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. Author manuscript; available in PMC: 2017 Apr 27.
Published in final edited form as: Cell Syst. 2016 Apr 27;2(4):283–288. doi: 10.1016/j.cels.2016.03.010

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

Pimkhuan Hannanta-anan 1, Brian Y Chow 1,*
PMCID: PMC4909256  NIHMSID: NIHMS776604  PMID: 27135540

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.

Graphical Abstract

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INTRODUCTION

Calcium-dependent transcription is a fundamental eukaryotic signaling mechanism that regulates critical functions in development, stress responses, and synaptic transmission (Clapham, 2007; Ghosh and Greenberg, 1995; Uhlén and Fritz, 2010; Uhlén et al., 2015). Propagating waves of elevated calcium encode information in their waveform in order to coordinate these systemwide transcriptional programs within individual cells and across cell populations. The mechanism by which cells decode this information and incorporate it into their decision making processes remains unclear.

It is known that the transcriptional activity of calcium-dependent transcription factors is enhanced by increasing calcium oscillation frequency (Bito et al., 1997; Dolmetsch et al., 1997, 1998; Parekh, 2011; Salazar et al., 2008; Smedler and Uhlén, 2014; Tomida et al., 2003). However, cells decode oscillatory biochemical signals based on not only the peak amplitude and frequency as principle components of the waveform, but also the duty cycle (or duty ratio). Here, duty cycle is defined as cumulative load of calcium concentration elevated above basal levels with respect to time, normalized to a steady-state load of equal peak amplitude (Salazar et al., 2008). Oscillation duty cycle can serve as a measure of cumulative load that influences transcriptional signaling (Purvis and Lahav, 2013) (Figure 1A).

Figure 1. Calcium Decoding by the NFAT Transcription Factor.

Figure 1

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(A) NFAT information processing principles. Calcium oscillations encode information in their frequency, peak amplitude, and duty ratio (left). Elevated calcium induces NFAT translocation and transcriptional activation at rate α (middle). Nuclear export at rate β deactivates transcriptional signaling (inactive form, NFAT and active form, NFAT*). Calcium signal processing is frequency-selective when transcription is enhanced at specific oscillation frequencies (absolute ν, or relative ω = ν/β) (right). Alternatively, NFAT is a signal integrator if the enhancement is attributable to duty ratio.

(B) Mathematical models of NFAT sensitivity to (i) calcium frequency and (ii) duty ratio suggest it is primarily an integrator of cumulative calcium delivered by pulsatile loads regardless of peak amplitude, as the duty ratio-response spans the whole activation range, whereas frequency dependence is more modest. Note that the models for 0.5 and 1.0 μM calcium overlap.

(C) Signaling pathways and genetic circuit diagram of a synthetic optogenetic transcription device for decoding calcium encoding in HeLa cells. The calcium oscillations are optically created by melanopsin-mediated store-operated release, activating NFAT via CN. The transcriptional activity of the specific oscillation is reported by luciferase under the NFAT promoter.

(D) Confocal micrograph of engineered HeLa cells stably expressing melanopsin, visualized by a C-terminal GFP tag.

To understand whether cells decode each dynamical component of the calcium signal and initiate responses that are specific to those components, it is necessary to understand how frequency and duty cycle of calcium oscillations each affect transcription independently (Kalo and Shav-Tal, 2013; Schuster et al., 2002). However, experimental analyses tend to stimulate cells with fixed calcium pulse-widths and, under those conditions, frequency and duty cycle change concomitantly. Thus, it remains unclear whether calcium-dependent transcription factors primarily act as signal integrators of cumulative load delivered by pulsatile signals or whether they selectively respond to specific oscillation frequencies. In principle, these “decoding principles” can be experimentally discerned by matching calcium duty cycle across frequencies, and vice versa, to respectively isolate the contributions frequency and duty cycle make to transcriptional activation levels.

Here, we use mathematical modeling, optogenetics, and synthetic biology to resolve the primary decoding principle for NFAT (Hogan et al., 2003), a calcium-dependent transcription factor crucial to immune cell function, development, stem cell differentiation, and tumor progression (Horsley et al., 2008; Macian, 2005; Mancini and Toker, 2009; Nguyen and Di Giovanni, 2008). To activate NFAT, increases in cytoplasmic calcium concentration activate the calcium-dependent phosphatase, calcineurin (CN). CN, in turn, dephosphorylates cytoplasmic NFAT to expose its nuclear localization signal; dephosphorylated (activated) NFAT then shuttles to the nucleus, binds its cognate promoter, and initiates transcription (Figure 1A; we call this nuclear translocation rate α). Activated NFAT is constitutively phosphorylated within the nucleus and subsequently exported from it in a phosphorylation-dependent manner (we call the nuclear export rate β in Figure 1A). When cytoplasmic calcium levels fall, CN activity decreases and the phosphorylated form of NFAT accumulates in the cytoplasm, halting NFAT-dependent transcription.

Because nuclear export is the rate-limiting step in returning NFAT activity to baseline (Colella et al., 2008; Kalo and Shav-Tal, 2013; Tomida et al., 2003; Yissachar et al., 2013), NFAT accumulates in the nucleus when the calcium oscillation frequency (we call this frequency ν in Figure 1A) exceeds export frequency (Salazar et al., 2008). Therefore, transcription under the NFAT promoter increases with increasing oscillation frequency, as originally demonstrated under duty cycle-unmatched conditions (Dolmetsch et al., 1997, 1998). However, NFAT may alternatively (or complementarily) integrate total elevated calcium concentrations, as originally proposed in a model of “integrative tracking” (Berridge, 2006; Berridge et al., 2003).

RESULTS

Using a calcium decoding model with an analytic solution (Salazar et al., 2008), we performed a sensitivity analysis to identify the individual effect of specific parameters on the NFAT response. We calculated the mean NFAT transcriptional activity as a function of either relative calcium oscillation frequency (ω) or duty ratio (γ) while holding the remaining components constant. The relative oscillation frequency is the determinant frequency parameter suggested by the model and is defined as oscillation frequency (ν) relative to the NFAT nuclear export rate (β). This analysis (Figure 1B) spanned a physiological range of peak calcium amplitude (A) at either an optimal duty ratio or an optimal relative frequency (calculated using Equations 1 and 2 in Supplemental Information).

This analysis revealed that frequency sensitivity over the physiologically relevant range (ω = 0–3) is confined to the lower range of relative frequency (ω < 1), with a maximum frequency-dependent enhancement of ~30% (Figure 1Bi). However, duty cycle has a more pronounced effect that spans the entire range of transcriptional activation regardless of oscillation amplitude (Figure 1Bii). The model thus suggests that NFAT is more sensitive to duty cycle than specific frequency and, consequently, is primarily an integrator of cumulative calcium load. Importantly, the analysis also informed our experimental design by identifying the parameter regimes in which the NFAT response is most sensitive to, as well as estimating the maximum change in NFAT activity as a function of these individual parameters.

Next, we tested these analytic results directly by optogenetically controlling intracellular calcium in HeLa cells, as described below (Figures 1C and D), and correlating the oscillation waveforms to transcriptional activity in multiplexed gene expression assays. Based on a mammalian synthetic optogenetic transcription device (Ye et al., 2011), the engineered HeLa cells stably expressed human melanopsin (hOPN4), a light-gated Gαq-coupled opsin useful for optogenetic control of store-operated release of calcium from the ER (Lin et al., 2008; Provencio et al., 1998; Qiu et al., 2005). Calcium release drove luciferase reporter expression under the regulation of the NFAT promoter. HeLa cells were chosen because they primarily express one isoform (NFAT4; Schaab et al., 2012) that dominates the frequency sensitivity (refer to Figures S1A and S1B), thereby simplifying modeling and model-to-data correlations.

Calcium waveforms in individual cells were evoked by optical stimulation and measured via calcium imaging with X-Rhod-1. We identified stimulation conditions that yielded calcium transients of identical peak concentrations (amplitudes) while varying areas-under-the-curve (AUCs) (Figure 2A). Figure 2A shows the three waveforms and the empirically derived optical pulse-trains to reliably generate them. Repeated short pulses (1 s-long every 5 s, blue dots in Figure 2A) were used to prevent photoreceptor bleaching by constant illumination. Transcriptional induction epochs are created by repeating individual calcium transients and adjusting the inter-peak period in order to either match duty cycle across varying frequencies or match frequency across varying duty cycles (Figure 2). The highest oscillation frequency that allows calcium elevation to completely decay to the baseline was ν ~8 mHz, and thus, opto-genetic calcium clamping had sufficient temporal precision to ensure NFAT nuclear accumulation. The matching peak amplitudes (~0.27 μM) exceed the half-saturation concentration for NFAT activation (Ks = 0.2 μM; Figure S1C).

Figure 2. Parametric Analysis of Calcium Oscillation Frequency and Duty Cycle on NFAT Transcriptional Signaling.

Figure 2

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(A) Calcium transients of identical peak amplitude, but varying AUC (presented as Rel. AUC, relative to the AUC of 0.5 s-stimulation) created by melanopsin stimulation, as measured by calcium indicator X-Rhod-1 imaging. The colored dots represent optical stimulation pulses. The data are represented as mean ± SEM (with 306–490 cells per averaged trace).

(B) Schematic of custom illuminator to deliver identical stimulation as used in calcium imaging experiments, using a variable potentiometer and microcontroller to respectively tune irradiance and control timing per row of LEDs (see also Figure S2).

(C) Schematic for multiplexed optogenetic transcription assays in a multiwell plate (blue illuminator, melanopsin stimulation and yellow illuminator, recapitulation of X-Rhod-1 imaging).

(D) Photograph of the experimental setup, here shown outside of the tissue culture incubator.

(E) Isolating the role of calcium frequency on NFAT transcriptional activity by optogenetic duty-cycle matching at fixed peak amplitude. The luciferase reporter expression under the transcriptional regulation of NFAT is optogenetically induced by melanopsin stimulation (refer to Figure 1C). The transcriptional activity is normalized to non-illuminated cells that are otherwise identical. No statistically significant difference is observed between frequencies at a given duty ratio (unpaired t test).

(F) Isolating the role of duty cycle under frequency-matched conditions at fixed peak amplitude. The transcriptional activity increases with duty ratio with statistical significance (unpaired t test, * = p < 0.05, and *** = p < 0.001), suggesting that NFAT is primarily an integrator of cumulative load.

In (E) and (F), data are represented as mean ± SD.

To isolate the specific contribution of frequency on NFAT transcriptional activity, we matched the duty ratio by adjusting the period between repeated peaks from Figure 2A. Two frequency windows of interest (1.65–5 and 0.70–2.1 mHz) encompassed the most frequency-sensitive regime (ω = 0.3–1.0; Figure 1Bi), based on reported 1/βNFAT4 of 3.0–7.6 min (Kar and Parekh, 2015; Yissachar et al., 2013). Multiplexed transcription studies were performed in 24-well plates using custom illuminators that reproduce the calcium imaging conditions, for both melanopsin stimulation and X-Rhod-1 excitation, by controlling light-emitting diodes (LEDs) via potentiometers (irradiance) and microcontrollers (timing) (Figures 2B–2D). No statistically significant difference in transcriptional activity was observed within either duty cycle-matched window (Figure 2E). Note that to limit cell variation and basal luciferase expression, all data within a particular frequency window were collected simultaneously, but the two distinct frequency windows draw from different cell passages. The duty cycle-matched assays suggest that NFAT is not primarily a frequency-selective decoder of calcium oscillations, as initially proposed by others (Dolmetsch et al., 1997, 1998).

To determine whether NFAT is instead primarily an integrator of cumulative load, we assessed the effect of duty cycle on transcriptional activity, at fixed frequency and amplitude (Figure 2F). All data in Figure 2F were acquired simultaneously, but the frequency-matched data set was generated with different cell passages than the duty cycle-matched one. The same waveforms in Figure 2A were used to span a 3-fold duty cycle range in a regime (γ = 0.1–0.3) chosen to maximize sensitivity based on the model in Figure 1Bii. Statistically significant differences were observed across this range. Similar-fold increases in cumulative load are sufficient to fundamentally alter cellular dynamics, including transitions between poised and actively proliferating stem cell states (Deng et al., 2015). The increased sensitivity to duty cycle over spike timing suggests that NFAT behaves as a signal integrator, as suggested by the mathematical model and hypothesized by others (Berridge, 2006; Berridge et al., 2003; Colella et al., 2008; Kar et al., 2012). Importantly, previous studies using paired pulses of calcium in rat basophilic leukemia cells (RBL) confirmed that NFAT activation indeed integrates calcium elevations that are coincident within an inter-pulse temporal cutoff (Kar et al., 2012). The results here reveal that such decoding-by-integration is in fact the dominant mechanism for NFAT.

The finding is also consistent with related studies on NFκB in immune cells (Song et al., 2012; Zhu et al., 2011) and the stimulus duration-dependent switching of cAMP-response element binding protein (CREB) in neurons (Bito et al., 1996, 1997), thus suggesting that decoding-by-integration is a general transcriptional principle applicable to many mammalian calcium-responsive activators. However, because these transcription factors differ from NFAT in terms of the presence of negative feedback loops mediated by IκB in NFκB signaling (Levine et al., 2013; Tay et al., 2010) and in terms CREB responsiveness to additional second messenger beyond calcium (Shaywitz and Greenberg, 1999), one should keep attuned to signaling network-specific differences in the decoding principles employed by different calcium-dependent transcription factors.

DISCUSSION

Systematically isolating the contributions of individual waveform parameters to calcium-dependent transcriptional activity clarifies the role of frequency in studies performed with steady-state loads and/or unmatched duty cycles. Previously observed enhancements with shortening periods were likely attributable to consequent differences in cumulative load. This distinction does not imply frequency insensitivity, since excited cells typically increase their calcium spike frequency (Schuster et al., 2002), but rather shifts the attributable source of enhancement to better reflect the decoding principle, from specific frequency or peak timing, to frequency-coupled cumulative load.

NFAT signaling relies on pulsatile calcium to coordinate with other regulators to direct diverse signaling outputs (Purvis and Lahav, 2013) and to achieve selectivity by temporal cutoff filtering (of isoform-specific translocation rates; Levine et al., 2013; Yissachar et al., 2013), in addition to distinct spatial calcium signatures within cells (Di Capite et al., 2009; Kar and Parekh, 2015; Mehta et al., 2014; Parekh, 2011). Single-cell studies on calcium-dependent transcription factors have shown that nuclear translocation occurs in pulsatile bursts of increasing output frequency with increasing steady-state calcium input (Cai et al., 2008; Kar and Parekh, 2015; Levine et al., 2013; Tay et al., 2010; Yissachar et al., 2013), so that coordinated gene expression under one promoter depends on the fraction of localization time, but not exact timing or true frequency (frequency-modulated [FM] coordination; Cai et al., 2008; Levine et al., 2013). Since fraction of localization time is also a duty ratio, the optogenetic findings here are consistent with the general FM-coordination principle, but now applied to the upstream, non-steady-state oscillatory input.

In total, this work defines a workflow for using mathematical modeling, optogenetics, and mammalian synthetic biology to isolate the decoding principles at work in dynamical regulation of transcription. By resolving this experimental confound in interpreting the roles of interdependent components of an oscillatory signal, this parameterized analysis refines a key decoding principle of frequency modulated calcium-dependent transcription to better explain how mammalian cells dynamically respond to complex stimuli.

EXPERIMENTAL PROCEDURES

A brief summary of experimental procedures is provided below (refer to Supplemental Information for detailed procedures).

Genetic Constructs and Transduction

Clonal populations of HeLa cell lines stably expressing human melanopsin (hOPN4) were created by lentiviral transduction under the CMV promoter. For transcriptional activation assays, cells were transfected with NFAT-luciferase reporter plasmid pGL4.30[luc2P/NFAT-RE/Hygro] (Promega E8481) using TransIT-LT1 transfection agent (Mirus Biotech) according to manufacturer protocols. Cells were plated on 1 cm coverslips for calcium imaging or black 24-well plates for gene expression assays, with collagen adhesion layers.

Calcium Imaging

Calcium imaging with X-Rhod-1 (Invitrogen X-14210) in phenol-free media was performed on a Leica DMI6000B fluorescence microscope (Chroma filters, λex < 575 nm and λem > 580 nm), equipped with MetaMorph software for automated optical stimulation (Lumencor LEDs, hOPN4 activation: λ = 470 nm at 3.8 mW/cm2, X-Rhod-1 imaging: λ = 570 nm at 2.8 mW/cm2), fluorescence imaging (pco.edge sCMOS camera), and fluid perfusion (Harvard Apparatus syringe pump and Autom-8 perfusion chamber). Absolute calcium concentrations of individual cells were quantified by perfusing calcium ionophore (Sigma-Aldrich C7522) and calcium-free (Fmin) and high calcium calibration (Fmax) buffers immediately after measuring optogenetically induced calcium transients and employing a calcium calibration standard (characterized in Supplemental Information, Section 7: Calcium calibration) for fluorescence-calcium conversion (refer to Supplemental Information, Section 6: Calcium imaging). Image processing, cell segmentation, and measurements of fluorescence intensity of individual cells were performed using ImageJ and MetaMorph. Mean AUC and peaks reported in Figure 2A are the average of all single-cell traces from six coverslips (three epochs per cell, 306–490 cells in total).

Gene Expression Assays

LED illuminators on custom circuit boards for optogenetic induction of luciferase reporter expression were aligned to 24-well plates (black-walled to prevent optogenetic cross-talk between wells). Optical stimulation conditions between gene expression assays and calcium imaging were matched via (1) an Arduino microcontroller to control LED timing and (2) variable potentiometers to tune LED irradiance (hOPN4 activation: λ = 470 nm at 3.8 mW/cm2, X-Rhod-1 imaging: λ = 570 nm at 2.8 mW/cm2). Cells were optogenetically stimulated for 6 hr in phenol-free media (Clontech) and chemically lysed with cell culture lysis reagent. Luciferase bioluminescence was quantified on a Tecan M200 plate reader according to assay manufacturer protocols (Promega E1501) and normalized to non-illuminated wells from the same plate. Refer to Supplemental Information Section 11: Illuminator construction and programming for schematics. Refer to a zip file containing all Solid-Works CAD files that will be permanently available for download at the University of Pennsylvania Scholarly Commons (http://www.repository.upenn.edu/).

Mathematical Modeling and Data Analyses

Mathematical modeling and sensitivity analysis was performed using MATLAB. Data were analyzed in MATLAB and plotted in R and Excel. Statistical analyses were performed in R.

Supplementary Material

1

Highlights.

  • Calcium oscillations precisely controlled by optogenetics in engineered HeLa cells

  • Calcium waveform-dependent NFAT activity assessed in transcription assays

  • NFAT activation is more sensitive to duty ratio than frequency of the oscillation

  • Consistent with mathematical modeling, NFAT integrates cumulative calcium load

Acknowledgments

The authors thank Arjun Raj, David Issadore, Ed Boyden, Daniel Schmidt, and the members of the Chow Lab for helpful discussion. The authors also thank the Boyden Lab (MIT) for the hOPN4 plasmid and the following laboratories at Penn for generously sharing reagents, cell lines, and equipment: Cremins, Gadue, Issadore, Lazarra, Meaney, and Raj. B.Y.C. acknowledges the support of NSF Biophotonics (CBET 126497), W.W. Smith Charitable Trust for the Heart, Penn Medicine Neuroscience Center, NIH/NIDA (1R21 DA040434-01), and DARPA (Living Foundries HR0011-12-C-0068). P.H. is on partial fellowship support from the Thai Ministry of Science and Technology Scholarship.

Footnotes

AUTHOR CONTRIBUTIONS

P.H. and B.Y.C. conceived of all experiments, conducted all data analysis, and wrote the paper. P.H. performed all experiments.

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Supplementary Materials

1
NIHMS776604-supplement-1.pdf (446.5KB, pdf)

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