Analysis of the Lotus japonicus nuclear pore NUP107-160 subcomplex reveals pronounced structural plasticity and functional redundancy

Abstract

Mutations in the Lotus japonicus nucleoporin genes, NUP85, NUP133, and NENA (SEH1), lead to defects in plant-microbe symbiotic signaling. The homologous proteins in yeast and vertebrates are part of the conserved NUP84/NUP107-160 subcomplex, which is an essential component of the nuclear pore scaffold and has a pivotal role in nuclear pore complex (NPC) assembly. Loss and down-regulation of NUP84/NUP107-160 members has previously been correlated with a variety of growth and molecular defects, however, in L. japonicus only surprisingly specific phenotypes have been reported. We investigated whether Lotus nup85, nup133, and nena mutants exhibit general defects in NPC composition and distribution. Whole mount immunolocalization confirmed a typical nucleoporin-like localization for NUP133, which was unchanged in the nup85-1 mutant. Severe NPC clustering and aberrations in the nuclear envelope have been reported for Saccharomyces cerevisiae nup85 and nup133 mutants. However, upon transmission electron microscopy analysis of L. japonicus nup85, nup133 and nena, we detected only a slight reduction in the average distances between neighboring NPCs in nup133. Using quantitative immunodetection on protein-blots we observed that loss of individual nucleoporins affected the protein levels of other NUP107-160 complex members. Unlike the single mutants, nup85/nup133 double mutants exhibited severe temperature dependent growth and developmental defects, suggesting that the loss of more than one NUP107-160 member affects basal functions of the NPC.

Keywords: Lotus japonicus; NUP107-160 subcomplex; nuclear pore complex; nucleoporins; plant nucleus.

Figure 1

Indirect immunolocalization of L. japonicus NUP133. 3D structured illumination microscopy (Z-stack) detected a punctated NUP133 signal around the nucleus, corresponding to individual NPCs or clusters of nuclear pores. No change to NUP133 localization was observed in the nup85-1 mutant. The α-NUP133 primary antibody was visualized with an Alexa Fluor® 488 labeled secondary antibody (green). DNA was stained with DAPI (red). wt, wild type. Scale bars = 5 μm.

Figure 2

NPC distance distribution. (A) TEM micrographs of ultrathin sections of L. japonicus root tips (N, nucleus; C, cytosol; M, nuclear membranes). White arrow heads indicate the position of nuclear pores. The lower images show a magnified view of individual pores. Scale bar = 500 nm. (B) Distance distribution between adjacent NPCs. (C) Box plot showing NPC distances. “^***” indicates significant difference from the wild type. (ANOVA revealed significant differences between average distances with F_{(3, 648)} = 7.63, p < 0.001. Dunnett test all vs. wild type on log transformed data identified nup133-1 as having a smaller mean than wild type; p < 0.001). The number of measured distances for each line is indicated in brackets below the x-axis.

Figure 3

Quantification of NUP85 and NUP133 protein levels. (A) Total protein extract from L. japonicus roots was separated by SDS-PAGE, blotted on membrane and immunolabelled with α-NUP85, α-NUP133, and α-histone H3 antibodies. (B) For quantification, signal intensities of NUP85 and NUP133 were normalized by the histone H3 signal and shown as percentages of wild type levels (wt). Mean protein levels were averaged from three independent experiments. Error bars show SD, “^***” indicate significant differences from the wild type (P ≤ 0.05, t-test).

Figure 4

Analysis of nup85/nup133 double mutants. The loss of both NUP85 and NUP133 caused severe growth and developmental defects in the double mutants. At low temperature (18°C) cotyledons and short roots could still develop, while at higher temperature (24°C), plant development was arrested at the early germination stages. Plants and undeveloped seeds were cultivated for 16 days. Scale bar = 1 cm.