Dynamic Response Diversity of NFAT Isoforms in Individual Living Cells Nissan Yissachar, Tali Sharar Fischler, Ariel A. Cohen, Shlomit Reich-Zeliger, Dor Russ, Eric Shifrut, Ziv Porat, Nir Friedman  Molecular Cell  Volume 49, Issue 2, Pages 322-330 (January 2013) DOI: 10.1016/j.molcel.2012.11.003 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 49, 322-330DOI: (10.1016/j.molcel.2012.11.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Frequency-Modulated Nuclear Localization Bursts of NFAT4 in Response to Extracellular Calcium (A) Schematics of NFAT dephosphorylation and nuclear translocation in response to IgE-mediated mast cell activation or calcium stimulation. (B) Fluorescence images of endogenous or GFP-tagged NFAT1 and NFAT4 in RBL-2H3 cells, following 50 mM calcium stimulation. Arrows indicate cells with nuclear NFAT4. Green, NFAT1/NFAT4; blue, Hoechst nuclear staining. (C) Fraction of cells showing nuclear NFAT with or without calcium stimulation. Cells were fixed at several time points following calcium addition. Totals of 730 and 1,270 cells were counted for NFAT1 and NFAT4, respectively, and the percentage of cells showing nuclear NFAT was calculated. (D) Filmstrip showing two RBL-2H3 cells expressing GFP-NFAT4 before and after addition of 50 mM extracellular calcium (yellow square). Cells show unsynchronized short nuclear localization bursts. Frames are separated by 4 min. (E–G) The nuclear localization value was calculated as the ratio between nuclear and cytoplasm intensities (Figure S1E). (E and F) Single-cell time traces showing NFAT4 nuclear localization in response to 25 mM (E) or 50 mM (F) extracellular calcium. (G) Single-cell time traces showing no change in NFAT1 localization in response to 25 mM (top) or 50 mM (bottom) extracellular calcium. (H) Frequency of NFAT4 nuclear localization bursts increases with calcium concentration. Error bars represent SEM. (I) Fraction of NFAT4 responding cells increases with calcium concentration. The criterion for a “responding cell” is nuclear localization observed at any time point throughout the experiment (Figure S1F). (H) and (I) show the average of two independent experiments. Molecular Cell 2013 49, 322-330DOI: (10.1016/j.molcel.2012.11.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Dynamic Diversity in the Response of NFAT1 and NFAT4 to IgE-Mediated Mast Cell Activation (A and B) Single-cell time traces showing NFAT1 (A) and NFAT4 (B) nuclear localization in response to activation with low or high antigen doses. (C and D) Population average time traces for NFAT1 (C) and NFAT4 (D) nuclear localization in response to activation with various antigen doses. Each line represents the average nuclear localization for a population of cells, activated with a given antigen concentration. The nuclear localization value in (A–D) was calculated as the ratio between nuclear and cytoplasm intensities (Figure S1E). (E) Antigen concentration modulates the amplitude of NFAT1, but not of NFAT4 nuclear localization. Dots represent the median response amplitude for NFAT1 (blue) and NFAT4 (red) in a population of cells activated with a given antigen concentration. Error bars represent SEM. (F and G) Antigen concentration modulates the response time of NFAT4 (G), but not of NFAT1 (F). Black and blue dots show measured response times for single cells from two independent experiments. Red dots represent the median response time. (H) Response duration of NFAT1 (blue) and NFAT4 (red) similarly grows with antigen dose. (I) Average autocorrelation function of NFAT1 (blue) and NFAT4 (red) responses to high doses of antigen concentration (50 ng/ml). Dashed lines: fit with one (NFAT1) or sum of two (NFAT4) exponential decay functions. Molecular Cell 2013 49, 322-330DOI: (10.1016/j.molcel.2012.11.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 NFAT1 Shows an Integral and Homogenous Impulse Response, Whereas NFAT4 Shows a Proportional, All-or-None Impulse Response (A) Representative single-cell traces showing the response of NFAT1 and NFAT4 to pulses of 2, 5, 10, and 30 min of antigen at two doses (low, 0.78 ng/ml; high, 50 ng/ml). Right column: Population average time traces of these impulse responses. Trace colors correspond to the different pulse durations, as represented by the colored bars. The nuclear localization value was calculated as the ratio between nuclear and cytoplasm intensities (see Figure S1E). (B) Fraction of cells showing nuclear localization of NFAT1 and NFAT4 in response to pulses of low and high antigen doses of different duration, and to a continuous signal. Error bars represent SD of the average of two independent experiments. (C and D) Nuclear import of endogenous NFAT1 (C) and NFAT4 (D) in response to 50 ng/ml of antigen. Cells were fixed 0, 2, 5, 10, 30, or 45 min following activation, and NFAT nuclear localization was analyzed using ImageStreamX flow-microscopy. The y axis shows the similarity index between NFAT staining and nuclear staining (Hoechst), which is a measure for NFAT nuclear localization (see Supplemental Experimental Procedures and Figures S3A and S3B). Mean similarity is presented at each time point (dots), normalized by the mean similarity at t = 0 (no antigen present). For exponential fit function and parameters (black line) see Supplemental Experimental Procedures. (E) Mean normalized similarity (NFAT to nuclear stain, as in [C] and [D]) of endogenous NFAT1 and NFAT4 30–45 min following stimulation with 0.78 or 50 ng/ml of antigen. Molecular Cell 2013 49, 322-330DOI: (10.1016/j.molcel.2012.11.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Dynamic Response Diversity of Isoforms Responding to the Same Signal can Allow for Increased Range of Temporal Signal Processing Logics by Cells In this schematic figure, input signal (top) represents four potential dynamic cases: a transient pulse (i), a step-like increase (ii), a short gap in signal (iii), and a noisy signal (iv). Middle: nuclear localization of NFAT1 (integral) and NFAT4 (proportional) in response to the signal. Bottom: expression level of a target gene for three cases. Bottom left, regulation only by NFAT1; bottom center, regulation by both isoforms; bottom right, regulation only by NFAT4. The range of possible dynamic responses is larger than that possible by each isoform alone. Combination of NFAT1 and NFAT4 dynamics can enable an asymmetric cellular response, which is sensitive and fast on the one hand, but can filter out signal gaps and noise on the other hand. Molecular Cell 2013 49, 322-330DOI: (10.1016/j.molcel.2012.11.003) Copyright © 2013 Elsevier Inc. Terms and Conditions