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. 2023 Apr 21;42(11):e113578. doi: 10.15252/embj.2023113578

Figure 4. Time‐lapse microscopy of EBOV VLPs at different pH.

Figure 4

  1. Schematic showing the VLP membrane and pHluorin‐VP40 facing the luminal side of the VLPs. Upon protonation, pHluorin loses its fluorescence properties and serves as a proxy for proton diffusion across the membrane.
  2. Overview confocal fluorescence microscopy image showing pleomorphic pHluorin‐labeled VLPs composed of VP40, pHluorin‐VP40 (ratio 10:1) and GP.
  3. Magnified images of representative VLPs acquired during time‐lapse microscopy at neutral pH and after acidification to approximately pH 5. Frames are exemplarily shown at 0, 1, 3, 5 and 10 min after lowering the external pH.
  4. Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH, low pH and in the presence of T‐X100 at low pH over time. Data was obtained from 3 independent VLP preparations. Number of technical replicates: n = 42 (pH 7.4); n = 19 (pH 4.5 and pH 5 + T‐X100).
  5. Plot showing the drop of pH inside VLPs over time after lowering the pH of the surrounding buffer to 5. The dots represent the mean values, and the dashed lines are the theoretical fit to Equation 3.
  6. Membrane permeability of VLPs (red) and HEK 293T cells expressing different ratios of VP40 and M2 (blue). The mean permeability obtained from 3 independent VLP preparations and 3 biological replicates of cell transfections is displayed on a logarithmic scale, error bars represent standard errors of the mean. Permeability coefficients: filamentous VLPs 1.2 ± 0.2 Å/s (n = 154), spherical VLPs 33 ± 9 Å/s (n = 66), cells expressing no M2 345 ± 71 Å/s (n = 44), cells expressing pHluorin‐VP40 and M2 at 1:0.002 molar ratio 409 ± 85 Å/s (n = 30), cells expressing pHluorin‐VP40 and M2 at 1:0.02 molar ratio 683 ± 263 Å/s (n = 28) and cells expressing pHluorin‐VP40 and M2 at 1:0.2 molar ratio 1940 ± 562 Å/s (n = 26).

Data information: Scale bars: B: 10 μm, C: 2 μm.

Source data are available online for this figure.