Optogenetic Control of Calcium Oscillation Waveform Defines NFAT as an Integrator of Calcium Load.
ABSTRACT: 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.
Project description:Calcium (Ca<sup>2+</sup>) plays a central role in mediating both contractile function and hypertrophic signaling in ventricular cardiomyocytes. L-type Ca<sup>2+</sup> channels trigger release of Ca<sup>2+</sup> from ryanodine receptors for cellular contraction, whereas signaling downstream of G-protein-coupled receptors stimulates Ca<sup>2+</sup> release via inositol 1,4,5-trisphosphate receptors (IP<sub>3</sub>Rs), engaging hypertrophic signaling pathways. Modulation of the amplitude, duration, and duty cycle of the cytosolic Ca<sup>2+</sup> contraction signal and spatial localization have all been proposed to encode this hypertrophic signal. Given current knowledge of IP<sub>3</sub>Rs, we develop a model describing the effect of functional interaction (cross talk) between ryanodine receptor and IP<sub>3</sub>R channels on the Ca<sup>2+</sup> transient and examine the sensitivity of the Ca<sup>2+</sup> transient shape to properties of IP<sub>3</sub>R activation. A key result of our study is that IP<sub>3</sub>R activation increases Ca<sup>2+</sup> transient duration for a broad range of IP<sub>3</sub>R properties, but the effect of IP<sub>3</sub>R activation on Ca<sup>2+</sup> transient amplitude is dependent on IP<sub>3</sub> concentration. Furthermore we demonstrate that IP<sub>3</sub>-mediated Ca<sup>2+</sup> release in the cytosol increases the duty cycle of the Ca<sup>2+</sup> transient, the fraction of the cycle for which [Ca<sup>2+</sup>] is elevated, across a broad range of parameter values and IP<sub>3</sub> concentrations. When coupled to a model of downstream transcription factor (NFAT) activation, we demonstrate that there is a high correspondence between the Ca<sup>2+</sup> transient duty cycle and the proportion of activated NFAT in the nucleus. These findings suggest increased cytosolic Ca<sup>2+</sup> duty cycle as a plausible mechanism for IP<sub>3</sub>-dependent hypertrophic signaling via Ca<sup>2+</sup>-sensitive transcription factors such as NFAT in ventricular cardiomyocytes.
Project description:Dynamic processes, such as intracellular calcium signaling, are hallmark of cellular biology. As real-time imaging modalities become widespread, a need for analytical tools to reliably characterize time-series data without prior knowledge of the nature of the recordings becomes more pressing. The goal of this study is to develop a signal-processing algorithm for MATLAB that autonomously computes the parameters characterizing prominent single transient responses (TR) and/or multi-peaks responses (MPR). The algorithm corrects for signal contamination and decomposes experimental recordings into contributions from drift, TRs, and MPRs. It subsequently provides numerical estimates for the following parameters: time of onset after stimulus application, activation time (time for signal to increase from 10 to 90% of peak), and amplitude of response. It also provides characterization of the (i) TRs by quantifying their area under the curve (AUC), response duration (time between 1/2 amplitude on ascent and descent of the transient), and decay constant of the exponential decay region of the deactivation phase of the response, and (ii) MPRs by quantifying the number of peaks, mean peak magnitude, mean periodicity, standard deviation of periodicity, oscillatory persistence (time between first and last discernable peak), and duty cycle (fraction of period during which system is active) for all the peaks in the signal, as well as coherent oscillations (i.e., deterministic spikes). We demonstrate that the signal detection performance of this algorithm is in agreement with user-mediated detection and that parameter estimates obtained manually and algorithmically are correlated. We then apply this algorithm to study how metabolic acidosis affects purinergic (P2) receptor-mediated calcium signaling in osteoclast precursor cells. Our results reveal that acidosis significantly attenuates the amplitude and AUC calcium responses at high ATP concentrations. Collectively, our data validated this algorithm as a general framework for comprehensively analyzing dynamic time-series.
Project description:The nuclear factor of activated T-cell (NFAT) transcription factors play an important role in many biological processes, including pathological cardiac hypertrophy. Stimulated by calcium signals, NFAT is translocated to the nucleus where it can regulate hypertrophic genes (excitation-transcription coupling). In excitable cells, such as myocytes, calcium is a key second messenger for multiple signaling events, including excitation-contraction coupling. Whether the calcium signals due to excitation-contraction and excitation-transcription coupling coincide or how they can be differentiated is currently unclear. Here we construct a mathematical model of NFAT cycling fitted to skeletal myocyte and baby hamster kidney cell data. The model replicates key behavior with respect to sensitivity to calcineurin overexpression and to calcium oscillations. Finally, we measure the sensitivity of the system to a simulated hypertrophic calcium signal, against a background excitation-contraction coupling calcium oscillation. We find that NFAT cycling is sensitive to excitation-transcription coupling even when both calcium signals are in the same cellular compartment, thus showing that separation of the signals may not be necessary in vitro.
Project description:The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-?, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phospho-inositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER.
Project description:The Kv1.3 potassium channel plays an essential role in effector memory T cells and has been implicated in several important autoimmune diseases including multiple sclerosis, psoriasis and type 1 diabetes. A number of potent small molecule inhibitors of Kv1.3 channel have been reported, some of which were found to be effective in various animal models of autoimmune diseases. We report herein the identification of clofazimine, a known anti-mycobacterial drug, as a novel inhibitor of human Kv1.3. Clofazimine was initially identified as an inhibitor of intracellular T cell receptor-mediated signaling leading to the transcriptional activation of human interleukin-2 gene in T cells from a screen of the Johns Hopkins Drug Library. A systematic mechanistic deconvolution revealed that clofazimine selectively blocked the Kv1.3 channel activity, perturbing the oscillation frequency of the calcium-release activated calcium channel, which in turn led to the inhibition of the calcineurin-NFAT signaling pathway. These effects of clofazimine provide the first line of experimental evidence in support of a causal relationship between Kv1.3 and calcium oscillation in human T cells. Furthermore, clofazimine was found to be effective in blocking human T cell-mediated skin graft rejection in an animal model in vivo. Together, these results suggest that clofazimine is a promising immunomodulatory drug candidate for treating a variety of autoimmune disorders.
Project description:Tomida2003 - NFAT functions Calcium
This model is described in the article:
NFAT functions as a working
memory of Ca2+ signals in decoding Ca2+ oscillation.
Tomida T, Hirose K, Takizawa A,
Shibasaki F, Iino M.
EMBO J. 2003 Aug; 22(15):
Transcription by the nuclear factor of activated T cells
(NFAT) is regulated by the frequency of Ca(2+) oscillation.
However, why and how Ca(2+) oscillation regulates NFAT activity
remain elusive. NFAT is dephosphorylated by Ca(2+)-dependent
phosphatase calcineurin and translocates from the cytoplasm to
the nucleus to initiate transcription. We analyzed the kinetics
of dephosphorylation and translocation of NFAT. We show that
Ca(2+)-dependent dephosphorylation proceeds rapidly, while the
rephosphorylation and nuclear transport of NFAT proceed slowly.
Therefore, after brief Ca(2+) stimulation, dephosphorylated
NFAT has a lifetime of several minutes in the cytoplasm. Thus,
Ca(2+) oscillation induces a build-up of dephosphorylated NFAT
in the cytoplasm, allowing effective nuclear translocation,
provided that the oscillation interval is shorter than the
lifetime of dephosphorylated NFAT. We also show that Ca(2+)
oscillation is more cost-effective in inducing the
translocation of NFAT than continuous Ca(2+) signaling. Thus,
the lifetime of dephosphorylated NFAT functions as a working
memory of Ca(2+) signals and enables the control of NFAT
nuclear translocation by the frequency of Ca(2+) oscillation at
a reduced cost of Ca(2+) signaling.
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Project description:Neurons recruited for local computations exhibit rhythmic activity at gamma frequencies. The amplitude and frequency of these oscillations are continuously modulated depending on stimulus and behavioral state. This modulation is believed to crucially control information flow across cortical areas. Here we report that in the rat hippocampus gamma oscillation amplitude and frequency vary rapidly, from one cycle to the next. Strikingly, the amplitude of one oscillation predicts the interval to the next. Using in vivo and in vitro whole-cell recordings, we identify the underlying mechanism. We show that cycle-by-cycle fluctuations in amplitude reflect changes in synaptic excitation spanning over an order of magnitude. Despite these rapid variations, synaptic excitation is immediately and proportionally counterbalanced by inhibition. These rapid adjustments in inhibition instantaneously modulate oscillation frequency. So, by rapidly balancing excitation with inhibition, the hippocampal network is able to swiftly modulate gamma oscillations over a wide band of frequencies.
Project description:A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+) signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+) oscillations in terms of the time constant of Ca(2+) spike amplitude decay and the Ca(2+) oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+) signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+) signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-?1, -?3, -?4, -?1, -?3 and -? were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-?1 and PLC-?4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-?1 and PLC-?4 resulted in specific changes in the characteristics of Ca(2+) oscillations, such as the time constant of the temporal changes in the Ca(2+) spike amplitude and the Ca(2+) oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+) release, can cause cell-to-cell variability in the patterns of Ca(2+) signals and that PLC-?1 and PLC-?4 contribute to generate cell-specific Ca(2+) signals evoked by G protein-coupled receptor stimulation.
Project description:Neurophysiological observations are clarifying how astrocytes can actively participate in information processing and how they can encode information through frequency and amplitude modulation of intracellular Ca2+ signals. Consequently, hardware realization of astrocytes is important for developing the next generation of bio-inspired computing systems. In this paper, astrocytic calcium oscillations and neuronal firing dynamics are presented by De Pittà and IF (Integrated & Fire) models, respectively. Considering highly nonlinear equations of the astrocyte model, linear approximation and single constant multiplication (SCM) techniques are employed for efficient hardware execution while maintaining the dynamic of the original models. This low-cost hardware architecture for the astrocyte model is able to show the essential features of different types of Ca2+ modulation such as amplitude modulation (AM), frequency modulation (FM), or both modes (AFM). To show good agreement between the results of original models simulated in MATLAB and the proposed digital circuits executed on FPGA, quantitative, and qualitative analyses including phase plane are done. This new neuromorphic circuit of astrocyte is able to successfully demonstrate AM/FM/AFM calcium signaling in its real operation on FPGA and has applications in self-repairing systems. It also can be employed as a subsystem for linking biological cells to artificial neuronal networks using astrocytic calcium oscillations in future research.
Project description:Maintenance of myeloid progenitor cells is controlled by complex regulatory mechanisms and is orchestrated by multiple different transcription factors. Here, we report that the activation of the transcription factor nuclear factor of activated T cells (NFAT) by calcium-sensing protein calcineurin inhibits the proliferation of myeloid granulocyte-monocyte progenitors (GMPs). Myeloid progenitor subtypes exhibit variable sensitivity to induced Ca(2+) entry and consequently display differential engagement of the calcineurin-NFAT pathway. This study shows that inhibition of the calcineurin-NFAT pathway enhances the proliferation of GMPs both in vitro and in vivo and demonstrates that calcineurin-NFAT signaling in GMPs is initiated by Flt3-L. Inhibition of the calcineurin-NFAT pathway modified expression of the cell cycle regulation genes Cdk4, Cdk6, and Cdkn1a (p21), thus enabling rapid cell cycle progression specifically in GMPs. NFAT inhibitor drugs are extensively used in the clinic to restrict the pathological activation of lymphoid cells, and our data reveal for the first time that these therapies also exert potent effects on maintenance of the myeloid cell compartment through specific regulation of GMP proliferation.