Tuesday 16th August 2022

A variable-gain stochastic pooling motif mediates information transfer from receptor

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INTRODUCTION

A limited number of transmembrane receptors expressed on the cell surface mediate crucial transmission of information between extracellular and intracellular signaling molecules. Key questions are understanding the mechanisms and limitations that underlie signal transmission, in particular for cytokine receptor signaling that is often deregulated in disease. The nuclear factor κB (NF-κB) signaling pathway is an archetypal molecular communication channel that transmits information about extracellular cytokines to regulate cellular adaptation through activation of the RelA transcription factor (13). When ligated with inflammatory molecules, such as tumor necrosis factor (TNF) and interleukin-1β (IL-1), many activated receptors converge on NF-κB signaling (4, 5).

Ligation of TNF to the TNF Receptor 1 (TNFR1) recruits adaptor proteins and enzymes to form a large multiprotein complex near the plasma membrane (69). Ubiquitin-modifying enzymes are critical components that assemble linear, branched, and mixed polyubiquitin scaffolds around the multiprotein complex (1013). The NF-κB essential modulator (NEMO) subunit of the cytoplasmic IκB kinase (IKK) complex is rapidly recruited via direct interaction with the polyubiquitin scaffold and accessory proteins, where IKK is activated through induced proximity with regulatory kinases (4, 1416). The fully assembled TNFR1 complex, referred to as “complex I” [CI; (6)], is a master regulator of inflammation-dependent NF-κB signaling. Although other inflammatory molecules such as IL-1 signal through CI-like complexes using different receptors, adaptor proteins (17, 18), and varying compositions of ubiquitin chain scaffolds (19, 20), all regulate NF-κB through IKK activation mediated by induced proximity with other signaling mediators that reside on CI (21).

When observed in single cells exposed to inflammatory stimuli, the RelA subunit of NF-κB encodes a dynamic transcriptional signal by translocating from the cytoplasm into the nucleus (2, 3, 2224). Models calibrated to single-cell RelA data (2326) have revealed numerous transcriptional mechanisms and emergent properties that place the NF-κB pathway among exemplars of dynamical biological systems (27, 28). Key to these findings are two mediators of negative feedback, IκBα and A20, which are transcriptionally regulated by NF-κB. IκBα restores NF-κB to its baseline cytoplasmic localization through nuclear export and sequestration, whereas A20 diminishes kinase activation upstream of NF-κB through disassembly of CI-like structures in addition to noncatalytic mechanisms (10, 23, 25, 26, 29). Dynamical regulation of transcription and feedback via NF-κB is strongly recapitulated between models and experiments; however, there is a dearth of quantitative single-cell data at the level of cytokine detection and dynamical properties of CI-like complexes to substantiate our understanding of upstream signal transmission.

Here, we develop genetically modified cells that endogenously express fluorescent protein (FP) fusions of NEMO and RelA, allowing same-cell measurements of CI-like structures and canonical NF-kB signaling from live-cell images. We establish differences between TNF and IL-1 responses in biophysical properties of NEMO complexes and demonstrate a continuum relating CI-like structures and downstream NF-kB responses in the same cell. By tracking single complexes, we demonstrate that (i) cytokine dosage and time-varying presentation modulate the timing and numbers of CI-like structures, (ii) single complexes have switch-like activation profiles where the aggregate of NEMO recruitment and time-varying properties of each complex are cytokine specific, and (iii) dynamics of formation and dissolution for single complexes during the primary cytokine response are independent of transcriptional feedback. Last, we characterize a signaling motif called a variable-gain stochastic pooling network (SPN) that encompasses our experimental observations. The variable-gain SPN (VG-SPN) motif has beneficial noise mitigation properties and provides a trade-off between information fidelity, ligand specificity, and resource allocation for intracellular signaling molecules. We propose that the VG-SPN architecture and its associated benefits to signal transmission are common mechanisms for receptor-mediated signal transduction.

RESULTS

Surface receptor expression is limiting for numbers of cytokine-induced signaling complexes

IKK activity is a convergence point for proinflammatory signals that regulate NF-κB downstream of many cytokine receptors (26, 30). Ligands that bind to multiple receptors with differing kinetics (31) and decoy receptors that sequester or antagonize signaling complexes (32) layer additional complexity to signal initiation at the plasma membrane. To establish expectations for numbers and types of IKK-activating complexes, we measured surface receptor expression in U2OS cells that were previously shown to form dynamic IKK puncta in response to TNF and IL-1 (19, 33). Using flow cytometry with reference beads for absolute quantification, we estimated the number of surface receptors per cell for TNFR1, TNFR2, IL-1R1, IL-1R2, and IL-1R3 (Fig. 1 and fig. S1). On average, each U2OS cell presented approximately 1300 TNFR1, 700 IL-1R1, and an abundance of IL-1R3 surface receptors. Only a small number of TNFR2 and IL-1R2 were detected on the cell surface. For reference, we measured surface receptors on HeLa and KYM1 cells (fig. S1) and found results consistent with previous reports for TNFRs (3436) and agreement with surface receptor expression in other cell lines (3740).

<a rel="nofollow" href="https://advances.sciencemag.org/content/advances/7/30/eabi9410/F1.large.jpg?width=800&height=600&carousel=1" title="Differential expression of cytokine receptors enables cells to selectively respond to their environment. (A) Schematic of cognate receptors for TNF and IL-1 cytokines. Monomeric receptors and receptors that engage with decoy receptors (IL-1R2) are inactive and do not transmit signals into the cytoplasm (left). Activated receptor complexes (right), consisting of a TNFR homotrimer bound to TNF or an IL-1R1–IL-1R3 heterodimer bound to IL-1, are capable of seeding CI-like complexes in the cytoplasm. (B) Quantification of surface receptor expression on single U2OS cells. The average of three to seven biological replicates is shown for each condition. Error bars represent SEM." class="fragment-images colorbox-load" rel="gallery-fragment-images-1850456189" data-figure-caption="

Fig. 1 Differential expression of cytokine receptors enables cells to selectively respond to their environment.

(A) Schematic of cognate receptors for TNF and IL-1 cytokines. Monomeric receptors and receptors that engage with decoy receptors (IL-1R2) are inactive and do not transmit signals into the cytoplasm (left). Activated receptor complexes (right), consisting of a TNFR homotrimer bound to TNF or an IL-1R1–IL-1R3 heterodimer bound to IL-1, are capable of seeding CI-like complexes in the cytoplasm. (B) Quantification of surface receptor expression on single U2OS cells. The average of three to seven biological replicates is shown for each condition. Error bars represent SEM.

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Fig. 1 Differential expression of cytokine receptors enables cells to selectively respond to their environment.

(A) Schematic of cognate receptors for TNF and IL-1 cytokines. Monomeric receptors and receptors that engage with decoy receptors (IL-1R2) are inactive and do not transmit signals into the cytoplasm (left). Activated receptor complexes (right), consisting of a TNFR homotrimer bound to TNF or an IL-1R1–IL-1R3 heterodimer bound to IL-1, are capable of seeding CI-like complexes in the cytoplasm. (B) Quantification of surface receptor expression on single U2OS cells. The average of three to seven biological replicates is shown for each condition. Error bars represent SEM.

Although activated TNFR1 and TNFR2 both form TNF-induced homotrimeric complexes, the TNFR2 subtype binds with lower affinity to soluble TNF and shows enhanced activation by membrane-bound TNF (31, 41). In contrast, ligand-activated IL-1 receptor (IL-1R1) forms a heterodimer with the IL-1R3 accessory protein, and dimerization can be inhibited through competitive sequestration by the IL-1R2 decoy (Fig. 1A) (32). Because surface expression of TNFR2 and IL-1R2 are comparably low in U2OS, receptor composition of TNF- and IL-1–induced oligomers will consist predominantly of TNFR1 trimers and IL-1R1–1R3, respectively. Together with known receptor-ligand stoichiometry (Fig. 1A), our results predict that single cells can simultaneously form a maximum of hundreds of IKK-recruiting complexes for saturating cytokine concentrations (approximately 400 and 700 for TNF and IL-1, respectively). Surface receptor expression is substantially lower than numbers for downstream signaling molecules such as NEMO that are expressed in orders of a million per cell (42).

Cytokine-specific and dose-specific modulation of NEMO complex features

We set out to investigate how cytokine receptors engage NEMO as an integration hub to regulate NF-κB signaling. To counteract effects of NEMO overexpression, which can strongly inhibit NF-κB…

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