Dynamics of HIV-1 Assembly and Release
Figure 5
Recruitment of Gag to the budding site.
(A) Scheme of the photoconversion experiment designed to determine the source of Gag molecules recruited to an assembly site. Recruitment can either occur from the surrounding plasma membrane (I) or directly from the cytosol (II). By calculating the ratio of fluorescence intensities in green and red channels at the assembly site with respect to the same ratio calculated from the local background, the origin of the recruited Gag molecules could be determined. (B–C) Live-cell images of a HeLa cell transfected with a mixture of pCHIV andpCHIVmEosFP before photoconversion (B) and directly after photoconversion (C). The scale bar signifies 5 µm. (D) Intensity trajectory from an individual Gag cluster that appeared 26 s after photoconversion. (E) The average fluorescence intensity with 488 nm excitation (green line) and with 561 nm excitation (red line) of the plasma membrane measured in the vicinity of 23 assembly sites that were detectable before photoconversion as a function of time after photoconversion. The standard deviation of the individual traces is given as a halo. (F) The ratio of fluorescence intensity after green excitation to the fluorescence signal after red excitation normalized to the same ratio determined from the local background measured for 102 different assembly sites in five cells.