Figure 9.

Mitotic PAM cells were identified and allowed to proceed until ∼60–90 min after telophase. At this time, both the nucleoplasm and lamina fluoresce. When daughter cell nuclei are photobleached at this time, the recovery in the nucleoplasm is so fast that a bleach zone cannot be detected in a subsequent confocal scan (a). However, the bleached areas in the lamina region recover much more slowly and remain detectable for up to 70 min and later. Only one daughter cell nucleus is shown due to the size of the nucleus. The overall nucleoplasmic fluorescence becomes less intense during this period of observation and the number of nucleoplasmic foci increase. Many of these foci are not continuous with the nuclear surface, as shown through a focus series of images using the confocal microscope (not shown). Bar, 10 μm. A live PAM cell in early G1 expressing GFP-lamin A was photobleached across the entire cell and an image was captured in the subsequent confocal scan (d). As expected for an early G1 cell, the bleach zone is apparent only at the lamina rim (arrows). This same cell was immediately extracted in IF buffer while being viewed on the microscope stage and an image was captured 10-s later (e). The nucleoplasmic fluorescence is almost completely extracted. Bar, 5 μm. FRET analysis was also performed on cells in G1 (e–j). Cells were doubly transfected with pCFP-LB1 and pYFP-LA and examined using a FRET filter setup, as described in Materials and Methods. In cells early in G1 (120–180 min after telophase), the CFP-LB1 (e) was able to activate YFP-LA, resulting in a FRET signal (f), implying these molecules interact. Furthermore, cells very early in G1 (<90 min after telophase), when lamin A is largely nucleoplasmic (YFP-LA; j) also have a FRET signal, indicating an interaction at this time (h and i). Bar, 10 μm.