Figure 4. Structural changes in VLPs throughout Gag accumulation.

(A) Example traces of wild-type Gag-GFP (black line, sf3 in Matrix of Gag) and Gag-∆p6 (grey line, missing ESCRT-I recruiting p6 domain) assembling into single VLPs. Images were collected every 30 s with excitation illumination polarized either perpendicular (${\displaystyle \hat{\mathbf{p}}}$) or parallel (${\displaystyle \hat{\mathbf{s}}}$) to the glass surface. Total Gag characterized by P+2S (top) and relative average dipole orientation by P/S (bottom). P+2S from wild-type Gag was fit to an exponential and used to predict an expected P/S (blue dashed line) assuming membrane bending throughout assembly. (B) Comparison of average P/S from all traces before VLP assembly (membrane background) and after VLP assembly (plateau region) for three different tagged versions of wild-type Gag. p6-GFP (N = 8), MA-sf3 (N = 9), and MA-sf11 (N = 7). Error bars represent s.d. (C) To compare the evolution of VLP structure to the assembly of Gag the time for each assembly trace was normalized from 0 (beginning of Gag assembly) to 1 (end of Gag assembly). A normalized time difference for each trace between Gag half assembly [½ (P+2S)_max] and the dipole half drop [½ (P/S)_max] was found and all normalized differences were compiled into a histogram. (D) Illustration of sphere budding from flat membrane. (E) Illustration of coordinate system with θ and ϕ representing position on the sphere and β and ψ representing orientation of excitation dipole. (F) Predicted P/S when β = 45°, background intensity is 45% of full assembly intensity, and evanescent field penetration depth is the same as the radius of the VLP.

Figure 4—figure supplement 1. Example traces of Gag accumulation (quantified as P+2S) and fluorophore polarization (quantified as P/S) during VLP assembly.

Three different tagging schemes were used: labeled with mEGFP after p6 domain at carboxy terminal of Gag (A), labeled with circularly permutated GFP variant three in matrix of Gag (B), labeled with circularly permutated GFP variant 11 in matrix of Gag (Pédelacq et al., 2006) (C).

Figure 4—figure supplement 2. Example traces of Gag-∆p6-mEGFP accumulation (quantified as P+2S) and fluorophore polarization (quantified as P/S) during VLP assembly.

Figure 4—video 1. Images of Gag-mEGFP were acquired with excitation perpendicular (P) or parallel (S) to the glass surface.

Download video file ^{(13.5MB, mp4)}

DOI: 10.7554/eLife.36221.019

Left side of video is calculated P+2S (quantification of Gag intensity) and right side is ratio image P/S (measurement of structure).

Figure 4—video 2. Example of individual puncta of Gag-mEGFP with P/S and P+2S images.

Download video file ^{(35.1KB, mp4)}

DOI: 10.7554/eLife.36221.020