Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells

Abstract

So far, POM121 and gp210 are the only known anchoring sites of vertebrate nuclear pore complexes (NPCs) within the lipid bilayer of the nuclear envelope (NE) and, thus, are excellent candidates for initiating the NPC assembly process. Indeed, we demonstrate that POM121 can recruit several nucleoporins, such as Nup62 or Nup358, to ectopic assembly sites. It thus appears to act as a nucleation site for the assembly of NPC substructures. Nonetheless, we observed functional NPCs and intact NEs in severely POM121-depleted cells. Double knockdowns of gp210 and POM121 in HeLa cells, as well as depletion of POM121 from human fibroblasts, which do not express gp210, further suggest that NPCs can assemble or at least persist in a POM121- and gp210-free form. This points to extensive redundancies in protein-protein interactions within NPCs and suggests that vertebrate NPCs contain additional membrane-integral nucleoporins for anchorage within the lipid bilayer of the NE. In Stavru et al., we describe such an additional transmembrane nucleoporin as the metazoan orthologue of yeast Ndc1p.

Figure 1.

Ectopic expression of POM121 at mitochondria. (A) EGFP was fused behind residues 1–70 of TOM20, expressed in HeLa cells, and thereby anchored to the outer mitochondrial membrane (Mito-GFP). (top) Colocalization of the fusion protein with the NPC marker mAb414. (bottom) Staining of NPCs with the Alexa 568–labeled Impβ^45–462 after digitonin permeabilization. Signals for mitochondria and NPCs do not overlap. (B) The POM121^73–797 fragment, lacking its natural membrane anchor and the FG repeat domain, was fused behind the Mito-GFP module (Mito-GFP-POM121) and expressed in HeLa cells. (top) Colocalization of the Mito-GFP-POM121 signal with mitotracker-stained mitochondria (images depict a transfected and a nontransfected cell). (bottom) Bright staining with Impβ^45–462 indicates recruitment of FG or GLFG repeat Nups to the ectopic POM121 fragment at mitochondria. Clustering of mitochondria is a side effect of this ectopic expression. The weak Mito-GFP-POM121 signal at the NE reflects the fact that targeting of the fusion protein to mitochondria is in competition with incorporation into bona fide NPCs.

Figure 2.

The central POM121 domain is sufficient to recruit a subset of Nups. Images show immunofluorescence colocalization of Mito-GFP-POM121^73–797 with indicated NPC markers. The ectopic assembly sites clearly recruited FG-repeat Nups (detected by mAb414) and stained positive for Nup358, Nup205, and Nup62 but remained negative for Nup153 and TPR. As indicated, expression was either in HeLa cells or in 3T3 fibroblasts, whose mitochondria show less clustering upon Mito-GFP-POM121^73–797 expression.

Figure 3.

POM121 is not limiting for NPC assembly. HeLa cells were transfected with POM121-specific siRNAs and fixed 60 h later. Triple immunofluorescences were performed with combinations of rabbit anti-POM121; mouse mAb414 or anti-Nup88; and guinea pig anti-Nup107, -Nup96, -Nup62, or -Nup358. Bright NE staining for POM121 is visible only in nontransfected cells that are shown for reference. POM121-depleted NPCs still contained wild-type levels of Nup107, Nup88, Nup358, Nup96, and Nup62 and stained normally with mAb414. White arrows indicate cells with <5% residual POM121. Red arrows label a virtually POM121-free pair of cells that just completed cytokinesis.

Figure 4.

Assembly of human NPCs in the absence of gp210. (A) Primary neonatal fibroblasts (5 × 10⁴ cells) and HeLa cells (2.5 × 10⁴ cells) were analyzed by immunoblotting with the indicated antibodies. Note that fibroblasts appear gp210 negative. (B) HeLa cells were transfected with a gp210-specific siRNA and fixed 60 h later. DNA was stained with Hoechst 33342. Triple immunofluorescence was with rabbit anti-gp210, guinea pig anti-Nup107, and mouse mAb414. Arrows exemplify a transfected cell with <5% residual gp210. The gp210 depletion did not impair assembly of Nup107 or mAb414-reactive Nups into NPCs.

Figure 5.

NPCs appear normal even in cells lacking gp210 and depleted of POM121. (A) HeLa cells were treated simultaneously with POM121- and gp210-specific siRNAs and fixed 60 h later. Triple immunofluorescence was with Alexa 647–labeled anti-POM121, Alexa 488 anti-Nup107, and Alexa 568 anti-gp210. Arrows point to cells almost completely depleted of gp210 and POM121. Comparison with nontransfected cells indicates that this codepletion affected neither NE assembly nor assembly of Nup107 into NPCs. (B) POM121 was depleted by RNAi from human primary fibroblasts that already lack gp210 (Fig. 4 A). Arrows indicate cells whose POM121 staining was reduced to background levels. NE or NPC assembly remained unaffected as judged by their normal mAb414 signal.