Journal: eLife

Article Title: Adaptor protein supersaturation drives innate immune signaling and cell fate

doi: 10.7554/eLife.107962

Figure Lengend Snippet: ( A ) Schematic diagram of experiment in HEK293T cells to reconstitute the apoptosome with optogenetic control, in either a non-supersaturable or supersaturable format. The non-supersaturable format comprises CASP9 activated by APAF1 CARD (as in the native apoptosome); the supersaturable format comprises chimeric CASP9 with CASP1 CARD replacing CASP9 CARD (CASP9 CASP1CARD ), activated by chimeric APAF1 with NLRC4 CARD in place of APAF1 CARD . 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 1 min 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. APAF1 CARD -Cry2 + CASP9-mScarlet-I, dark n=163, pulse n=375, Mann-Whitney U=11,362 (p<0.0001). NLRC4 CARD -Cry2 + CASP9 CASP1CARD , dark n=46, pulse n=305, Mann-Whitney U=4,253 (p<0.0001). ( C ) Coefficient of variation (CV) of fluorescence distribution in HEK293T cells expressing the indicated protein pairs after a single 1 min 488 nm laser activation. Top, APAF1 CARD -Cry2, and CASP9-mScarlet-I display rapid cluster formation that dissociates by 20 min. Bottom, NLRC4 CARD -Cry2 and chimeric CASP9 CASP1CARD cluster less rapidly but the clusters continue to grow indefinitely. ( D ) Representative images from experiment in C. Clusters of APAF1 CARD -Cry2 and CASP9-mScarlet-I form then dissociate, while NLRC4 CARD -Cry2 and CASP9 CASP1CARD clusters only get larger. Scale bar 10 µm. ( E ) Quantification of cell death of the HEK293T chimeric cells (as in A) using Annexin V-Alexa 488 staining, either 2 hr after a single 1 min pulse of 488 nm laser, or after 2 hr of ‘constant’ stimulation whereby cells were subjected to a 1 s pulse every 1 min. p-Values derived from t-test.

Article Snippet: Finally, the doxycycline-controlled lentiviral vectors were cloned via Gibson assembly with the respective coding sequences from PYCARD, CASP9, CASP1, and mScarlet-I into pCW57.1 (Addgene #41393).

Techniques: Control, Activity Assay, Fluorescence, Expressing, MANN-WHITNEY, Activation Assay, Staining, Derivative Assay

Journal: eLife

Article Title: Adaptor protein supersaturation drives innate immune signaling and cell fate

doi: 10.7554/eLife.107962

Figure Lengend Snippet: ( 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 CASP1 FL exhibits a high C sat in the mid-micromolar range, * D. rerio CASP1 FL exhibits a high C sat in the mid-micromolar range based on prior calibrations of DAmFRET plots , which greatly exceeds the nanomolar concentration expected for endogenous procaspase-1 , 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 (ΔΔ G (nucleus - solute) ) declines sharply with concentration beyond C sat ( ; ), 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 (ΔΔ G (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 ( ; ). 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.

Article Snippet: Finally, the doxycycline-controlled lentiviral vectors were cloned via Gibson assembly with the respective coding sequences from PYCARD, CASP9, CASP1, and mScarlet-I into pCW57.1 (Addgene #41393).

Techniques: Protein-Protein interactions, Bacteria, Concentration Assay, Binding Assay, Sublimation, Amplification, Battery, Activation Assay