Characterization of extravagations formed in transfected HeLa cells.

(A) Ultrastructural analysis. HeLa cells were co-transfected with plasmids encoding the indicated proteins. At 18 hours post transfection, cells were fixed and processed for TEM as described in Materials and Methods. HeLa cells co-transfected with plasmids encoding EGFP-pUL31, pUL34 and pUs3 formed extravagations that were not observed in control transfections receiving pCI-neo (see Fig 6). White arrowheads indicate extravagations comprised of both outer nuclear membrane (ONM) and inner nuclear membrane, white arrows indicate extravagations comprised of ONM only and white asterisks indicate zippered nuclear membranes, which were also not observed in control transfections. Nu indicates the nucleus. (B) Fluorescence recovery after photobleaching analysis. HeLa cells were co-transfected with EGFP-pUL31, pUL34 and pUs3. Live cells displaying extravagations were imaged at 18 hours post transfection and selected images of cells immediately before, immediately after, or at a later time after the region indicated by the arrowhead was photobleached are shown. Scale bar is 10 μm. The corresponding quantification of fluorescence intensity over time of the photobleached region as well as a control region not subjected to photobleaching is shown beside the images. Error bars are standard error of the mean.