Figure 4

Relative loss of the Env signal in the particle population induced by cell contact. VLPs labeled with Env.YFP and MA.mCherry were bound to fibronectin coated chambered coverslips and incubated under live cell imaging conditions at 37°C. Particles bound to the cover slip were visualized by epifluorescence microscopy. DFJ-8 cells were added, and the moment of attachment of the cells to the coverslip was defined as time point 0. Incubation was continued at 37°C, and images were recorded at 1 frame/min. Please note that due to the experimental setup only single slices within the focal plane are depicted. (A) shows individual images of a cell on a VLP layer carrying Env.YFP recorded at the indicated time points. The outline of the cell as determined by bright field microscopy is indicated in white. Note that for both time points the same cell is shown, but the cellular morphology is changing in the early phase of attachment. (B) shows a control experiment, using fusion impaired VLPs double labeled with Env.YFP.H8R and MA.mCherry. (C) shows a control experiment, using double labeled VLPs deficient in the viral protease. (D) shows a control experiment using the same double labeled VLPs as in (A) in the presence of 30 mM NH₄Cl. Scale bars in all depicted images correspond to 10 μm. (E) Color separation of double labeled particles over time. Images recorded at the indicated time points were evaluated using an automated tracking software. The number of red and green punctuated signals, originating from MA.mCherry and YFP-labeled Env, respectively, were determined for at least 400 single particles in three independent experiments, and the total number of red and green signals per image was quantified. The plot shows the ratio between the number of green and red signals determined as a measure for the bulk amount of double labeled particles. Quantification in regions covered by cells is shown for particles carrying Env.YFP in the absence (green) and presence of NH₄Cl (grey), for particles carrying Env.YFP.H8R (orange) and for Env.YFP.PR(-) particles (red), respectively. As control, the same quantitative analysis was performed for the background signal of particles in areas where no cells had settled (black).