Figures and data
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.
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).
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.
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.
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.
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.
