D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle

Abstract

Current fluorescent monitors of free [Ca2+] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca2+. “D4cpv-Casq1,” expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective KD of 222 µM and a dynamic range [(Rmax − Rmin)/Rmin] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, Rmax, and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca2+]SR in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca2+]SR was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca2+ concentration.

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

“…2B shows a raw confocal image of the fluorescence of Casq1 fused with EYFP in cells of a calsequestrin-null mouse. EYFP-Casq1 fluorescence forms two bands per sarcomere, consistent with the location previously found for native Casq1 (23), and for Casq1 fused with D4cpV (24). The agreement indicates that Casq1 fused to either tag traffics in the same way as native Casq1, to SR terminal cisternae (TC).…”

“…4C is a 3D rendering of the deblurred stack. The images confirm the observation (24) that in cells at rest, D4cpv-tagged Casq1 resides mostly in two bands per sarcomere. Fluorescence costaining (23,24) has shown that the bands correspond to triads, each with two TC not resolved in these images.…”

“…The images confirm the observation (24) that in cells at rest, D4cpv-tagged Casq1 resides mostly in two bands per sarcomere. Fluorescence costaining (23,24) has shown that the bands correspond to triads, each with two TC not resolved in these images. The narrow and wide spaces between these calsequestrin bands correspond respectively to the I and A bands of the sarcomere (24).…”

“…Fluorescence costaining (23,24) has shown that the bands correspond to triads, each with two TC not resolved in these images. The narrow and wide spaces between these calsequestrin bands correspond respectively to the I and A bands of the sarcomere (24).…”

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

“…Regarding the data obtained at 22 [19] to near 1 mM [20], with most of the obtained figures around 400-800 M [21][22][23][24][25][26][27]. On the other hand, NMR measurements [21], skeletal muscle cells [25,27] and rabbit heart cells [28] The new probe has also a very important technical and experimental advantage over the previous aequorin probes. All the [Ca 2+ ] ER measurements carried out before with these probes were performed in cells previously depleted of Ca 2+ , because the high Ca 2+ content of the ER precluded reconstitution with the coelenterazine cofactor.…”