Biology subjects

Miller, T.

Publications and source records attributed to Miller, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Batch Effect Correction to Enhance Utility of a Publicly Available ALS Proteomics Dataset

Large, publicly available databases of patient data have been successful in identifying associations between neurodegenerative diseases and biological features that might otherwise go unnoticed. The Answer ALS Consortium is one such database that has generated a rich repository of multi-omic data from people with amyotrophic lateral sclerosis (ALS). However, large studies can suffer from batch effects that obscure genuine biological signal and prevent experiment reproducibility. In this study, we identified a batch effect in the Answer ALS 1-2 proteomic dataset (508 samples x 3,643 proteins) and corrected the effect using both the internal controls provided by Answer ALS and the batch effect-correction algorithm (BECA) ComBat. We identified a batch effect in the Answer ALS Dataset 1-2 proteomic dataset that, uncorrected, may obscure biology. Proteins MOS and H4 were outliers of expression in the dataset that correlated with the batch effect. Removing MOS and H4 did not correct the effect fully but did reveal new principal component (PC) contributing proteins related to motor neurons and potentially ALS: AINX, NFL, and NFM. The batch differential controls (BDCs) provided by Answer ALS were used to correct samples in their respective batches. ComBat, a well-characterized BECA, was applied to the dataset both before (ComBat-BDC) and after (BDC-ComBat) correction with the BDCs. BDC-ComBat was the only method that successfully reduced batch effect across the entire dataset. This method also preserved four of the top six PC-contributing proteins from the original dataset, including three neuronal proteins, suggesting that the corrected dataset recapitulates biology as well as the variability of the original dataset. Altogether, we produced a version of the Answer ALS proteomic dataset (354 samples by 3,641 proteins) with globally reduced batch effect and protein-level reduced batch effect. This version of the dataset recapitulates the individual protein variability and important neuronal proteins of the original. We also created a workflow for identifying and correcting batch effects that can be applied to other large data repositories.

bioinformatics↗

bioRxiv2026

Pangenome Analysis Reveals Gene Content Variation and Evolutionary Dynamics Across the Culex pipiens Species Complex

The Culex pipiens species complex contains the principal northern-hemisphere vectors of West Nile virus, lymphatic filariasis and several arboviruses; yet, the genomic basis of its diversity remains poorly resolved. We present the first pangenome characterization of the four widely recognized Cx. pipiens complex forms (Cx. pipiens, Cx. pallens, Cx. quinquefasciatus, Cx. molestus), with Cx. tarsalis as outgroup. From chromosome-scale assemblies (533-790 Mb) and unified annotations transferred from the Cx. quinquefasciatus reference, we identified 16,568 orthogroups. Restricted to the ingroup, 92,821 genes (98.2 %) were partitioned into 11,284 core (71.1 %), 3,726 shell (23.5 %) and 871 cloud (5.5 %) orthogroups. Form-specific cloud orthogroups were unevenly distributed ({chi}{superscript 2} = 71.9, p = 1.7 x 10-{superscript 1}), with Cx. molestus carrying the largest set. A concatenated 8,067-locus phylogeny placed Cx. pallens and Cx. quinquefasciatus as sisters with full bootstrap support but only 58.9 % gene-tree and 45.1 % site concordance at the deep ingroup node, a pattern consistent with prior evidence that Cx. pallens arose through hybridization between Cx. pipiens and Cx. quinquefasciatus. CAFE5 detected 582 gene families with significant rate departures, and inferred expansions outnumbering contractions on every lineage (counts across all orthogroups with an inferred size change; Cx. quinquefasciatus greatest, 3,733 expanded vs. 729 contracted). Whole-genome ANI ranged 93.1-94.7 % (skani); synteny was conserved at the chromosome scale (74-77 % collinear anchors) with 258-294 inversions [≥] 100 kb per pair. Transposable elements occupied a uniform genome fraction (52.7-54.4 %). These results refine the divergence framework for the complex, provide evidence consistent with a hybrid origin of Cx. pallens, and establish a pangenomic baseline for future population-level resequencing. HighlightsO_LIFirst chromosome-scale pangenome of the Culex pipiens species complex. C_LIO_LI16,568 orthogroups partitioned into core, shell and cloud compartments. C_LIO_LIPhylogenomic discordance supports a hybrid origin for Cx. pallens. C_LIO_LICAFE5 reveals strong, lineage-wide gene-family expansion across the complex. C_LIO_LIConserved synteny and [~]53% transposable-element content across all genomes. C_LI

genomics↗

bioRxiv2026

ZATT/ZNF451 promotes release of stalled TOP2 cleavage complexes

Topoisomerase II (TOP2) resolves DNA topological constraints through a tightly regulated cycle of DNA double-strand cleavage and religation. Nearby DNA damage or chemotherapeutic agents such as etoposide block the DNA religation step, stabilizing TOP2-DNA cleavage complexes (TOP2ccs) at DNA double-strand breaks (DSBs). The SUMO E3 ligase ZATT (ZNF451) has recently emerged as a key effector of TOP2cc repair, but its mechanism of action remains poorly understood. Here, we show that ZATT is sufficient to resolve TOP2ccs independently of TDP2, TOP2 proteolysis, and canonical DSB repair pathways. Using Xenopus egg extracts and biochemical reconstitution, we find that ZATT salvages trapped TOP2 by promoting TOP2 release from its stalled cleavage complex. Structural modeling and targeted mutagenesis in Xenopus egg extracts and human cells identify a highly conserved hydrophobic pocket in the tower domain of TOP2 where the ZATT coiled-coil "hooks on" to promote TOP2cc resolution. Our findings reveal a new strategy to resolve TOP2ccs that bypasses the exposure of dangerous DNA breaks.

molecular biology↗

bioRxiv2026

Evaluating Spike Antigenicity across Endemic Human Coronavirus Models using Flow Virometry

While SARS-CoV-2 research has advanced rapidly since COVID-19, endemic human coronaviruses (HCoVs) remain comparatively understudied. Tools to phenotype spike (S), the primary antigenic target on coronaviruses, at the single-virion level could improve vaccine design by capturing variation in epitope availability and spike abundance. Here, we establish a calibrated flow virometry (FV) platform to quantify S antigenicity on native endemic (HCoV-229E, HCoV-OC43) and epidemic (SARS-CoV-2) coronaviruses directly in cell culture supernatants. FV revealed cell line-dependent differences in S antigenicity, including receptor-induced changes in epitope accessibility. Comparison of virion-associated S with recombinant stabilized S by ELISA and biolayer interferometry showed consistent binding for HCoV-OC43, MERS-CoV, and SARS-CoV-2, but differences for HCoV-229E, with FV resolving heterogeneity not captured by bulk assays. Finally, FV showed that HCoV-229E from patient-derived air-liquid interface cultures exhibited reduced antibody binding and distinct S antigenicity compared to cell line-derived virions. Together, these findings establish FV as a platform for single-virion analysis of HCoV antigenicity.

microbiology↗

bioRxiv2026

Intracellular TDP-43 amyloid nucleates from arrested nascent condensates

TDP-43 is a model protein for pathophysiological phase transitions, forming a multitude of intracellular assemblies with different physical properties. Physiological condensation is widely presumed to precede pathological aggregation, but the causal relationships between different modes of assembly in vivo are still unclear. Here we use Distributed Amphifluoric FRET (DAmFRET) and complementary approaches to map the phase space of TDP-43 self-assembly in yeast cells. We discovered that the low-complexity C-terminal domain (CTD) on its own partitions into soluble clusters that dynamically arrest en route to liquid-liquid phase separation. These clusters uniquely supported amyloid formation, and only when templated by pre-existing amyloids of other proteins. Self-interacting modules outside the CTD, whether in the full-length TDP-43, pathological C-terminal fragments, or fusion partners, all suppressed amyloid nucleation. They did this by promoting CTD condensation beyond the arrested state. Stress and cotranslational self-association had the same effect. We leveraged this property of condensation to stop CTD amyloid formation by co-expressing an oligomeric binder in cells. Our findings reveal that TDP-43 amyloid formation occurs only under very specific physical and biological circumstances that present new opportunities for therapeutic control.

biophysics↗

bioRxiv2025

Identification and performance of environmentally-driven recruitment relationships in state space assessment models

Environmentally-driven recruitment relationships are important for understanding fisheries responses to climate change; however they are difficult to estimate due in part to large variability in the recruitment process. State space models provide a promising way forward in allowing the characterization of multiple sources of stochastic variability. Here we conducted a large simulation-estimation study using environmentally-driven recruitment relationships to evaluate the effects of operating model characteristics on state space assessment model performance. We generally find low parameter and assessment bias across operating and estimating model combinations; however, some assessment and parameter bias is present under conditions of high recruitment variability and low spawning stock biomass contrast. Model identifiability for the correct functional form of the stock recruit relationship and the environmental relationship was generally poor, and projections were insensitive to assumed values of the environmental driver. We recommend the use of random effects on recruitment in state space assessments and caution against explicit stock-recruitment relationships. We encourage future work on environmental nonstationarity, which will be of increasing importance as exploited fish stocks experience accelerating rates of environmental change.

ecology↗

bioRxiv2025

High-frequency Wus2-integrated transformation toolkit enhances Cas9 editing efficiency and expands capability with SpRY in sorghum

Sorghum bicolor L. (Moench), the fifth most important cereal crop internationally, is used as food, feed, forage, and fuels. Importantly, sorghums natural tolerance to environmental stresses leads to tolerance to climate variation. To optimize sorghum germplasm for crop improvement requires highly efficient genetic transformation and genome editing; however, sorghum has historically been recalcitrant to these genetic approaches. In this study, we report a high-efficiency engineering toolkit, optimized for genome editing via Agrobacterium-mediated transformation. Using CRISPR/Cas9-based editing machinery, the genetic tools led to editing efficiencies up to 95.7% when monocistronic guide RNAs targeted the phytoene desaturase (SbPDS) gene. We also tested a PAM (protospacer adjacent motif)-broadened Cas9 variant, intronized SpRY (ZmSpRYi), achievling comparable editing efficiencies. Using this toolkit permits exploitation of advanced editing tools, like prime editors, base editors, and targeted knock-in methods. These genetic advances offer new methods for sustainable crop improvement in sorghum and potentially other cereals. Technology readinessThis study demonstrates the development of an integrated genome engineering platform that combines morphogenic gene-assisted transformation with CRISPR/Cas9- and SpRY-mediated mutagenesis for multiplex genome editing, establishing a Technology Readiness Level (TRL) of 4/5. This workflow enables the generation of allelic variants through editing of both functional and regulatory elements in the plant genome. Advancing toward TRL 5 will require additional validation in relevant sorghum genotypes, including elite varieties. Moreover, the PAM-flexible editor SpRY is still under optimization, particularly at non-canonical PAM sites, which may limit its application for the modification of certain cis-regulatory elements. Further improvements in precision genome editing, such as the integration of prime editing or targeted knock-in technologies, will also be necessary to enable desired and predictable modifications. With continued optimization and incorporation of these advanced tools, this robust genome engineering platform has the potential to accelerate plant breeding and enable basic research supporting plant-based solutions to address climate change.

plant biology↗

bioRxiv2025

A tunable affinity fusion tag for protein self-assembly

The concentrations of individual proteins vary between cells, both developmentally and stochastically. The functional consequences of this variation remain largely unexplored due to limited experimental tools to manipulate the relationship of protein concentration to activity. Here, we introduce a genetically encoded tool based on a tunable amyloid that enables precise control of protein concentration thresholds in cells. By systematically screening dipeptide repeats, we identified poly-threonine alanine (poly-TA) as an ideal candidate due to its unique ability to form amyloid-like assemblies with a negligible nucleation barrier at arbitrarily chosen concentration thresholds. We demonstrate that the saturating concentration (Csat) of poly-TA can be finely tuned by adjusting the length of uninterrupted TA repeats, even while maintaining length and composition, providing a modular system for manipulating protein solubility. This tool offers a powerful approach to investigate the relationship between protein concentration, phase separation, and cellular function, with potential applications in cell-, developmental-, and synthetic biology.

cell biology↗

bioRxiv2025

A tool to dissect heterotypic determinants of homotypic protein phase behavior

Proteins commonly self-assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each proteins sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self-assembly in cells. Here, we extend DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether and thereby recruit candidate modifier proteins to mEos3-fused query proteins. This accessorization allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valency of mEosNb-fused modifiers. We show that our system recapitulates known effects of multivalency on liquid-liquid phase separation and can discriminate between nucleation mechanisms of amyloid and amyloid-like assemblies. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions. Lay summaryProtein self-assemblies are essential for cellular function, but can also contribute to disease. We develop a new tool to study how their formation is influenced by other cellular factors. This tool allows us to control the location and number of interactions between a protein of interest and other proteins that may influence it. Our results provide new insight into mechanisms of self-assembly and will aid research toward treating diseases associated with aberrant assembly.

biophysics↗

bioRxiv2024

Simultaneous mapping of RNA secondary, tertiary, and quaternary structure in living cells by multi-site DMS probing

Identifying tertiary structures and protein binding sites on RNA molecules remains a key challenge in RNA biology. We report a new chemical probing strategy termed multi-site DMS-MaP (msDMS-MaP) that exploits base tautomerization during mutational profiling reverse-transcription to measure typically invisible DMS-induced modifications at the N7 position of G. These N7-G modifications are now resolved concurrently with conventional N1 and N3 modifications with only minor modifications to established protocols, providing a multi-dimensional, single-molecule readout of RNA structure. We show that N7-G reactivity specifically reports on RNA tertiary and quaternary structure, enabling identification of diverse, functionally significant motifs such as cooperatively folding tertiary domains and protein binding sites in living cells. We apply msDMS-MaP to obtain a map of the quaternary structural ensemble of the 7SK non-coding snRNP, revealing unique protein binding sites across three 7SK structural isoforms. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.

biochemistry↗

bioRxiv2024

Satellite glial contact enhances differentiation and maturation of human induced sensory neurons

Sensory neurons generated from induced pluripotent stem cells (iSNs) are used to model human peripheral neuropathies, however current differentiation protocols produce sensory neurons with an embryonic phenotype. Peripheral glial cells contact sensory neurons early in development and contribute to formation of the canonical pseudounipolar morphology, but these signals are not encompassed in current iSN differentiation protocols. Here, we show that terminal differentiation of iSNs in co-culture with rodent Dorsal Root Ganglion satellite glia (rSG) advances their differentiation and maturation. Co-cultured iSNs develop a pseudounipolar morphology through contact with rSGs. This transition depends on semaphorin-plexin guidance cues and on glial gap junction signaling. In addition to morphological changes, iSNs terminally differentiated in co-culture exhibit enhanced spontaneous action potential firing, more mature gene expression, and increased susceptibility to paclitaxel induced axonal degeneration. Thus, iSNs differentiated in coculture with rSGs provide a better model for investigating human peripheral neuropathies.

neuroscience↗

bioRxiv2024

Lipid-siRNA conjugate accesses a perivascular transport mechanism and achieves widespread and durable knockdown in the central nervous system

Short-interfering RNA (siRNA) has gained significant interest for treatment of neurological diseases by providing the capacity to achieve sustained inhibition of nearly any gene target. Yet, efficacious drug delivery throughout deep brain structures of the CNS remains a considerable hurdle for intrathecally administered therapeutics. We herein describe an albumin-binding lipid-siRNA conjugate that transports along meningeal and perivascular CSF pathways, leading to broad dispersion throughout the CNS parenchyma. We provide a detailed examination of the temporal kinetics of gene silencing, highlighting potent knockdown for up to five months from a single injection without detectable toxicity. Single-cell RNA sequencing further demonstrates gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for neurological disease-modifying therapies.

bioengineering↗

bioRxiv2024

Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine

KRAS, a small GTPase involved in cell proliferation and differentiation, frequently gains activating mutations in human cancers. For KRAS to function, it must bind the plasma membrane (PM) via interactions between its membrane anchor and phosphatidylserine (PtdSer). Therefore, depleting PM PtdSer abrogates KRAS PM binding and activity. From a genome-wide siRNA screen to identify genes regulating KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatases: myotubularin-related proteins (MTMR) 2, 3, 4, and 7. Here, we show that silencing MTMR 2/3/4/7 disrupts KRAS PM interactions by reducing PM PI 4-phosphate (PI4P) levels, thereby disrupting the localization and operation of ORP5, a lipid transfer protein maintaining PM PtdSer enrichment. Concomitantly, silencing MTMR 2/3/4/7 elevates PM PI3P levels while reducing PM and total PtdSer levels. We also observed MTMR 2/3/4/7 expression is interdependent. We propose that the PI 3-phosphatase activity of MTMR is required for generating PM PI, necessary for PM PI4P synthesis, promoting the PM localization of PtdSer and KRAS. eTOC summaryWe discovered that silencing the phosphatidylinositol (PI) 3-phosphatase, MTMR, disrupts the PM localization of PtdSer and KRAS. We propose a model, where MTMR loss depletes PM PI needed for PM PI4P synthesis, an essential phospholipid for PM PtdSer enrichment, thereby impairing KRAS PM localization.

cell biology↗

bioRxiv2023

Protein supersaturation powers innate immune signaling

How minute pathogenic signals trigger decisive immune responses is a fundamental question in biology. Classical signaling often relies on ATP-driven enzymatic cascades, but innate immunity frequently employs death fold domain (DFD) self-assembly. The energetic basis of this assembly is unknown. Here, we show that specific DFDs function as energy reservoirs through metastable supersaturation. Characterizing all 109 human DFDs, we identified sequence-encoded nucleation barriers specifically in the central adaptors of inflammatory signalosomes, allowing them to accumulate far above their saturation concentration while remaining soluble and poised for activation. We demonstrate that the inflammasome adaptor ASC is constitutively supersaturated in vivo, retaining energy that powers on-demand cell death. Swapping a non-supersaturable DFD in the apoptosome with a supersaturable one sensitized cells to sublethal stimuli. Mapping all DFD nucleating interactions revealed that supersaturated adaptors are triggered to polymerize specifically by other DFDs in their respective pathways, limiting potentially deleterious crosstalk. Across human cell types, adaptor supersaturation strongly correlates with cell turnover, implicating this thermodynamic principle in the trade-off between immunity and longevity. Profiling homologues from fish and sponge, we find nucleation barriers to be conserved across metazoa. These findings reveal DFD adaptors as biological phase change materials in thermal batteries to power cellular life-or-death decisions on demand.

immunology↗

bioRxiv2023

Monitoring plasma nucleosome concentrations to measure of disease response and progression in dogs with hematopoietic malignancies.

BackgroundHematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients. MethodsCirculating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded. ResultsPlasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein. ConclusionsPlasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

cancer biology↗

bioRxiv2022

Wildflower phenological escape differs by continent and spring temperature

Temperate understory plant species are at risk from climate change and anthropogenic threats that include increased deer herbivory, habitat loss, pollinator declines and mismatch, and nutrient pollution. Recent work suggests that spring ephemeral wildflowers may be at additional risk due to phenological mismatch with deciduous canopy trees. The study of this dynamic, commonly referred to as "phenological escape", and its sensitivity to spring temperature is limited to eastern North America. Here, we use herbarium specimens to show that phenological sensitivity to spring temperature is remarkably conserved for understory wildflowers across North America, Europe, and Asia, but that canopy trees in North America are significantly more sensitive to spring temperature compared to in Asia and Europe. We predict that advancing tree phenology will lead to decreasing spring light windows in North America while spring light windows will be maintained or even increase in Asia and Europe in response to projected climate warming.

ecology↗

bioRxiv2022

CD47-SIRPα controls ADCC killing of primary T cells by PMN through a combination of trogocytosis and NADPH oxidase activation.

Immunotherapies targeting the "dont eat me" myeloid checkpoint constituted by CD47 SIRP interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRP therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies, and the strong ADCC effector function of Poly Morpho Nuclear Cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRP checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRP blockade and ROS induction. Although previous studies had concluded that disruption of SIRP signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRP also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRP immune checkpoint and to design agents able to enhance myeloid cells function while limiting adverse effects to healthy cells able to participate in the anti-tumor immune response. SynopsisDose-limiting toxicities demonstrate the need to investigate anti-CD47-SIRP therapy effects on non-tumor CD47-bearing cells. We demonstrate that anti-CD47 mAbs stimulate potent killing of T cells by PMN through a novel combination of trogocytosis and ROS regulated by SIRP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/483323v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@14fac48org.highwire.dtl.DTLVardef@1b7bd14org.highwire.dtl.DTLVardef@1de8ec7org.highwire.dtl.DTLVardef@6fd5d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

A nucleation barrier spring-loads the CBM signalosome for binary activation

Immune cells activate in a binary, switch-like fashion that involves proteins polymerizing into large complexes known as signalosomes. The switch-like nature of signalosome formation has been proposed to result from large energy barriers to polymer nucleation. Whether such nucleation barriers indeed drive binary immune responses has not yet been shown. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, a key determinant of transcription factor NF-{kappa}B activation in both innate and adaptive immunity. We found that the adaptor protein BCL10 encodes an intrinsic nucleation barrier, and that this barrier has been conserved from cnidaria to humans. Using optogenetic tools and a single-cell transcriptional reporter of NF-{kappa}B activity, we further revealed that endogenous human BCL10 is supersaturated even in unstimulated cells, indicating that the nucleation barrier operationally stores energy for subsequent activation. We found that upon stimulation, BCL10 nucleation by CARD9 multimers triggers self-templated polymerization that saturates NF-{kappa}B activation to produce a binary response. Pathogenic mutants of CARD9 that cause human immunodeficiencies eliminated nucleating activity. Conversely, a hyperactive cancer-causing mutation in BCL10 increased its spontaneous nucleation. Our results indicate that unassembled CBM signalosome components function analogously to a spring-loaded mousetrap, constitutively poised to activate NF-{kappa}B through irrevocable polymerization. This finding may inform our understanding of the root causes and progressive nature of pathogenic and age-associated inflammation.

cell biology↗