensin (4–50 μM) and ouabain (30 μM) retards posttetanus calcium decline, suggesting that efficient calcium clearance depends on a low level of axonal [Na]i. Posttetanus calcium clearance is not affected by K-mediated depolarization. To further examine coupling between axonal [Na]i and [Ca2+]i, the resting axonal [Ca2+]i was monitored as axonal [Na+]i was elevated with ouabain, veratridine, and monensin. In all cases, elevation of axonal [Na+]i evokes a calcium influx into axons. This influx is unrelated to activation of calcium channels but is consistent with calcium influx via reversal of the Na/Ca exchanger expected as a consequence of axonal [Na+]i elevation. In conclusion, this study demonstrates that calcium homeostasis in the axons of the optic nerve is strongly coupled to axonal [Na+]i in a manner consistent with the Na/Ca exchanger playing a major role in extruding calcium following nerve activity.