Figures and data
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Differentiated BoNT/A reporter cell line, Red SNAPR, is highly sensitive to BoNT/A intoxication.
(A) Schematic diagram of the BoNT/A reporter construct, SNAPR. Representative images of ReNcell VM cell line, Red SNAPR, stably expressing SNAPR and incubated with 100nM BoNT/A for 48 hours. (B) Western blot of cell lysates from (A) probed with tRFPT and tGFP antibodies. (C) Schematic diagram of the BoNT/A intoxication assay. (D) Red SNAPR differentiated in normal or ReDS medium, then incubated with 0, 10 and 100 nM BoNT/A for 48 hours. Quantification of EC50 dose response of BoNT/A in ReD SNAPR cells incubated with 0 - 100nM BoNT/A in normal and ReDS medium using GFP/RFP ratio readout. (E) Western blot of cell lysates from (D) probed with tRFPT and tGFP antibodies.
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Genome-wide RNAi screen of BoNT/A-treated Red SNAPR cell line.
(A) Schematic diagram of assay pipeline. (B) Cells treated with non-targeting control siRNA (siNT3) and positive control siRNA (siTXNRD1), then incubated with BoNT/A for 48 hours. Cells were stained with DAPI and TXNRD1 antibody post-fixation. (C) ScreenSifter Z-score analysis of GFP/RFP ratio index of whole genome siRNA library plates. (D) R-squared analysis of both genome-wide RNAi screen replicates. Blue dots = No toxin control, Red dots = Toxin control, Green dots = Positive control (siTXNRD1). (E) Descending Z-score of control-normalized and averaged GFP/RFP ratio of genome-wide screen to determine cut-off values of +1.5 for positive regulators and -3 for negative regulators. (F) Chronological order of genome-wide control-normalized screen reflecting the positive regulators (red) and negative regulators (blue) derived from (E).
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Surface expression of the BoNT/A receptor SV2 is regulated by endocytic trafficking and signaling genes, forming a cohort of the positive regulators of the genome-wide screen.
(A) Detecting surface SV2 using a specific antibody against the extracellular loop of SV2. (B) siVAMP2 as a positive control for surface SV2 expression. ReNcell VM treated with siNT3, siVAMP2 and siTXNRD1 for 3 days and stained with SV2A antibody after fixation without membrane permeabilization. Quantification of surface SV2 per cell where whole image SV2A MFI was divided by total nuclei count (DAPI) from 3 fields of view and 3 independent replicates. (C) Replicate screen results for surface SV2 regulators from genome-wide positive hits. Purple box = surface SV2 repressors e.g. siCTLC (clathrin light chain); Brown box = surface SV2 enhancers e.g. siRab11b. siNT3 and siTXNRD1 does not regulate surface levels of SV2. (D) STRINGS analysis of surface SV2 repressors. (E) STRINGS analysis of surface SV2 enhancers.
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Protein network analysis of genome-wide hits reveals known and novel regulators of BoNT/A intoxication.
ScreenSifter analysis revealed (A) TXNRD1-associated genes. (B) Ganglioside synthesis-associated genes. (C) Src-associated genes. (D) Hsp70-associated genes. (E) Retromer-associated genes. (F) Translocon-associated genes. (G) STRINGS network analysis of connected hits (non-connected hits are excluded). Associated genes tied to respective cellular molecular complex/processes are bounded and annotated. (H) Summary diagram of all positive and negative hits mapped to their intracellular localities.
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BoNT activity is first detected in the neuronal soma then emanate to the axons
(A) Schematic diagram of STAB. Split-mNG detection system consisting of ReNcell VM expressing mNG1-10 and mNG11-tagged BoNT/A. Reconstitution and fluorescence occurs at site of BoNT/A translocation in the cytosol. (B) Stable expression of mNG1-10 in the cytosol of ReNcell VM detected by HA antibody. Neurons are stained with Tuj1 antibody to depict cell boundaries. (C) ReNcell VM stably expressing mNG1-10 and incubated with BoNT/A-mNG11 for 0-48 hr. Insets show representative cells of interest (arrowhead) from each timepoint. (D) Quantification of cells from C where fluorescence intensities of soma and axons from 60 neurons across 3 independent replicates are shown. Graph of Pearson’s correlative coefficient between BoNT/A LC-mNG and Tuj1 antibody staining across the intoxication time points. (E) VPS-35-depleted cells are incubated with BoNT/A-mNG11 for 0-48 hrs. Inset shows representative cell of each condition, depicting extensive reconstituted mNG fluorescence in control cells. Quantification of MFI/cell shown on the right. Graphs are obtained from at least 300 cells across 3 independent experiments.
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BoNT/A is retrogradely trafficked through Golgi and ER membranes and LC translocation is dependent on SEC61.
(A) Split-mNG constructs targeted to Golgi and ER membranes are stably expressed in ReNcell VM and incubated with 50 nM of BoNT/A-mNG11. Control cells (without BoNT/A-mNG11) are shown at top panels of each condition (B) Cells expressing Golgi-mNG1-10 and showing reconstituted mNG fluorescence. Overlap of HA antibody and mNG signals in the Golgi (arrowhead). (C) ER-mNG1-10 expressing cells showing mNG fluorescence and distinct overlap of HA antibody and mNG in the ER. (D) Sec61G-mNG1-10 expressing cells showing mNG fluorescence distinct overlap of HA antibody and Sec61G signals. (E) Quantification of MFI/cell from B-D showing increased mNG fluorescence in Golgi, ER and Sec61G-expressing cells after addition of BoNT/A-mNG11. (F) Representative image of SEC61G depletion in ReNcell VM. (G) Golgi-mNG1- 10, ER-mNG1-10 and Cytosol-mNG1-10-expressing cells are depleted with siSEC61G and incubated with BoNT/A-mNG11 for 48 hours. (H) Quantification of MFI/cell from G. Graphs are obtained from at least 300 cells across 3 independent experiments.