Nuclear Pores in Plant and Animal Cells

Abstract

Trafficking between the nucleoplasm and the cytoplasm occurs through the nuclear pore complex (NPC), which consists of large multiprotein complexes. Over the last several years, major progress has been made in both structural determination of the entire assembly of the NPC in yeast and animal cells. By contrast, the plant NPC has long been neglected. Components of the NPC in Arabidopsis thaliana have been identified recently using an interactive proteomic approach. The Arabidopsis nucleoporins are homologous to human nucleoporins, except for a single protein called Nup136. Nup136 is involved in flowering and pollen development, suggesting that Nup136 plays a physiological role in plant reproduction. Nup136 also regulates morphology of the nucleus. Overexpression of Nup136-GFP was found to induce elongation of nuclei in various tissues, whereas deficiency of Nup136 caused a reduction in the size of nuclei. Nup136 is thought to be a functional homologue to animal Nup153, although they have no sequence homology. The mechanism underlying the regulation of nuclear morphology by Nup136, which is thought to be unique to higher plants, is discussed.

The nucleus is a definitive feature of eukaryotic cells, having twin bilamellar membranes, the inner and outer nuclear membranes that separate the nucleoplasm from the cytoplasm. The nuclear pore complex (NPC), which spans two nuclear membranes, regulates the exchange of macromolecule trafficking between the nucleus and the cytoplasm. In animal cells, the nuclear lamina that tightly underlies the inner nuclear membrane is associated with the NPC.1,2 The lamina composed of type V intermediate filament proteins forms a mesh-like structure throughout the inner periphery of the nuclei. A similar structure has been observed in tobacco nuclei,3,4 although plants have no intermediate filaments. However, very little information is available about the physiological function and the biochemical components of this structure. An important question is how plants establish and maintain nuclear structure.

Morphological Features of Arabidopsis Nuclei

The morphology of nuclei varies in size and shape depending on the tissues, even within individual organisms. Arabidopsis thaliana, a model plant, in which nuclei are morphologically differentiated throughout the mature organs, provides an ideal material to study nuclear morphology, involving variation in size and shape.5,6 To visualize the nucleus in living Arabidopsis plants, Chytilova and colleagues generated transgenic plants expressing nuclear localized green fluorescent protein (GFP).5 They found that nuclear shape varies widely from a spherical morphology to a highly elongated morphology. This finding and results from related studies indicate that nuclear morphology is diverse and it depends on particular types of tissues and cells.6

The root tissue of Arabidopsis has been studied as a model for cellular differentiation because of its simple structure (Fig. 1A). Figure 1 shows that nuclear morphology varies according to the stage of cell differentiation in the root. In the meristematic zone, where undifferentiated cells undergo mitosis actively, the nuclei are uniformly small and spherical (Fig. 1D). After mitosis, the cells start to expand in the elongation zone, where nuclei increase their volume by maintaining a spherical shape (Fig. 1C). Highly elongated nuclei with almost rod-like shapes are evident in the stationary zone, within which the cells have already differentiated to epidermal cells (Fig. 1B). These results indicate that nuclei change their shape and size along with growing cells.

The Plant NPC

The NPC is the sole gateway between the nucleoplasm and the cytoplasm, mediating the traffic of proteins and RNAs.7,8 This nucleocytoplasmic transport of macromolecules is an important event in eukaryotic cells. The NPC is the largest multiprotein channel, consisting of multiple copies of approximately 30 different nucleoporins (Nups) of yeast9 and vertebrates.10 Nups are classified into two categories:11 the first includes scaffold Nups, which consist mainly of the Nup107-Nup160 and Nup93 subcomplexes, and the second includes peripheral Nups, which consist mainly of the Nup62 subcomplex, a nuclear basket and cytoplasmic filaments.

Knowledge of the functions of individual components and the overall structure of the NPC in plants lags behind that of vertebrates10 and yeast.9 Until very recently, only eight Nups of plants had been characterized.8 Using interactive proteomics, we recently revealed and characterized Arabidopsis NPC structure.12 Transgenic Arabidopsis expressing GFP-tagged RAE1 (RNA export factor 1), a known plant Nup,13 was generated. Then, to isolate proteins interacting with the GFP-tagged nucleoporin, immunoprecipitation was performed with anti-GFP antibody using the transgenic Arabidopsis tissues. The immunoprecipitates were subjected to mass spectrometry analysis using an LTQ-Orbitrap for the identification of proteins present. The identified candidates for nucleoporins were cloned and subcellular localization analyses were performed by expression of GFP fusion in the cells. Twenty-five nucleoporins were identified in immunoprecipitates from transgenic Arabidopsis plants expressing RAE1-GFP. These nucleoporins were further subjected to interactive proteomics with RAE1-GFP to explore other nucleoporins that make up the plant NPC. By repeating these analyses, a total of 30 Arabidopsis Nups were identified,12 22 of which had not been previously annotated (Fig. 2).

Most of the Arabidopsis Nups exhibited high sequence homology to vertebrate Nups. A Nup unique to plants was found and designated as Nup136, which was reported as Nup1.14 Neither yeast nor vertebrates have homologs of Nup136. T-DNA insertion mutants of Nup136 had pleiotropic phenotypes, such as defects in pollen development, flowering time and nuclear morphology,12 suggesting that Nup136 plays an important role in the NPC.

Nup136 and Nuclear Morphology

To explore the function of Nup136 in greater detail, the knockout mutant nup136 was established and three T3 lines of transgenic Arabidopsis plants were generated that stably overexpress Nup136-GFP under the 35S promoter. The transgenic plants show no significant whole-plant phenotype compared to wild-type plant. The nuclei in wild-type and transgenic plants were inspected with a confocal laser microscope. Guard cells of wild-type plants had spherical nuclei (Fig. 3A); trichomes had spindle-shaped nuclei (Fig. 3E); and leaf epidermal cells had both types of nuclei (Fig. 3C). On the other hand, transgenic plants overexpressing Nup136-GFP had elongated nuclei in guard cells, leaf epidermal cells and trichomes (Fig. 3B, D and F). In particular, nuclei in trichomes of the overexpressor formed extremely elongated structures (Fig. 3F). To quantify the morphology of nuclei, a circularity index was applied (Fig. 3G) and the length of the major axis was measured (Fig. 3H) with ImageJ software (1.40 g, NIH, USA). Nuclei of the three overexpressor lines had a lower circularity index (Fig. 3G) and a longer major axis (Fig. 3H) in comparison to wild-type plants. In contrast, nuclei of the knockout mutant nup136 had a higher circularity index and a shorter major axis.12 Taken together, these results indicate that the quantity of Nup136 on the nuclear envelope determines nuclear shape in Arabidopsis.

How does overexpressed Nup136 affect nuclear morphology? The DNA content is one factor influencing nuclear size. During differentiation, a substantial portion of Arabidopsis cells undergoes endoreduplication, which involves replication of chromosomal DNA without cell division.15 For example, the nuclei in guard cells are diploid16 and are smaller and more spherical in shape compared to the surrounding pavement cells that undergo multiple rounds of endoreduplication. This raises the possibility that Nup136 is involved in endoreduplication.

Alternatively, it is possible that Nup136 interacts with structural factors responsible for the maintenance of nuclear morphology. In-lens field emission scanning electron microscopy (feSEM) of tobacco nuclei has shown that an organized filamentous structure interconnects NPCs under the inner nuclear membrane.3,4 The organization and dimensions of this filamentous structure are similar to those of the nuclear lamina in Xenopus oocytes.17 These results suggest that plant NPCs interact closely with lamin-like structures at inner nuclear envelopes as seen in animal cells. It is known that the nuclear lamina controls spatial distribution of the NPC on the nuclear envelope in Caenorhabditis18 and Drosophila19 by physical association with the NPC through the C-terminal domain of Nup153.20,21 Arabidopsis Nup136 is thought to be a functional homolog to Nup153, although they have no sequence homology. Thus, it is assumed that Nup136 in Arabidopsis is a key determinant of nuclear structure through the regulation of the interaction between NPCs and lamin-like structures.

Outlook

It is likely that nuclear morphology correlates closely to the state of cellular differentiation in Arabidopsis. However, the mechanism by which plant cells control their nuclear shape is largely unknown. In the study of progeria syndrome, it has been shown that mutated lamin A gene causes a defect in NPC-mediated nuclear import and nuclear morphology.22 This raises the possibility that nuclear structure is involved in the regulation of NPC functions according to the state of cellular differentiation. Further study of Nup136 is necessary for understanding how plants regulate nuclear structure.

Figures and Tables

Involvement of the nuclear pore complex in morphology of the plant nucleus

Published online:

01 May 2011

Figure 1 Shape and size of nuclei vary among developmental stages in root cells of Arabidopsis thaliana. (A) Bright field image of Arabidopsis root tissues in a 7-day-old seedling. Circles indicate regions inspected for nuclear morphology. (B–D) Nuclei in root epidermal cells of the stationary zone (B), elongation zone (C) and meristematic zone (D) were visualized with DAPI. Images were obtained by confocal laser scanning microscopy. Bars = 20 µm.

Figure 1 Shape and size of nuclei vary among developmental stages in root cells of Arabidopsis thaliana. (A) Bright field image of Arabidopsis root tissues in a 7-day-old seedling. Circles indicate regions inspected for nuclear morphology. (B–D) Nuclei in root epidermal cells of the stationary zone (B), elongation zone (C) and meristematic zone (D) were visualized with DAPI. Images were obtained by confocal laser scanning microscopy. Bars = 20 µm.

Involvement of the nuclear pore complex in morphology of the plant nucleus

Published online:

01 May 2011

Figure 2 Comparison between the NPCs of plants and vertebrates. Subcomplexes are shown as single units.

Figure 2 Comparison between the NPCs of plants and vertebrates. Subcomplexes are shown as single units.

Involvement of the nuclear pore complex in morphology of the plant nucleus

Published online:

01 May 2011

Figure 3 Overexpression of Nup136-GFP altered Nuclear Morphology. (A–F) The nuclei in guard cells (A and B), rosette leaf epidermal cells (C and D), trichome cells (E and F) of wild-type (WT) or transgenic plants expressing Nup136-GFP (Nup136-GFPox) were visualized with Hoechst 33342 (1 µg/ml). Images were obtained by confocal laser scanning microscopy (CLSM) and differential interference contrast microscopy (DIC). Bars = 10 µm (A and B), 25 µm (C–F). (G and H) The circularity index (G) and major axis (H) of nuclei were measured in the wild type (WT) and three independent T3 lines (#1-4, #2-1 and #3-3) overexpressing Nup136-GFP.

Figure 3 Overexpression of Nup136-GFP altered Nuclear Morphology. (A–F) The nuclei in guard cells (A and B), rosette leaf epidermal cells (C and D), trichome cells (E and F) of wild-type (WT) or transgenic plants expressing Nup136-GFP (Nup136-GFPox) were visualized with Hoechst 33342 (1 µg/ml). Images were obtained by confocal laser scanning microscopy (CLSM) and differential interference contrast microscopy (DIC). Bars = 10 µm (A and B), 25 µm (C–F). (G and H) The circularity index (G) and major axis (H) of nuclei were measured in the wild type (WT) and three independent T3 lines (#1-4, #2-1 and #3-3) overexpressing Nup136-GFP.

Extra View to: Tamura K , Fukao Y , Iwamoto M , Haraguchi T , Hara-Nishimura I . Identification and characterization of nuclear pore complex components in Arabidopsis thaliana. Plant Cell 2010; 22:4084 - 4097; PMID: 21189294; http://dx.doi.org/10.1105/tpc.110.079947

Nuclear Pores in Plant and Animal Cells

Source: https://www.tandfonline.com/doi/abs/10.4161/nucl.2.3.16175

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