Nuclear pores: Two-sided approach

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Nuclear pore complexes (NPCs) are inserted into the nuclear envelope from both the nucleoplasmic and cytoplasmic sides in a Ran guanosine triphosphate-dependent manner.

How are nuclear pore complexes (NPCs) inserted into the nuclear envelope (NE)? And do NPCs form from existing pores or assemble de novo? A report in Science by Martin Hetzer and colleagues now sheds light on both questions.

By pre-assembling nuclei in vitro from Xenopus laevis chromatin, cytosol and membranes, and subsequently incubating the nuclei, Hetzer and co-workers could monitor NPC insertion into NEs by immunofluorescence. During the incubation period, the NE expanded and the number of NPCs increased.

As the small guanosine triphosphatase (GTPase) Ran drives nucleocytoplasmic transport, the authors investigated whether Ran-mediated transport is required for pore formation. In the presence of a Ran mutant that blocked the generation of RanGTP (by inhibiting the Ran nucleotide-exchange factor RCC1), NPC formation was indeed inhibited. However, when nuclear transport was blocked in the presence of wild-type Ran, new NPCs were efficiently inserted into the NE. So, NPC insertion in vitro requires RanGTP but can occur in the absence of nuclear-transport activity. The presence of the nuclear-transport receptor importin- beta , which is known to inhibit NPC insertion into the reforming NE at the end of mitosis, also reduced NPC insertion in vitro.

So, from which side(s) of the NE are NPCs inserted? The authors found that the incubation of nuclei with a cytosolic mutant of Ran, which was excluded from the nucleus, inhibited NPC insertion. Insertion could be restored by the addition of RCC1. A nucleoplasmic mutant of Ran also inhibited NPC insertion, which indicates that Ran-dependent NPC insertion in vitro takes place on both sides of the NE.

The authors then stained nuclei with antibodies against several nucleoporins, the structural components of NPCs. All showed strong staining of the NE rim and antibodies against the Nup107–Nup160 complex showed an additional nucleoplasmic signal. When NPC insertion was blocked, the rim signal was reduced, whereas the nucleoplasmic Nup107–Nup160 signal increased. The reduced rim signal and the nuclear accumulation of the Nup107–Nup160 complex could be reverted by the addition of fresh cytosol, but not by the incubation of the nuclei with Nup107–Nup160-depleted cytosol. Together, these data indicate that the Nup107–Nup160 complex is incorporated from both sides of the NE.

Finally, by fluorescently labelling nuclei with a green dye that stained the pre-existing pores and then incubating the nuclei with a red dye to stain newly formed pores, the Hetzer group showed that the newly formed NPCs appeared as red dots that lacked the green signal of the previously existing pores. This indicates that NPC assembly occurs de novo, and the authors found that this is also the case in living cells.

Arianne Heinrichs

References

D'Angelo, M. A. et al. Nuclear pores form de novo from both sides of the nuclear envelope. Science 312, 440–443 (2006) | Article | PubMed |