Nuclei exhibiting the lpin-1(RNAi)-induced mild phenotype give rise to paired nuclei following mitosis because of defects in NEBD. (A) L4-staged worms (strain OCF3) expressing NPP-1::GFP (green) and histone H2B::CR (red) grown on bacteria expressing control dsRNA for 48 hours. Live imaging of a four-cell-stage embryo from such a worm using confocal microscopy. Merged images of multiple focal planes from the indicated time point (in seconds) are shown. The nucleus labeled with an arrow is in prometaphase at time 0 seconds, in metaphase at 80 seconds, and in anaphase by 160 seconds. (B) L4-staged worms (strain OCF3) expressing NPP-1::GFP (green) and histone H2B::CR (red) were grown on bacteria expressing lpin-1 dsRNA for 36 hours and a four-cell embryo with cells exhibiting a mild phenotype (for example, see arrow) was imaged at the indicated time points, as described above. The timing of prometaphase, metaphase and anaphase is similar to that shown in A. (C) L4-staged worms (strain OCF2) expressing tubulin::GFP (green) and histone H2B::CR (red) were grown on bacteria expressing lpin-1 dsRNA for 36 hours and embryos were taken for live imaging as described above. In the example shown, a two-cell-stage embryo contains one cell with a normal nucleus (top right) and one cell with paired nuclei (bottom left). Chromosome segregation in the normal nucleus precedes that of the paired nuclei. In the case of the paired nuclei, the spindle poles align on both sides of the interface between the two DNA clusters before spindle elongation (arrow). Note the greater width of the spindle in the cell that has paired nuclei (arrowhead, time 480 seconds) compared with the spindle in the cell that has a single nucleus (arrowhead, time 280 seconds). (D,E) L4-staged worms (strain OCF4) expressing YFP::LMN-1 (green) and histone H2B::CR (red) were grown on bacteria expressing either control dsRNA (D) or lpin-1 dsRNA (E) for 24 hours at 24°C, and embryos from these worms were imaged by live microscopy as described above. In the case of the control RNAi, the nuclear lamina is at the nuclear periphery; it dissociates at the 160 second time point, during the early stages of anaphase. By contrast, in the case of lpin-1(RNAi) the lamina is present at the periphery of each of the paired nuclei, and is also hanging off the edges of the interface between the two nuclei (time point 520 seconds, arrowhead). As mitosis progresses, the nuclear lamina persists, and it is stretched as the distance between the segregating chromosomes increases. Note that the nuclear lamina appears to separate the DNA that had originated from each of the paired nuclei, presumably preventing their mixing.