Cells can modify their future behavior based on prior exposures, a phenomenon known as cellular adaptation. In innate immunity, such adaptive responses are critical for fine-tuning host defense, enabling trained immunity or tolerance. These states have been largely attributed to long-lasting epigenetic or metabolic reprogramming. Whether prior microbial encounters can rapidly lower receptor signaling thresholds to selectively enhance responsiveness of individual innate immune receptors remains unknown. Here, we identify a previously unrecognized, non-genetic form of cellular adaptation. We show that TLR4 activation by LPS, while inducing classical endotoxin tolerance, simultaneously primes macrophages for enhanced responses to subsequent activation of the RNA sensor TLR7. Because TLR4 and TLR7 use the same signaling machinery, this indicates that they are selectively reprogrammed in this process. TLR7 training requires type I interferons and is marked by a strong increase in Myddosome assemblies at endosomal membranes, without changes in TLR7 abundance or ligand uptake. These findings reveal that innate immune training can be rapidly encoded at the level of receptor-proximal signaling, linking microbial priming to enhanced nucleic acid immunity and potentially to TLR7-driven autoimmunity in genetically predisposed individuals. Short summary LPS priming drives receptor-proximal immune adaptation in macrophages, inducing classical TLR4 endotoxin tolerance while simultaneously sensitizing the RNA sensor TLR7 by enhancing Myddosome nucleation at endosomal membranes. This compartment- and receptor specific reprogramming may link prior infection history to elevated risk of TLR7-driven autoimmunity. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, MA 9812/3-1 to O.M. Max Planck Society, https://ror.org/01hhn8329 Cincinnati Children's Hospital Medical Center (O.M.)

Protein polymer scaffolds composed of death fold (DF) proteins are critical to the formation of signalosomes in immune signaling. The biophysical properties that these polymeric scaffolds require for signal transduction are not clearly defined. Here, we engineered single-component DF signalosomes. We found that functionality depends on the stability provided by the DF polymer, which could also be achieved with a bacterial DF domain, a synthetic filament-forming domain, and amyloid-like sequences. This demonstrates the importance of polymer stability and inducibility irrespective of the motif's origin. By varying the number of included TRAF6 interaction motifs, we demonstrate that avidity is a tunable property that can control the amplitude of signaling outputs. This work lays out a reductionist framework to elucidate the required signaling properties through polymeric scaffolds by adjusting their assembly kinetics, stability, and avidity.

A key feature of innate immune signaling is the compartmentalization of signaling effectors into cellular structures referred to as signalosomes. Critical to the formation of these compartments are protein polymers composed of Death Domains (DD). However, the biophysical properties these polymeric scaffolds require for signal transduction are not clearly defined. Here, we engineered a single-component signalosome, referred to as Chimeric Higher-order Assemblies for Receptor Mediated Signaling (CHARMS). We found that CHARMS functionality depends on the stability provided by the DD polymer, which could also be achieved with bacterial DDs and synthetic filament-forming domains. This demonstrates the importance of kinetic stability and inducibility, irrespective of the origin of the motif. By varying the multiplicity of TRAF6 interaction motifs, we demonstrate that avidity is a tunable property that can control the amplitude of signaling outputs. This work lays out a reductionist framework to dissect the required properties of signaling through polymeric scaffolds by adjusting their assembly kinetics, stability and avidity.

We developed a system for optogenetic release of single molecules in live cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for the controlled delivery of functional proteins to cytosol and plasma membrane in amounts compatible with single molecule imaging, greatly simplifying access to single molecule microscopy of any protein in live cells. Furthermore, we could reconstitute cellular functions such as ion conductance by delivering BK and VRAC ion channels to the plasma membrane. Finally, we could induce NF-kB signaling in T-Lymphoblasts stimulated by IL-1 by controlled release of a signaling protein that had been knocked-out in the same cells. We observed light induced formation of functional inflammatory signaling complexes that could trigger IKK phosphorylation in single cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single molecule level.

The Myddosome is an oligomeric protein complex composed of MyD88 and members of IL-1 receptor-associated kinase (IRAK) family that transduce signals from Toll-like and IL-1 family receptors. The molecular dynamics of Myddosome formation and how the Myddosome organizes downstream signaling reactions provide insight into how TLR/IL-1Rs activate a decisive cellular response critical for the induction of inflammation. Supported lipid membranes formed on a continuous glass coverslip have been extensively used to study the molecular dynamics of receptor signaling. Here, we describe a protocol for the formation of IL-1-functionalized support lipid membrane that can be used to visualize the molecular dynamics of Myddosome formation and signaling in live cells.

IL-1 receptor (IL-1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, how these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. During IL-1R signaling, Myddosomes dynamically reorganize into multi-Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF-kB activation. Myddosomes function as scaffolds that assemble an NF-kB signalosome consisting of E3-ubiquitin ligases TRAF6 and LUBAC, K63/M1-linked polyubiquitin chains, phospho-IKK, and phospho-p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10-fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL-1R signaling.

The controlled oligomerization of signaling proteins is an essential feature of many inflammatory signaling pathways. An example is IL-1 receptor signaling, which relies on the oligomerization of the Death Domain (DD)-containing proteins MyD88 and IRAK family kinases. This process leads to the assembly of the Myddosome signaling complex, and disrupting assembly holds potential for anti-inflammatory treatments. However, IRAKs’ signaling activity is also regulated by auto-/trans-phosphorylation, and it is unclear if these processes operate at or downstream of Myddosome assembly. Here, we find that the initial stage of Myddosome assembly is solely controlled by MyD88:IRAK4 DD interactions. In later stages, IRAK4 auto-phosphorylation serves as a switch, regulating IRAK1/2/3 incorporation and DD oligomerization. Small molecule inhibitors of IRAK4 kinase activity block this later stage of assembly, explaining how they dampen inflammatory signaling. Our data reveals IRAK4 auto-phosphorylation as an energy-dependent switch activating the heterotypic assembly of IRAKs’ DDs and downstream inflammatory IL-1 signaling. This highlights how a signaling cascade integrates phosphorylation and protein oligomerization steps.### Competing Interest StatementMJT has research projects sponsored by Kymera Therapeutics.

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than fourMyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than 4 MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.

One of the great challenges in biology is to understand the emergence of spatial and temporal order in cellular processes. This challenge is particularly relevant to cell signaling, where the localized assembly and activation of protein complexes is used to control everything from cell fate to hormone release. While several decades of research have yielded many of the players in signaling pathways, most of the fundamental, conceptual questions of how signaling works at a molecular level remain mysterious. Immune cell activation represents an ideal system for understanding the spatial and temporal dynamics of cell signaling since the immune system faces an extreme version of many of the problems that all signaling systems must face: how to discriminate between closely related signals and activate the appropriate pathway even when the amount of signal is vanishingly small. In this paper, we have used high-resolution Total Internal Reflection Fluorescent (TIRF) imaging combined with functionalized supported lipid bilayers and engineered receptors and cell lines to visualize the molecular dynamics of immune signaling pathways. We will give details of our experiments that have revealed how the T cell receptor and IL1 receptor signaling networks activate an immune response.

Programmed cell death-1 (PD-1) is a coinhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). By titrating PD-1 signaling in a biochemical reconstitution system, we demonstrate that the co-receptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1-recruited Shp2 phosphatase. We also show that CD28, but not the TCR, is preferentially dephosphorylated in response to PD-1 activation by PD-L1 in an intact cell system. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways play key roles in regulating effector T cell function and responses to anti-PD-L1/PD-1 therapy.

T cells mount an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.

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T cells mount an immune response based upon the strength of the interaction between its T cell receptor (TCR) and peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells become activated by some pMHCs, but not others with slightly shorter bound times, remains an unanswered question. Here, we developed a finely tunable synthetic receptor-ligand system in which the extracellular domains of the TCR and pMHC were replaced with two short hybridizing strands of DNA. Remarkably, the T cell signaling response can discriminate between DNA ligands differing by a single base pair. Single molecule imaging reveals that ligated receptors with longer bound times promote the binding of more ligands nearby to create clusters; stable clusters of 2-4 ligated receptors then trigger receptor phosphorylation and signaling. These results reveal how T cells convert the bound time of an extracellular receptor-ligand interaction into a physical reorganization of molecules in the membrane and intracellular signaling.

Precise control over interfacial chemistry between nanoparticles and other materials remains a major challenge that limits broad application of nanotechnology in biology. To address this challenge, we used 'steric exclusion' to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping each QD with a functionalized oligonucleotide. We demonstrated the utility of these QDs as modular and nonperturbing imaging probes by tracking individual Notch receptors on live cells.

Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin-enhanced green fluorescent protein (EGFP) and actin-binding protein-EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.

Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.