Optogenetic Control of Calcium Oscillation Waveform Defines NFAT as an Integrator of Calcium Load

Abstract

SUMMARY It is known that the calcium-dependent transcription factor NFAT initiates transcription in response to pulsatile loads of calcium signal. However, the relative contributions of calcium oscillation frequency, amplitude, and duty cycle to transcriptional activity remain unclear. Here, we engineer HeLa cells to permit optogenetic control of intracellular calcium concentration using programmable LED arrays. This approach allows us to generate calcium oscillations of constant peak amplitude, in which frequency is varied while holding duty cycle constant, or vice versa. Using this setup and mathematical modeling, we show that NFAT transcriptional activity depends more on duty cycle, defined as the proportion of the integrated calcium concentration over the oscillation period, than on frequency alone. This demonstrates that NFAT acts primarily as a signal integrator of cumulative load rather than a frequency-selective decoder. This approach resolves a fundamental question in calcium encoding and demonstrates the value of optogenetics for isolating individual dynamical components of larger signaling behaviors.

Optogenetic toolkit for precise control of calcium signaling

“…Naturally-existing photosensitive melanopsin, a member of the opsin subgroup of GPCRs first characterized in retinal ganglion cells [65], can be expressed in other types of mammalian cells to drive Ca 2+ dependent reactions when illuminated by blue or green light. Vertebrate melanopsin (or opsin 4, OPN4) and its variants have been used as intrinsic bistable optogenetic switches to generate both sustained and transient Ca 2+ responses to control Ca 2+ -dependent transgene expression and GPCR pathways [48, 66, 67]. By taking advantage of a light-gated human melanopsin (hOPN4) to generate Ca 2+ oscillations with programmable frequency, peak amplitude, and duty cycle, Hannanta-anan et al resolved the decoding principle for NFAT by quantitatively assessing the impact of these parameters on NFAT-dependent transcriptional outputs [66].…”

“…Vertebrate melanopsin (or opsin 4, OPN4) and its variants have been used as intrinsic bistable optogenetic switches to generate both sustained and transient Ca 2+ responses to control Ca 2+ -dependent transgene expression and GPCR pathways [48, 66, 67]. By taking advantage of a light-gated human melanopsin (hOPN4) to generate Ca 2+ oscillations with programmable frequency, peak amplitude, and duty cycle, Hannanta-anan et al resolved the decoding principle for NFAT by quantitatively assessing the impact of these parameters on NFAT-dependent transcriptional outputs [66]. Results from both optogenetic studies and mathematical modeling converge to support the conclusion that NFAT acts as a signal integrator of Ca 2+ load rather than a frequency-selective decoder.…”

Systematic Characterization of Dynamic Parameters of Intracellular Calcium Signals

“…Many theories have been proposed to establish how information can be encoded. Some of these involve encoding information on the basis of calcium binding cooperativity (Larsen et al, 2004), amplitude and frequency modulation (De Pitta et al, 2009), changes in spike time variation (Thurley et al, 2014), and signal integration (Hannanta-anan and Chow, 2016). In order to reconcile these theories and establish a universal syntax for calcium-encoded information, tools such as this algorithm will aid in the large-scale analysis of experimental data sets required for the validation of mathematical models.…”

Optogenetic Immunomodulation: Shedding Light on Antitumor Immunity

“…However, how the Ca 2+ oscillation amplitude, frequency, and duty cycle contribute to gene transcription remains unclarified. By using a light-gated G-protein coupled receptor (human melanopsin; hOPN4) as the optical actuator, along with NFAT-dependent luciferase gene expression as readout, Hannanta-anan et a.l generated a set of Ca 2+ oscillation waveform inside HeLa cells and further built a mathematical model to define NFAT as an integrator of accumulative Ca 2+ load, rather than a decoder that is selective to frequency [27]. One caveat in this study is that photo-stimulation of hOPN4 ultimately leads to the generation of two secondary messengers, Ca 2+ and diacyl glycerol (DAG), the latter of which further activates protein kinase C and the activated protein-1 (AP-1).…”