Fig 1.
Creation and schematic design of a TEV protease-sensitive α-synuclein-GFP fusion protein.
(Panel A) A schematic diagram of the fusion protein construct: mouse α-synuclein, 6x His, TEV protease site, and GFP. (Panel B) Schema of the protease protection assay. Pre-formed fibrils (black) containing GFP (green) are incubated with cells for 1 h to permit endocytosis into cells. TEV protease (orange) is added and incubated with cells for 30–60 min for optimal fusion protein cleavage. GFP is cleaved from PFFs exposed to TEV protease within the media and on cell surfaces. Internalized α-syn-TEV-GFP PFFs are protected from TEV cleavage. (Panel C) Coomassie-stained gel showing α-syn-TEV-GFP PFFs incubated in vitro with and without TEV protease. Sonicated PFFs were diluted to 75 μg/ml in 100 μl of dPBS in two separate tubes. Five μl of TEV protease stock or buffer were added, and tubes incubated for 1 h at 37°C. One hundred μl of 2x sample buffer were then added and samples heated prior to SDS-PAGE and Coomassie staining.
Fig 2.
Characterization of α-syn-TEV-GFP pre-formed fibrils and seeding efficacy.
(Panel A) Electron microscopy images of negatively-stained α-synuclein monomers, intact fibrils, sonicated fibrils, and sonicated fibrils following TEV treatment. (Scale bar = 200 nm). (Panel B) Histogram of fibril size distribution after sonication with and without TEV treatment (bin size 20 nm; >200 fibrils counted for each condition). (Panel C) α-syn-TEV-GFP PFFs are competent to seed the fibrillation of α-synuclein monomers. Thioflavin T fluorescence, generated following fibril formation, is shown.
Fig 3.
Incubation with TEV protease selectively removes GFP fluorescence from the cell surface.
(Panel A) Confocal images of Neuro2A cells after incubation with α-syn-TEV-GFP PFFs (green) for 1 h, followed by incubation with (bottom panels) or without TEV protease (top panels) or for 30 min. Images are presented as the average of 3 slices from the middle of a confocal Z-stack (Scale bar = 5 μm). (Panel B) Quantification of the percent of fluorescence remaining after trypan blue quenching. The total fluorescence signal in the 3D confocal images was measured both pre- and post-trypan-blue treatment in four independent experiments; the average for each experiment is shown as a single data point. Data are presented as a ratio of final post-trypan blue-quenched fluorescence to pre-trypan blue fluorescence for each individual experiment. *, p = 0.036; ns, not significant (p>0.05); Sidak’s multiple comparison test.
Fig 4.
Western blotting provides a quantitative index of internalized intact PFFs.
(Panel A) Anti-α-synuclein Western blot, showing the total level of intact α-syn-TEV-GFP per well when α-syn-TEV-GFP PFF-treated cells are incubated with or without TEV protease. (Panel B) Quantification of Western blot (triplicate samples, mean ± SD).
Fig 5.
Exposure of cells to extracellular TEV protease is not cytotoxic.
Neuro2A cells were exposed to TEV-P for 30 min and cell viability measured following an overnight growth period using the WST-1 cytotoxicity assay. Percent survival is plotted for treatment with TEV-P; 1% Triton X-100; 1x TEV-P buffer; and for untreated cells, by normalizing TEV-treated cell absorbance values to untreated cell absorbance values. TEV-P treatment does not result in significant cell death when compared to either buffer-treated or untreated cells. (****, p<0.0001; ns, not significant; one-way ANOVA and Tukey’s multiple comparisons tests).
Fig 6.
TEV-P-treated cells can be used for fluorescent imaging of internal markers as well as staining.
(Panel A) Neuro2A cells with Lysotracker. Neuro2A cells incubated with a-syn-TEV-GFP PFFS in the absence of TEV treatment exhibit large quantities of GFP-positive objects that obscure the detection of Lysotracker-positive objects. TEV P-treated Neuro2A cells exhibit a much lower GFP signal, permitting greatly enhanced detection of internalized α-syn-TEV-GFP PFFs colocalized with Lysotracker; white arrows indicate colocalization. Scale bar = 5 μm. Right portion: Quantitation of GFP-positive internalized objects indicates that co-localization of Lysotracker and GFP is greatly enhanced by TEV treatment. (Panel B) Comparison of Neuro2A cells incubated with PFFs either lacking or containing a TEV site. Confocal images were obtained after incubation of cells with either α-syn-TEV-GFP PFFs or α-syn-GFP PFFs (green) for 1 h, followed by incubation with TEV protease or buffer for 30 min. Images are presented as the average of 3 slices from the middle of a confocal Z-stack (scale bar = 5 μm). Quantitation of the total fluorescence signal in 3D confocal images was measured for both α-syn-TEV-GFP PFFs and α-syn-GFP PFFs, with and without TEV-P treatment, and shows a clear dependence on the presence of a TEV site (*, p<0.05; ns, not significant; one-way ANOVA and Tukey’s multiple comparisons tests). (Panel C) Differentiated primary rat hippocampal cells were fixed and stained with anti-GFP antiserum (red) 4 days post-exposure to 5 μg/ml α-syn-TEV-GFP PFFs (green) and treatment with either buffer (top row) or 100 U/ml TEV-P (bottom row). Images were taken at 40x; scale bar, 10 μm.