A select subset of DFDs have intrinsic nucleation barriers enabling persistent supersaturation.

A. Schematic diagram illustrating two models for signal amplification through protein selfassembly. Top left: Extrinsic model, where D/PAMP-binding coupled with nucleotide hydrolysis stabilizes active assemblies (red glow) relative to solute precursors (blue glow). This model is exemplified by localized actin polymerization downstream of many cell surface receptors,117 but could also occur indirectly by, for example, phosphorylation-mediated release of solubilizing factors. Bottom left: DFDs that function in this way will assemble promptly and monotonously above their saturation concentration (Csat). Top right: Intrinsic model, where the protein is supersaturated at rest but prevented from assembling by a sequence-encoded nucleation barrier. D/PAMPbinding eliminates the barrier, releasing the energy of supersaturation to drive assembly. The models are not mutually exclusive. Bottom right: DFDs that function in this way will remain soluble above Csat until stochastic nucleation, creating a discontinuous relationship of assembly to concentration across a population of cells. B. Illustration showing how the concentration-dependence of self-assembly as classified by DAmFRET relates to the subcellular morphology of self-assemblies classified by highthroughput confocal microscopy. “Continuous” and “discontinuous” classifications describe the relationship of self-assembly (AmFRET, y-axis) to expression level (x-axis) for each DFD. Discontinuous DFDs exhibit a range of concentrations where selfassembly occurs stochastically, indicating an intrinsic nucleation barrier. The four instances of visible assemblies despite no AmFRET-positive cells are presumed to result from those DFDs partitioning with other cellular components or endogenous condensates wherein they remain too dilute to FRET. Cells in the matrix are colored according to the log2 ratio of observed to expected frequencies. The indicated p-values (adjusted for multiple hypotheses) were obtained with an exact multinomial test using the total frequencies of each morphology. C. Distribution of DAmFRET classifications across the four subfamilies of DFDs. D. Schematic diagram of our experimental design to assess the ability of each DFD to seed itself. Top: Biological activation of an exemplary signalosome -- the AIM2 inflammasome -- occurs when the receptor AIM2 oligomerizes on the multivalent PAMP, dsDNA, and then templates the assembly of the adaptor protein, ASC. Bottom: Experimental paradigm to test for supersaturation mimics biological activation, by expressing each DFD in trans with the same DFD expressed as a fusion to μNS, a modular self-condensing protein. AmFRET-positivity will only occur if the μNS fusion templates subsequent self-assembly by the non μNS-fused DFD. E. Representative DAmFRET data contrasting two self-assembling DFDs -- one that is supersaturable (left) and the other that is not (right). The plot for the supersaturated protein exhibits a discontinuous distribution of AmFRET across the expression range (top and bottom). The discontinuity is eliminated, with all cells moving to the AmFRETpositive population, by expressing the protein in the presence of genetically encoded seeds (middle). The dashed horizontal lines approximate the mean AmFRET value for monomeric mEos3. Procedure defined units (p.d.u.). F. Contingency table showing that discontinuous DFDs tend to be self-seedable. Each DFD was co-expressed with an orthogonally fluorescent μNS-fused version of the same DFD. Fisher’s exact test revealed an association between continuity and self-seedability (p < 0.001). G. Boxplot comparing the Csat values (as approximated by C50seeded) of continuous and discontinuous DFDs. Discontinuous DFDs have significantly lower Csat, indicating greater stability of the assemblies. Mann-Whitney U = 457, ncontinuous = 26, ndiscontinuous = 20 (p < 0.001). H. Boxplot comparing supersaturability, represented as the fold change reduction in C50 by seeding (C50stochastic - C50seeded), of continuous and discontinuous DFDs. The C50 values were more strongly reduced by seeding for discontinuous DFDs than for continuous DFDs. Mann-Whitney U = 164, ncontinuous = 58, ndiscontinuous = 21 (p < 0.001). See also Figures S1, S2, S5 and Table S1.

Nucleation barriers are a characteristic feature of inflammatory signalosome adaptors.

A. Boxplot of DFD-containing protein abundances in monocytes, showing that discontinuous DFDs have higher endogenous expression levels. Mann-Whitney U = 53, ncontinuous = 26, ndiscontinuous = 8 (p = 0.039). Protein abundance values are from PAXdb.28 B. Scatter plot of DFD gene expression in monocytes (normalized transcripts per million) and Csat values. Spearman R = -0.285 (p = 0.03). Adaptor DFDs are labeled. Dataset obtained from the Human Protein Atlas. C. Top: box plots of degree centrality (left) and betweenness centrality (right) of continuous and discontinuous DFDs in the endogenous network of physically interacting DFD proteins, showing that the latter are more centrally positioned. Degree centrality MannWhitney U = 242.0 (p = 0.010); betweenness centrality Mann-Whitney U = 274.0 (p =0.030); ncontinuous = 46, ndiscontinuous = 18. Bottom: box plots of centrality measures of nonseedable and seedable DFDs, showing that the latter are more centrally positioned. Degree centrality Mann-Whitney U = 167.5 (p = 0.022); betweenness centrality Mann- Whitney U = 172.5 (p = 0.023); nnon-seedable = 35, nseedable = 16. D. Visualization of how the DAmFRET profiles of isolated DFD domains (left) change in their full-length contexts (right), showing that only adaptor proteins (green connections) tend to retain discontinuous transitions in their full-length context. E. Subnetworks of prominent signalosome adaptor proteins that were found to be supersaturable. Edges connect nodes with experimentally determined physical interactions with confidence > 0.9 in STRING. All proteins shown have DFDs except TRAFs. Each adaptor’s node size is proportional to its supersaturability score. F. Comparison of protein abundances at the whole body level for the signalosome components in Figure 2E (left) and Figure 2G (right), showing that adaptors are more highly expressed for the former. Protein abundance values are from PAXdb.28 P-values are from Mann-Whitney test. For supersaturable signalosomes: nsensor = 13, nadaptor = 6, neffector = 4; sensors and adaptors, U = 4.0 (p < 0.001); sensors and effectors, U = 6.0 (p = 0.023); adaptors and effectors, U = 18.0 (p = 0.257). For non-supersaturable signalosomes: nsensor = 3, nadaptor = 2, neffector = 2; sensors and adaptors, U = 1.0 (p = 0.400); sensors and effectors, U = 0.0 (p = 0.200); adaptors and effectors, U = 0.0 (p = 0.333). G. Subnetworks of signalosomes lacking supersaturable DFDs. Edges connect nodes with experimentally determined physical interactions with confidence > 0.9 in STRING. All proteins shown have DFDs except TRAF6. See also Figures S3, S4 and S5, and Tables S1 and S2.

Nucleation barriers may facilitate signal amplification in human cells.

A. Schematic diagram of experiment in HEK293T cells to reconstitute the apoptosome with optogenetic control, in either a non-supersaturable or supersaturable format. The nonsupersaturable format comprises CASP9 activated by APAF1CARD (as in the native apoptosome); the supersaturable format comprises chimeric CASP9 with CASP1CARD replacing CASP9CARD (CASP9CASP1CARD), activated by chimeric APAF1 with NLRC4CARD in place of APAF1CARD. Blue light triggered assembly in both cases, but subsequent continued assembly in the dark only occurred for the supersaturated format. B. Caspase 3/7 activity reporter fluorescence intensities in the absence of stimulation or after one minute of 488 nm stimulation for cell lines expressing the non-supersaturable or supersaturable pairs, showing that both pairs comparably activate caspase 3/7 while oligomerized. APAF1CARD-Cry2 + CASP9-mScarlet-I, dark n = 163, pulse n = 375, Mann-Whitney U = 11362 (p < 0.0001). NLRC4CARD-Cry2 + CASP9CASP1CARD, dark n = 46, pulse n = 305, Mann-Whitney U = 4253 (p < 0.0001). C. Coefficient of variation (CV) of fluorescence distribution in HEK293T cells expressing the indicated protein pairs after a single one minute 488 nm laser activation. Top, APAF1CARD-Cry2 and CASP9-mScarlet-I display rapid cluster formation that dissociates by 20 min. Bottom, NLRC4CARD-Cry2 and chimeric CASP9CASP1CARD cluster less rapidly but the clusters continue to grow indefinitely. D. Representative images from experiment in C. Clusters of APAF1CARD-Cry2 and CASP9mScarlet-I form then dissociate while NLRC4CARD-Cry2 and CASP9CASP1CARD crusters only get larger. E. Quantification of cell death of the HEK293T chimeric cells (as in A) using Annexin VAlexa 488 staining, either two hours after a single one minute pulse of 488 nm laser, or after two hours of “constant” stimulation whereby cells were subjected to a one second pulse every one minute. P-values derived from t-test. See also Figure S6, Table S3 and Movie S1, S2.

Innate immune adaptors are endogenously supersaturated.

A. Time course of apoptotic cell death of THP-1 cells following exposure to AIM2 ligand, poly(dA:dT). P-value obtained from ANOVA followed by pair comparison. B. Schematic diagram of the experiment to transiently optogenetically stimulate AIM2PYD to monitor ASCPYD assembly. This experiment was conducted in HEK293T cells because they do not undergo pyroptosis. C. Top, Time course of fluorescence intensity distribution in THP-1 cells following 10 seconds of optogenetic activation, showing that WT AIM2PYD forms clusters (high CV) that persist and induces cell death, while the F27G solubilizing mutant23 forms clusters that subsequently disperse. Bottom, normalized Sytox Orange fluorescence intensity for the experiment in the top panel. D. Representative confocal microscopy images from a timelapse of THP-1 monocytes showing that transient optogenetic stimulation of WT but not F27G mutant of AIM2PYD causes it to form puncta that coincide with cell death. Sytox Orange was used for this experiment because it can be excited without activating Cry2. E. Time course of cell death of THP-1 cells when subjected to a blue light pulse every 5 minutes (“repeated”), showing rapid cell death (violet trace) only when AIM2PYD is WT and when ASC is present. The absence of ASC results in slower death (green trace), consistent with apoptosis. The F27G mutation of AIM2PYD blocks cell death irrespective of ASC (black and golden traces). F. Coefficient of variation (CV) of fluorescence distribution of AIM2PYD-Cry2 and ASCmScarlet-I in THP-1 PYCARD-KO cells following a 10 s blue light pulse. This shows that AIM2PYD and ASC-mScarlet-I (with slightly delayed kinetics) rapidly form clusters that persist well after stimulus removal. ASC-mScarlet-I was induced to only ∼20% of the ASC expression in WT cells using 1.0 µg/mL doxycycline (dox). G. Quantification of CellTox staining in individual ASC-mScarlet-I THP-1 PYCARD-KO cells 30 minutes after a 10 second blue laser pulse, at different levels of dox-induced ASC-mScarlet-I expression. Green dotted line indicates 95% confidence interval (CI) for background fluorescence intensity, above which cells were considered CellTox-positive. Error bars denote standard deviation. Control, n = 37. 0.25 µg/mL dox, n = 36. 0.5 µg/mL dox, n = 47. 0.75 µg/mL dox, n = 113. 1 µg/mL dox, n = 180. H. Top: The metastability of supersaturation implies that cells will occasionally inflame and/or die from stochastic (without D/PAMPs) DFD nucleation, which creates a tradeoff between innate immunity and life expectancy. Bottom: Scatter plot showing the relationship between geometric mean of adaptor supersaturation including ASC, FADD, BCL10, TRADD, MAVS (as approximated by the ratio of transcription levels and Csat values) and mean lifespan for each cell type in the human body for which data is available.73 Cell types with greater DFD supersaturation have shorter mean lifespans. The red line represents the best-fit power-law regression, obtained by performing linear regression in log-log space. The shaded region represents the 95% confidence interval for the trend line. Spearman R = -0.8375 (two-tailed p = 0.000027). See also Figure S6 and Table S3.

The nucleating interactome is highly specific.

A. Matrix of all nucleating interactions (gray-shaded circles) detected in a comprehensive DAmFRET screen of > 10,000 DFD pairs. Each DFD-mEos3 (columns) was separately expressed with each DFD-μNS seed (rows). Darker shading of the circle denotes increased seedability. Interactions among members of the same signaling pathway (in legend) appear in color shaded squares. Asterisk denotes seeds that were screened in a separate experiment from the rest. The matrix was clustered on seedability values, on a log scale, using the SciPy.cluster.hierarchy v1.11.1 linkage and dendrogram Python packages, using the Ward variance minimization algorithm to calculate distances. Procedure defined units (p.d.u.). B. Circos plot of the nucleating interactions summarized by DFD subfamily. Each subfamily is represented with a segment proportional to the number of DFDs with a nucleating interaction, as indicated by ribbons within and between segments. Inner stacked bars around the perimeter show the numbers of DFDs in each subfamily seeded by the subfamily in that segment. Middle stacked bars around the perimeter show the numbers of DFDs in each subfamily that seed the subfamily in that segment. Outer stacked bars around the perimeter show total nucleating interactions involving the subfamily in that segment. C. Nucleating interactions involving DFDs in extrinsic apoptosis and pyroptosis, with blue edges highlighting the direct nucleating effect of AIM2 on FADD and ASC that is explored in Figure 4. The network was created in Cytoscape with node size corresponding to betweenness centrality and grouped by reported function. Interactions between FL proteins (Table S2) were included. Edge darkness indicates the seedability score of the corresponding interaction. See also Figure S7 and Table S4.

DFD nucleation barriers are deeply conserved.

A. Phylogenetic tree illustrating evolutionary relationships between DFD signaling pathways from bacteria to humans. B. DAmFRET classifications for DFD-only and FL components of the DISC from the model sponge, Amphimedon queenslandica, and of the inflammasome from the model fish Danio rerio, showing that adaptors are specifically supersaturable. *D. rerio CASP1FL exhibits a high Csat in the mid-micromolar range, *D. rerio CASP1FL exhibits a high Csat in the mid-micromolar range based on prior calibrations of DAmFRET plots,16 which greatly exceeds the nanomolar concentration expected for endogenous procaspase1,118 making it unlikely to supersaturate at endogenous concentrations. C. Physical logic of DFD function. Left: Cells experience thermodynamic perturbations either from stochastic fluctuations (noise) or D/PAMP binding to innate immune receptors. These perturbations can nucleate supersaturated signaling proteins (dashed horizontal lines) with a probability that depends on the type of phase transition and specifically, whether it is accompanied by structural ordering. Middle: For phase separation in the absence of structural ordering (LLPS), the nucleation barrier (ΔΔG(nucleus - solute)) declines sharply with concentration beyond Csat,103,119 which increases its susceptibility to noise. This limits the level of supersaturation that can be maintained by a cell (vertical dashed line), and therefore, the extent to which assembly (ΔΔG(solute -- assembly)) can power signal amplification (tiny battery schematic). Right: For phase separation with structural ordering (paracrystallization as in adaptor DFD assemblies), the dependence of nucleation on concomitant intramolecular fluctuations buffers the barrier against concentration (as indicated by a shallower curve relative to LLPS), which allows cells to maintain much higher levels of supersaturation.16,21 Following nucleation, the assemblies grow and deplete soluble protein until it is no longer supersaturated, driving amplification (diagonal orange arrow) through proximity-dependent effector activation. The intrinsic nucleation barriers encoded by solution phase DFD ensembles therefore allow them to function as phase change batteries (giant battery schematic) to power innate immune signal amplification. See also Figure S8 and Table S2.