NF-κB Dynamics Discriminate between TNF Doses in Single Cells

Abstract

Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune their cytokine responses beyond the decision to activate.

Keywords: NF-κB; TNF; information theory; live-cell imaging; signal transduction; single cell; transcription factor.

Figure 1. Population-level data cannot distinguish between switch-like and graded response mechanisms

(A) Schematic for (M1) a switch-like mechanism for activation where a cytokine dose increases the probability of an all-or-nothing response in each cell; (M2) a graded mechanism for single-cell activation in which each cell’s response is graded in proportion with increasing cytokine dose; and (M3) a combined threshold with graded mechanism where single cells responses are graded in proportion with cytokine dose only if the dose is greater than the cell’s threshold for activation. (B) Simulated responses of single cells modeled with extrinsic noise (described in Figure S1). Each cell is initialized in a unique ‘cell state’ that approximates its responsiveness to cytokine. Responses to 4 doses are compared in each model plotted as a raw response (left), or a normalized response that divides each cell’s response by its ‘cell state’ (middle). The fraction of non-responder cells is quantified for each dose (right). (C) Three mechanistically distinct models are indiscernible when using the average of single cell responses to approximate a population-level measurement. See also, Figure S1.

Figure 2. Heterogeneity of responses to TNF between cell lines and single cells

(A) Time courses for average nuclear RelA from fixed cells are shown for a panel of human cell lines exposed to indicated concentrations of TNF continuously or as a single 1-minute pulse (solid orange curve). On average, 11374 single cells were measured across the time points for each cytokine condition. Light colored lines indicate the standard deviation of approximately 1274 cells measured at each time point. Time points for fixation included 0, 10, 30, 60, 90, 120, 180, 240, and 360 minutes following exposure to TNF. (B) Descriptors used to quantify the response of cells to a cytokine. F_i, F_max, and F_f respectively describe the initial, maximal, and final amount of nuclear RelA fluorescence. AUC defines the area under the curve for the cytokine response and ΔAdapt quantifies the deviation from a perfect adaptive response. Rate_in and Rate_out quantify the maximal rate of nuclear entry and exit respectively for average of fixed-cell data. (C) Heatmaps for each descriptors quantified in a panel of cell lines exposed to indicated cytokine conditions. Formulae used to calculate ΔAdapt and fold change (Fold) are shown. See also, Figure S2. (D) Time-lapse images of FP-RelA stably expressed in KYM1 cells exposed to a single 1-minute pulse of 1ng/mL TNF; scale bar, 10 μm, see also Movie S1. (E) Time courses of nuclear FP-RelA density measured in single cells treated with a 1-minute pulse of 1 ng/mL TNF. See also, Figure S2 and Table S1.

Figure 3. Information Transmission Capacity of the TNF-NF-κB pathway

(A) Density plots of single-cell FP-RelA time courses for responses to TNF with indicated concentration and duration. Median of single-cell responses for each condition is shown in blue. Inset numbers indicate the total number of single-cell time courses collected (black), the number of cells with a significant amount of FP-RelA translocation (red or pink), and the fraction of non-responders (NR) for each condition. (B) Channel capacity values calculated for each data set: (dark blue) ‘Raw’ and ‘Fold’ data sets where each single-cell time course is represented in arbitrary units or fold change (Figure S3A); (light blue) ‘NRR’, data sets where time courses for non-responder cells are removed, the ‘Fold cont.’ data set only includes conditions from the Fold-NRR with continuous exposure to TNF (bottom row of panel A); (red) Average of 20 subsample control data sets where the same number of cell trajectories are removed from the ‘Fold’ data set as in the NRR, but cells were either ‘Randomly Selected’ (Fold RS) or ‘Responding cells were targeted for Removal’ (Fold RR) (See STAR methods). For all data sets, conditions with fewer than 100 responder cells (pink numbers in panel A) were removed from channel capacity calculations; p < 10⁻¹², t test. (C) Channel capacity values for scalar descriptors of FP-RelA dynamics (p ≪ 10⁻¹³, t test). Error bars represent standard deviation. See also Figure S3.

Figure 4. Repeat TNF Stimulation Reveals a Graded Mechanism of Dose Discrimination in Single Cells

(A) Time-lapse images of FP-RelA in live cells stimulated with a 1 minute reference pulse of 0.2 ng/mL TNF, followed by a 1 minute pulse with 5x the reference dose (1 ng/mL TNF). Same cells are marked; scale bar, 10 μm; see also Movie S2. (B) Representative single-cell time courses for the cells labeled in (A). Inset shows schematic of area under the curve calculations for single-cell responses to the first (AUC1) and second (AUC2) TNF pulse (C) Density plots of single-cell FP-RelA time courses for cells exposed to a reference TNF pulse followed by a range of increasing test doses. Median of single-cell responses is shown in blue and inset numbers indicate the number of single cell time courses collected in each condition. (D) Scatter plots showing AUC2/AUC1 stratified along AUC1 across the range of test conditions. Colored bar along top depicts bins of single cells based on AUC1 into an approximately equal number of cells per condition. (E) Average response of single cells (AUC2) to increasing ‘test pulse’ concentrations (0, 0.2, 1, 10, or 100 ng/mL TNF) for each bin in panel D; inset describes hill coefficients and goodness of fit for logistic regression. See also, Figure S4 and Table S2.