Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

Abstract

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.

Functionally redundant control of cardiac hypertrophic signaling by inositol 1,4,5-trisphosphate receptors

“…GCaMP6s has been shown to be more sensitive to calcium than other genetically encoded calcium indicators and synthetic calcium dyes such as Fura-2 [22, 40]. One limitation of GCaMP6s is the relatively slow kinetics which could preclude the possibility of imaging fast calcium release events [40].…”

“…One limitation of GCaMP6s is the relatively slow kinetics which could preclude the possibility of imaging fast calcium release events [40]. However, using cytosolic GCaMP6s we can detect cytosolic events not detected by Fura-2 (Fig.…”

Genetic tools to study complexity of striatal function

“…In order to improve sensitivity and kinetics of calcium indicators, several GCaMP derivatives have been generated ranging from the GCaMP3 to GCaMP5 (Akerboom et al, ). GCaMP6 are among the most recent showing substantial improvements regarding brightness and have three forms (GCaMP6s, 6m, and 6f) for slow, medium, and fast kinetics (Garcia, Chen, & Boehning, ).…”

Long-Term Imaging of Calcium Dynamics Using Genetically Encoded Calcium Indicators and Automatic Tracking of Cultured Cells

“…Agonists are manually added to the imaging chamber or automatically applied via peristaltic pump (e.g.,Nemesys pump, Cetoni [Korbuen, Germany]) at the bottom of the imaging chamber and the total volume is regulated by a separate pump placed above the imaging solution. This experimental approach allows imaging of cytosolic calcium dynamics in cultured cells for more than 8h (Figure 1B and [7]) without any detectable photobleaching or ectopic localization of GCaMP reporter within subcellular compartments. Longer imaging time may also be possible but may require better CO 2 /O 2 and humidity control and periodic application of fresh media.…”

“…Here we present an efficient and robust approach for long-term calcium imaging in cultured cells. This approach employs genetically encoded calcium indicators instead of calcium dyes [7] and uses semi-automatic cell detection and tracking of cultured cells.…”