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. 2018 Apr 24;115(17):E3969-E3977.
doi: 10.1073/pnas.1719398115. Epub 2018 Apr 9.

Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

Sasikumar Rajoo  1   2 , Pascal Vallotton  1 , Evgeny Onischenko  1 , Karsten Weis  3
Affiliations

Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

Sasikumar Rajoo et al. Proc Natl Acad Sci U S A. .

Abstract

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

Keywords: NPC composition; nuclear pore complex; nucleoporins; quantitative fluorescence microscopy; stoichiometry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Analysis of the relative NPC stoichiometry. (A) Outline of the nuclear rim intensity measurement (NuRIM) technique. (A, i–vi) Cells coexpressing dsRed-HDEL and various GFP-tagged Nups were imaged sequentially in bright-field, dsRed, and GFP channels. (A, i) Bright-field channel shows localization of immobilized yeast cells. (A, ii) Exclusion of the overcrowded regions (light areas) based on the bright-field images. (A, iii) dsRed-HDEL channel shows localization of dsRed-HDEL marking the NEs. (A, iv) Production of binary NE contour masks based on the dsRed images. (A, v) GFP channel showing localization of endogenously GFP-tagged Nups. (A, vi) Background-subtracted Nup-GFP intensities are measured and averaged across thousands of binary NE contours. (Scale bars: 2 μm.) (B) Linearity of GFP-based intensity measurements. Cells expressing indicated yEGFP-tagged Nups alone or in pairwise combinations were imaged in parallel. Corresponding NE signal intensities were quantified using NuRIM. Gray bars represent corresponding arithmetic sums of the NE signal intensities of singly labeled strains. Mean ± SD. n, number of image frames analyzed. (C) Robustness of GFP-based intensity measurements against microenvironmental variability. Intensity values obtained using NuRIM for the indicated Nups with C-terminal yEGFP tag separated by 6- or 102-aa linker sequence. Intensity values were normalized to Nup84-yEGFP containing a short linker. Mean ± SD. n, number of image frames analyzed. (D) Schematic representation of the NPC. Nup subgroups are depicted as colored cartoons. (E) Relative abundance of yeast Nups. NE intensities for the indicated yEGFP-tagged Nups were quantified using NuRIM and normalized for the average value obtained for the six components of the Y complex. *Relative abundance for GFP-Nic96 was obtained from independent measurements normalized to Nup120-GFP. Mean ± SD. n, number of independent experiments performed on different days.
Fig. 2.
Fig. 2.
Absolute stoichiometry of yeast Nups. (A) Representative intensity histograms and Gaussian fits (smooth lines) obtained for single yEGFP molecules, single NPCs in regions between separating nuclei in Nup84-yEGFP–expressing yeast cells (arrowhead), and for the yeast-expressed 120-mer VP2-yEGFP particles (arrowhead). (Scale bars: 2 μm.) (B) Intensity histograms and fits are displayed similar to A for single NPCs in Nup84-sfGFP–expressing yeast cells, and for purified SF12 and SF24 particles containing 12 and 24 sfGFP molecules, respectively. (C, i) Representative fluorescence microscopy image showing localization of Nup84-yEGFP–labeled NPCs. (C, ii) Computational model of the NE. NPCs are represented as green dots, dsRed-HDEL as red dots, and dim sources simulating background fluorescence as blue dots. (C, iii) Simulated image of the NE produced computationally from the model ii. Note good resemblance to genuine Nup84-yEGFP localization (compare with i). Inset compares computed PSF with experimentally acquired PSF. (C, iv) Representative 3D rendering of image stack shows heat map of intensities in Nup84-yEGFP cells illustrating high-intensity spots or “speckles” (red) produced due to stochastic overlap of the ∼120 NPCs. (Scale bar: 200 nm.)
Fig. 3.
Fig. 3.
Analysis of Nup stoichiometry in yeast mutants. (A, C, and E) Representative images illustrating Nup-yEGFP localization in actively growing yeast cells with specified genotypes. The corresponding NE intensities quantified using NuRIM are shown in B, D, and F, respectively. Mean ± SD. n, number of image frames analyzed for B, D, and F. (Scale bars: 2 μm.) (G) Representative intensity histograms and Gaussian fits obtained for single NPCs in NUP170-yEGFP and NUP170-yEGFP nup170Δ URA3::NUP170-yEGFP strains.
Fig. 4.
Fig. 4.
Modular organization of the NPC. (A) Schematic of the NPC scaffold. (B–D) Illustration depicting the experimental outcomes shown in Fig. 3.
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