Hole formation in the lamina could be explained by two alternative but not necessarily exclusive mechanisms. The close correlation with nuclear shrinking ( Figures 2B and 2E) suggests that the increased condensation rate triggered by NEBD provides a driving force for nuclear shrinking. Immediately after permeabilization, the condensation rate dramatically increased more than 3-fold and maximum condensation was reached within ∼2–3 min ( Figure 2F). As can be seen in Figures 2C–2F, chromosome condensation progressed gradually before NEBD ( Figure 2E). To investigate if nuclear shrinking after NEBD could be caused by condensing chromatin, we also quantitated chromosome condensation during G2/M transition from 4D imaging experiments. Hole formation reproducibly coincided with a rapid decrease in nuclear volume ( Figure 2B), suggesting a link between permeabilization and nuclear shrinking. In quantitative 4D analyses of the lamina, the appearance of gaps was directly compared with the change of inferred nuclear volume (see Experimental Procedures). Hole formation occurred simultaneously for the nuclear lamina and INM or nuclear pores as determined in cells coexpressing lamin B1 with LBR/POM121 ( Figures 3A and 3B). Once a hole opened, it expanded rapidly over the nuclear surface ( Figure 2A, reconstruction). In all the cells observed (n = 28), the hole formed in a region of the NE not in contact with chromosomes and distal from the centrosomes, whose position was evident from the distortions of the lower surface of the nuclear lamina ( Figure 2A projection). Quantitative reconstruction and dynamic visualization of such sequences showed that NEBD starts with one to three holes in the surface of the nuclear lamina ( Figure 2A reconstruction, compare Figures 1A–1D). Next, we investigated the precise structural nature of the permeabilization event by high resolution 4D imaging of lamin B1 and chromosomes. All three nuclear membrane proteins equilibrated with the ER, while lamin B1 appeared to be soluble in the cytoplasm, consistent with our previous results ( These results demonstrate that except for LBR, all NE proteins tested, including B type lamins, were dispersed only after NEBD. After NEBD, lamin B1 was solubilized rapidly within 5–10 min, and the transmembrane proteins POM121 and LAP2β equilibrated with the ER over a similar time course ( Figures 1G and 1H Supplemental Figure S2E). Importantly, no soluble lamin B1 could be detected before NEBD with a sensitivity of better than 5% of the total cellular protein ( Figure 1G). The nuclear rim concentration of LAP2β, POM121, and lamin B1, however, was unchanged at the time the first gap appeared in the NE, and their dispersal started only after the nucleus was permeabilized ( Figures 1C and 1D Supplemental Figure S2A). The redistribution of the INM protein LBR-YFP into the ER consistently started ∼8 min before NEBD, with the majority of the protein already residing in the ER at the time of permeabilization ( Figures 1B and 1F). To address the question of whether lamin depolymerization was a prerequisite for nuclear membrane disassembly, we investigated how the kinetics of NE protein dispersion were correlated to NEBD. LAP2β, POM121, and Lamin B1 Remain Bound in the NE until NEBD
After gap formation, chromosomes rapidly completed their condensation and congressed toward the metaphase plate while remnants of NE proteins remained associated with centrosomes until early metaphase (Supplemental Figures S1A, S1C, and S1D). Finally, the nucleus was permeabilized, evidenced by the influx of cytoplasmic molecules ( Figure 1A) and the appearance of gaps in the nuclear rim ( Figures 1A–1D, arrowheads see also Supplemental Figure S1). NE folds then matured into invaginations severely distorting the NE ( Figure 1C). At the same time, chromosomes started to condense and appeared as defined structures at the NE (Supplemental Figure S1A see Supplemental Data section below). ∼20 min before NEBD, centrosomes separated and started to migrate apart with well formed mitotic asters. The earliest indication of exit from G2 was the appearance of folds in the NE close to centrosomes up to one hour prior to NEBD (not shown). Next, the distinct morphological stages of G2/M transition from early prophase until metaphase were characterized in NRK cells expressing proteins of the NE (lamin B receptor, LBR lamina associated polypeptide β, LAP2β pore membrane protein 121 kDa, POM121 lamin B1), spindle apparatus (α-tubulin), and chromosomes (histone 2B, H2B) tagged with several spectrally distinguishable fluorescent proteins (CFP, GFP, YFP see Experimental Procedures).
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