Figures & data
Figure 1. Top: Interphase diagram showing known chromatin-nuclear envelope tethering mechanisms, along with the role proposed for PRR14. Model depicts PRR14 as a non-membrane protein, tethering between Lamin A/C and heterochromatin-bound HP1. Bottom: Telophase model depicting a role for PRR14 in specifying heterochromatin destined for reassembly at the nuclear lamina at mitotic exit. Telophase model is based on the dynamic behavior of tagged PRR14.
Figure 2. Confocal imaging showing localization of GFP-tagged PRR14 at the nuclear lamina and the peripheral heterochromatin compartment as detected by anti-H3K9me3 staining.
Figure 3. Confocal imaging showing nuclear lamina defects after PRR14 knockdown. Cells were transfected with the indicated siRNAs and monitored by confocal imaging 72h post-transfection. Scale bars, 10 μm.
Figure 4. Further evidence for HP1-dependent localization of PRR14 on chromatin. For these experiments, HeLa cells stably expressing both GFP-PRR14 and RFP-HP1α were used. Left: Cells were treated with the indicated drugs for 3h, and images were acquired using confocal microscopy. Mock indicates untreated cells progressing to metaphase. Aurora B kinase inhibitor ZM447439 treatment resulted in prometaphase arrest and co-retention of PRR14 and HP1 on chromatin. Right: Cells were transfected with indicated siRNAs 72h prior to drug treatment. Cells were then treated with both nocodazole and ZM447439 for 3 h, and confocal images were acquired. As shown, only knockdown of all three HP1 isoforms resulted in PRR14 release from chromatin.
Figure 5. Snapshots from the 3D-renderings generated by Imaris software, showing localization of Lamin A/C, GFP-PRR14, and DAPI (DNA) in HeLa cells at various stages of mitosis. Videos 1–5 of 3D renderings correspond to interphase, prophase, prometaphase, anaphase, and telophase stages, respectively. The interphase nucleus shown was chosen to highlight the localization of PRR14 with nuclear channels.
