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Fig. 2
Figure 2. pETX Binds to hMAL-GFP Expressed in Zebrafish ISVs in a Time-Dependent Manner
(A) Schematic depicting zebrafish transformation strategy, toxin injection site, and microscopic focus. Plasmids containing hMAL-GFP or hBENE-GFP were co-injected with tol2 transposase into the cell of 1-cell-stage zebrafish embryos. Positive fish 3–4 days post fertilization(DPF) were injected with Alexa 647-tagged pETX into the duct of Cuvier for systemic infection. The blue box indicates the region imaged in (B).
(B) The 40× confocal z stack images of zebrafish ISVs in hMAL-GFP-expressing fish versus hBENE-GFP-expressing control fish. Scale bar, 25 μm. ISV, intersegmental blood vessel; DLAV, dorsal longitudinal anastomotic vessel.
(C) Quantitative metric of co-localization, “Percent of ETX Material Co-localized in GFP Vessel,” indicates the percentage of pETX voxels co-localized in hMAL-GFP fish ISVs (n = 6 fish) versus hBENE-GFP control ISVs (n = 7 fish) above the automatically set threshold at 1 h post injection. Data are represented as mean ± SEM. Statistical analysis: unpaired, two-tailed Student's t test (**p < 0.01).
(D) Equal volumes of GFP along the walls of ISVs were rendered into Imaris surfaces, and sum GFP and 647 intensities within the surface were tracked over the course of ∼90 min at 30-s intervals per fish. The ratio of 647-pETX/GFP was plotted over time for hMAL and hBENE transients. Individual ratios of every fish at each time point are represented by opaque circles, and the average ratio at each time point is represented by the darker line.
(E) Snapshot images of 647-pETX or 647-H149A pETX and “merged” images with receptor at three time points post injection. Blue arrow highlights toxin accumulation. Scale bar, 20 μm.
(F) Flow cytometry analysis of pETX or H149A pETX binding to hMAL, rMAL, or GFP CHO cells.
See also Videos S1, S2, and S3. Statistical analysis: two-way ANOVA with Tukey’s test (∗∗∗∗p < 0.0001).