Fig. 2: SNIPRs access a distinct activation mechanism.
From: Engineered receptors for soluble cellular communication and disease sensing
a, Schematic of potential activation pathways for soluble or conventional SNIPRs, along with chemical inhibitors to perturb these reactions. b, Relative activation of primary human T cells by endogenous ligands, or Jurkat T cells by synthetic ligands, in the presence of the chemical inhibitors described in a. T cells expressing soluble or conventional SNIPRs, a conventional synNotch receptor or a proteolysis-independent chemically inducible promoter were selected as a representative panel. Data are mean of n = 3 technical replicate measurements after 24 h of incubation normalized to the activation of an inhibitor-free control sample. c, Confocal maximum intensity projection images of Jurkat T cells expressing mCherry-fused TGFβ SNIPRs in the presence of recombinant TGFβ1 labelled with AF647. Data are representative of n = 25 cells (30 min) or n = 27 cells (24 h) across three independent experiments. Scale bar, 5 μm. d, Left, confocal maximum intensity projection images of TGFβ SNIPR Jurkat T cells 24 h after exposure to AF647-labelled TGFβ1, costained with LysoTracker. Scale bars, 20 μm (left); 5 μm (right). Right, Pearson’s correlation coefficient between TGFβ1 and LysoTracker. n = 10 cells; mean ± s.d. Statistics computed using Welch’s unpaired two-tailed t-test. e, Left, confocal images of HeLa cells expressing an orthoSNIPR fused to GFP incubated with mCherry-labelled C6-101A orthoLigand and stained with LysoTracker marker for 30 min before imaging. Scale bar, 50 μm. Right, Pearson’s correlation coefficient between C6-101A orthoLigand and LysoTracker. n = 4 images per time point with at least 10 cells per image; mean ± s.e.m. Statistics computed using Welch’s unpaired two-tailed t-test. f, Top, schematic of dimerSNIPR architecture. A DmrA homodimerizing domain was inserted into a TGFβ-responsive SNIPR between the juxtamembrane domain and the transcription factor. The activation assay was performed in the absence of the extracellular TGFβ ligand. Bottom, activation of primary dimerSNIPR T cells by titration of the small molecule AP1903 (n = 2 technical replicates).
