FIG. 7.
Time-lapse four-dimensional confocal fluorescence imaging of a living HIV Gag-iGFP-expressing cell reveals a temporal sequence of Gag localization. Confocal z stacks were acquired at 10-min intervals for 14.5 h. (A through H) Montage of fluorescence images illustrating changes in the fluorescence pattern of Gag-iGFP at 2-h intervals. x-y cross-sections through the middle of the cell (top) and x-z profiles (bottom) are indicated. Triangles in panels C and D highlight early membrane accumulations of Gag. Arrows in panels G and H highlight intracellular vesicular accumulations of Gag. (I) The total integrated fluorescence intensity of the cell is graphed over time. Letters A through H indicate the time point at which each representative image was acquired. (J) Gag localization in 293T cells progresses through four morphological stages: (i) diffuse cytoplasmic; (ii) diffuse cytoplasmic and punctate membrane staining; (iii) diffuse cytoplasmic, strong membrane, and vesicular staining; and (iv) loss of nuclear exclusion. Time-lapse imaging of the 293T cell is representative of more than three independent long-duration imaging experiments in which one or more cells were successfully captured over time (see Movie S2 in the supplemental material).