Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation

Abstract

Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-Protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways.

New insights into mammalian signaling pathways using microfluidic pulsatile inputs and mathematical modeling

“…5C (i)). These results suggest selective high pass filtering of downstream signal activation, similar to calcium-NFAT signaling in the absence of receptor regulatory kinetics 13 . Thus, oscillatory signals are integrated at different paces downstream, resulting in distinct sets of transcriptional activation and fate regulation.…”

“…Time scales for these bursts vary widely from milliseconds to several hours. Consequently, downstream activation of effector molecules may vary significantly for time varying inputs as compared to step changes 1,13,14 . Microfluidics allows the study of cells under controlled fluid flow, in particular, under controlled ligand stimulation 5,15 .…”

“…Several studies have shown that transcription factor activation and gene transcription vary significantly between step changes and pulsatile stimulation 1,13,14,22 . The dynamics of activation and deactivation of transcription factors, or nuclear translocation and back-translocation, may determine whether a continuous or a pulsatile stimulation leads to higher activity.…”

“…Several mammalian 1,4,13,14,21,23,24,35,42–46 as well as non-mammalian systems including bacteria 47–49 , amoeba 50 , yeast 51,52 , and nematodes 22,53 have been investigated. Here we discuss in detail a few recent examples from mammalian signaling pathways, but also mention briefly examples from non-mammalian systems.…”

“…To gain mechanistic insights into the downstream signal regulation, we utilized pulsatile stimulation of HEK293 cells stably expressing M3 receptors to generate synchronized calcium oscillations of varying frequencies (fast (R = 24s), intermediate (R = 72 s) and slow (R = 144 s)) 13 . Using two downstream readouts – cytoplasmic calcium and calcium induced NFAT (nuclear factor of activated T-Lymphocytes) nuclear translocation, we investigated the response dynamics under frequency and amplitude-modulated signals (Fig.…”