Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis

Authors


*(fax +82 2 312 5657; e-mail hspai@yonsei.ac.kr).

Summary

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1, NbRae1, which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1-silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro. Inhibition of NbRae1 expression in BY-2 cells using a β-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.

Introduction

The nuclear pore complex (NPC) is a large, intricate macromolecular structure within the nuclear envelope which mediates the transport of macromolecules such as RNA and proteins between the nucleus and the cytoplasm (Rout et al., 2003; Suntharalingam and Wente, 2003; Akhtar and Gasser, 2007). Based on the previous characterization, the function and overall structure of the NPC, as well as its subunits, seem to be conserved in diverse eukaryotes (Bapteste et al., 2005). In yeast, NPC is composed of approximately 30 different nucleoporins (Nups), some of which contain docking sites for transport complexes on the NPC (Lim and Fahrenkrog, 2006; Tran and Wente, 2006). The Nups form a channel-like structure of eight-fold symmetry, which is divided into three regions: a nuclear basket, a central pore, and cytoplasmic fibrils. The RNAs are exported as ribonucleoprotein complexes (RNPs), and nuclear export receptors interact with both the RNP particles and Nups to direct their cargo through the NPC to the cytoplasm (Stutz and Izaurralde, 2003).

One of the mRNA export factors on the NPC is Rae1 in Schizosaccharomyces pombe, Gle2p in Saccharomyces cerevisiae, and Rae1/mrnp41 in metazoans. Temperature-sensitive mutations in Rae1/Gle2 block poly(A) RNA export in mammals and yeast (Brown et al., 1995; Murphy et al., 1996; Bharathi et al., 1997). In addition, the NPC and the nuclear envelope (NE) structure are disturbed in yeast temperature-sensitive gle2 mutants, including the NPC clusters, membrane-herniated structure, and additional layers of double membrane beneath the NE (Murphy et al., 1996). However, the disruption of Rae1 function in S. pombe blocks mRNA export without affecting these structures (Brown et al., 1995). Rae1 binds to poly(A)-containing mRNA (Kraemer and Blobel, 1997), and is a shuttling mRNA export factor that interacts with the Gle2p-binding sequence (GLEBS) motif of Nup98 at the NPC (Pritchard et al., 1999). Recently, it was proposed that Rae1/Gle2p delivers TAP/NXF1 to Nup98 by direct binding, which could be the first step in the interaction between the mRNA export complex and Nups (Blevins et al., 2003).

A dual function of Rae1 was first indicated in a temperature-sensitive Rae1 mutation in S. pombe, which resulted in nuclear accumulation of poly(A) RNA and cell-cycle arrest at the G2/M boundary (Whalen et al., 1997). Arrest occurs before the formation of the mitotic spindle, and the spindle pole bodies do not separate (Whalen et al., 1997). Later, it was found that Rae1 is a microtubule-associated protein and that the Rae1-containing RNP is required for mitotic spindle assembly (Blower et al., 2005). Furthermore, Rae1 is an essential mitotic checkpoint regulator that cooperates with the mitotic checkpoint protein Bub3 to prevent chromosome missegregation and premature separation of sister chromatids (Babu et al., 2003). Rae1 and Nup98 form a complex with Cdh1-activated anaphase-promoting complex (APC) and inhibit degradation of securin, the anaphase inhibitor, to prevent premature separation of sister chromatids and aneuploidy (Jeganathan et al., 2005). Rae1 plays an important role in the progression through the G1 phase of the cell cycle in Drosophila (Sitterlin, 2004).

Rae1 homologs are present in diverse plant genomes, but the functions of these genes in plants have not been investigated. In this study, we addressed the in vivo function of a Rae1 homolog of Nicotiana benthamiana, designated NbRae1, using virus-induced gene silencing (VIGS) and β-estradiol-inducible RNAi techniques. NbRae1 was enriched in the nuclear rim during interphase of the cell cycle, and NbRae1-deficient cells exhibited defects in the export of poly(A)+ RNAs. During mitosis, NbRae1 was associated with mitotic microtubules, and depletion of NbRae1 resulted in disrupted spindle organization and chromosome missegregation. These results provide new insights into the multiple cellular functions of NbRae1 in acentrosomal plant cells.

Results

Identification of NbRae1

We have carried out functional genomics using tobacco rattle virus (TRV)-based VIGS in N. benthamiana (Ahn et al., 2004; Park et al., 2005; Kim et al., 2006). The screening revealed that gene silencing of a homolog of human Rae1 caused growth arrest and abnormal leaf development. The full-length cDNA encoding NbRae1 (accession number ABG29731) is 1501 bp in length and encodes a 347 amino acid polypeptide with a molecular mass of 38 350.04 Da (Figure S1). The predicted NbRae1 protein contains four WD40 repeats and is highly homologous to the Rae1 proteins in human (accession number NP_003601), yeast (NP_011033), Arabidopsis (NP_178182), and rice (NP_001061119) (Figure S1). The NbRae1 transcript level in N. benthamiana seedlings was not significantly changed in response to diverse stresses or phytohormone treatments (Figure S2, Text S1).

Virus-induced gene silencing of NbRae1 and suppression of the endogenous transcripts

To induce silencing of NbRae1, we cloned three different cDNA fragments of NbRae1 into the TRV-based VIGS vector pTV00 (Kim et al., 2006), and infiltrated N. benthamiana plants with Agrobacterium tumefaciens containing each plasmid (Figure 1a). TRV:Rae1(N) and TRV:Rae1(C) contained 0.50 kb N-terminal and 0.49 kb C-terminal coding regions of the NbRae1 cDNA, respectively, while TRV:Rae1(F) contained the entire coding region. Virus-induced gene silencing with each construct resulted in growth arrest and abnormal leaf morphology (Figure 1b). This phenotype has been observed in all of the N. benthamiana plants (> 150) that have been subjected to NbRae1 VIGS to date. The effect of the NbRae1 gene silencing on the levels of endogenous NbRae1 mRNA in the leaves of the infiltrated plants was analyzed by semi-quantitative RT-PCR using three different sets of NbRae1 primers, Rae1-F, Rae1-N, and Rae1-C (Figure 1a,c). The RT-PCR using the Rae1-F primers detected significantly reduced levels of the endogenous NbRae1 mRNA in both the TRV:Rae1(N) and TRV:Rae1(C) lines compared with the TRV control, while the same primers detected high levels of viral genomic transcripts in the TRV:Rae1(F) lines. Similarly, the Rae1-N and Rae1-C primers revealed lower levels of the endogenous transcripts in the TRV:Rae1(C) and TRV:Rae1(N) lines, respectively. The transcript levels of actin, which serve as the control, remained constant (Figure 1c).

Figure 1.

 NbRae1 virus-induced gene silencing (VIGS) constructs, phenotypes, and NbRae1 silencing.
(a) Schematic of the three VIGS constructs containing different regions of the NbRae1 cDNA (marked by bars). The positions of the primer sets used for RT-PCR analyses (Rae1-F, Rae1-N, and Rae1-C) are shown.
(b) Effect of NbRae1 VIGS on plant morphology. Photographs of the leaves and whole plants were taken at 20 days after infection with Agrobacterium tumefaciens containing the Tobacco Rattle Virus (TRV) construct (control) or the TRV:Rae1 constructs.
(c) Effect of NbRae1 VIGS on NbRae1 transcript levels as measured by semi-quantitative RT-PCR. The RNA was extracted from the fourth leaf above the infiltrated leaves from Nicotiana benthamiana plants infected with TRV, TRV:Rae1(F), TRV:Rae1(N), and TRV:Rae1(C). The Rae1-F, Rae1-N, and Rae1-C primers were used. Actin mRNA levels served as a control.

Enrichment of the NbRae1 protein at the nuclear periphery

We examined the subcellular localization of NbRae1 by expressing NbRae1:GFP, in which the full-length NbRae1 (M1–K347) protein was fused to green fluorescent protein (GFP). The DNA construct encoding NbRae1:GFP under the control of the CaMV35S promoter was introduced into protoplasts isolated from N. benthamiana seedlings, and gene expression was examined by confocal laser scanning microscopy (Figure 2a). The green fluorescent signal of NbRae1:GFP was highly enriched around the NE, but was also weakly detected in the nucleoplasm and the cytosol at longer exposure (Figure 2a, top; data not shown). We also transformed the NbRae1:GFP construct under the control of the promoter of the Arabidopsis Rae1 homolog (At1g80670) into Arabidopsis protoplasts, and obtained a similar localization pattern (data not shown). As a positive control, localization of a GFP-fusion protein containing Arabidopsis WPP1, an MFP1 attachment factor 1 (MAF1) homolog that is primarily associated with the NE (Patel et al., 2004), was examined, and the GFP signal was clearly detected around the NE and in the cytoplasm (Figure 2a, middle). Green fluorescent protein alone was detected in the cytoplasm and the nucleus (Figure 2a, bottom).

Figure 2.

 Enrichment of NbRae1 in the nuclear envelope (NE).
(a) Nicotiana benthamiana protoplasts were transformed with the NbRae1:GFP fusion construct and examined 24 h later by confocal laser scanning microscopy. Chlorophyll autofluorescence, bright-field and merged images are shown. The NE localization of WPP:GFP fusion protein (Patel et al., 2004) and the cytosolic and the nuclear localization of GFP are shown as a control. The inset shows the nucleus, n. Scale bars = 10 μm.
(b) Immunoblot analysis of the expression of hemagglutinin (HA):NbRae1 fusion proteins in transgenic BY-2 cells using anti-HA antibody. Fifty micrograms of total protein isolated from the transgenic BY-2 cell lines 4 and 5 were loaded in each lane. Immunoblotting with anti-α-tubulin antibody was performed as a control for equal loading.
(c) BY-2 cells (line 4) expressing HA:NbRae1 fusion protein were immunolabeled with anti-α-tubulin (green) and anti-HA (red) antibodies, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), before observation under confocal laser scanning microscopy. The arrow shows the nucleus, n. Scale bars = 10 μm.
(d) The transgenic BY-2 cells (line 4) were immunolabeled with anti-γ-tubulin (red) and anti-HA (green) antibodies. Scale bars = 10 μm.

To confirm the NE-enriched localization of NbRae1, we transformed tobacco BY-2 cells with hemagglutinin (HA):NbRae1 constructs in which the N-terminus of the full-length NbRae1 protein coding region was fused to a HA tag under control of the CaMV35S promoter. NbRae1 protein products (∼40 kDa) were detected in the most rapidly growing transgenic BY-2 cell lines (nos four and five) by western blot analysis with anti-HA antibody (Figure 2b). The HA:NbRae1 protein in the transgenic cells was immunolabeled using anti-HA antibody and observed using confocal laser scanning microscopy (Figure 2c). Cortical microtubules and DNA were visualized by anti-α-tubulin antibody and 4′,6-diamidino-2-phenylindole (DAPI) staining, respectively. In interphase nuclei, NbRae1 was concentrated predominantly in the nuclear envelope, but was also present in the nucleoplasm and the cytosol in a punctuate pattern (Figure 2c). Cell lines 4 and 5 exhibited the same patterns of subcellular distribution of NbRae1. We next compared the NbRae1 localization with that of γ-tubulin in BY-2 cells using anti-HA and anti-γ-tubulin antibodies (Figure 2d). Consistent with the previous observations (Erhardt et al., 2002; Seltzer et al., 2007), γ-tubulin was highly enriched at the nuclear surface, and distributed in punctuate patterns in the cytosol and near the plasma membrane. There was no significant co-localization of NbRae1 and γ-tubulin in the perinuclear region: the red fluorescence of γ-tubulin was detected at the surface of the NE surrounding the green fluorescence of NbRae1 (Figure 2d).

The essential role of NbRae1 in mRNA export

In vertebrate and yeast cells, Rae1 is involved in mRNA export, and defects in Rae1 result in the accumulation of mRNA within the nucleus (Brown et al., 1995; Murphy et al., 1996; Bharathi et al., 1997). To determine the potential role of NbRae1 in mRNA export, we performed in situ hybridization to locate the poly(A) signals in the leaf cells from the control TRV and the NbRae1 VIGS plants (Figure 3). The abaxial epidermal layers of TRV, TRV:Rae1(N), and TRV:Rae1(C) plants were hybridized with a 45-mer oligo(dT) probe that was end-labeled with fluorescein for green fluorescence. To localize the nuclei, the epidermal layers were briefly stained with propidium iodide (PI) that fluoresces red. A low level of green fluorescence was detected in both the cytoplasm and the nuclei in the TRV epidermal cells. In contrast, the epidermal cells from both of the TRV:Rae1 lines accumulated much stronger poly(A) RNA signals in the nuclei than did the TRV control cells, suggesting that NbRae1 deficiency leads to a defect in mRNA export.

Figure 3.

 Nuclear accumulation of poly(A) RNAs in NbRae1-silenced leaves.
Nicotiana benthamiana leaves were subjected to in situ hybridization with fluorescein-tagged poly(dT) oligonucleotides, and nuclei were counterstained with propidium iodide (PI). Note the accumulation of green fluorescence, which corresponds to poly(A) RNA, in the nuclei of the TRV:Rae1(N) and TRV:Rae1(C) plants. TRV, tobacco rattle virus.

Ultrastructural study of the cellular structures including the NE and the NPC

We examined the nuclear morphology including the structure of the NE and NPC using transmission electron microscopy of the transverse leaf sections (Figure 4). Many of the nuclei in the TRV:Rae1 lines appeared to be larger and more elongated than those of the TRV control (Figure 4). Furthermore, the NE was occasionally altered in the NbRae1 VIGS lines: the NE appeared to be abnormally extended and contained additional membrane fragments beneath it (Figure 4e–g,; cf. control in a–c). However, the NPC clusters and the membrane-herniated structure shown in the yeast gle2 mutant were not observed in TRV:Rae1 lines (Figure 4h, cf. control in d). Apart from the apparent nuclear abnormality, there was no marked difference in the cellular structures, including the chloroplasts, between the control and NbRae1 VIGS lines. However, iodine staining indicated that TRV:Rae1 leaves accumulated less starch than TRV leaves (data not shown).

Figure 4.

 Ultrastructural analysis of cell morphology.
(a–h) Representative transmission electron micrographs of the nuclear envelope (NE) (a–c, e–g) and the nuclear pore complex (NPC) (d, h) from Tobacco Rattle Virus (TRV) control (a–d) and TRV:Rae1 (e–h) lines. The arrows indicate the NE (a), abnormal NE structures (e–g) and the NPC (d, h). Scale bars = 2 μm in (a–c, e, f), 1 μm in (g), and 0.2 μm in (d, h).
(i–l) Representative transmission electron micrographs of the leaf mesophyll cells (i, k) and the mesophyll chloroplasts (j, l) from TRV control (i, j) and TRV:Rae1 (k, l) lines. c, chloroplast; s, starch. Scale bars = 5 μm in (i), 1 μm in (j, l), and 20 μm in (k).

Reduced cell division activities in the TRV:Rae1 shoot apex

The Arabidopsis mutants cryophyte/los4 (DEAD box RNA helicase) and sar1/atnup160 (Nup160), both of which are defective in mRNA export, have a reduced stature and early flowering time but do not exhibit severe defects in growth and development (Gong et al., 2004; Dong et al., 2006). In N. benthamiana, VIGS of Nup85, Nup160, and NUCLEAR PORE ANCHOR (NUA) resulted in only mild effects on plant growth, in contrast to NbRae1 VIGS (data not shown). The mutation of NUA also causes defective mRNA export in Arabidopsis (Xu et al., 2007). These results indicate that the severe growth retardation in the NbRae1 VIGS plants may be caused by defects in other functions of NbRae1, separate from the mRNA export machinery. Based on these findings, we investigated if NbRae1 plays a role in the control of cell division in plants.

In plants, reduced cell division is frequently compensated by an increase in cell expansion, and cell enlargement has been used as an indicator for reduced cell division (Donnelly et al., 1999; De Veylder et al., 2001; Kessler and Sinha, 2004). We found that the leaves of the TRV:Rae1 plants were small, curled and distorted (Figure 1b). Light microscopic examination revealed that the morphology of the epidermal cells of the TRV:Rae1 leaves remained normal, but the cell size was enlarged about 1.5-fold compared with those of the TRV control (Figure 5a,c; cf. control in b). In contrast, the average number of cells in the epidermis decreased to ∼60% of the TRV control (Figure 5d). Transverse leaf sections also showed that the number of cells decreased and the cell size increased in every layer in the TRV:Rae1 leaves, compared with the TRV control leaves (Figure 5f,g; cf. control in e). The typical dorsoventral organization of the palisade and mesophyll cells was mostly maintained in the moderately affected tissues (Figure 5f), but in the severely affected tissues cell enlargement was accompanied by somewhat irregular cell morphology (Figure 5g).

Figure 5.

 Reduced mitotic activities and increased ploidy in NbRae1-silenced leaves.
(a, b) Representative light micrographs of the abaxial leaf epidermis of the tobacco rattle virus (TRV) control (a) and the TRV:Rae1 line (b). Scale bars = 150 μm.
(c, d) Comparison of cell sizes and relative cell numbers between TRV and TRV:Rae1 lines. The cell size (c) and relative cell number (d) of the epidermal cells (abaxial side) from the fourth leaf above the infiltrated leaf of TRV and TRV:Rae1 plants were compared by light microscopy and displayed in the bar graphs.
(e–g) Representative light micrographs of leaf transverse sections from the TRV control (e) and the moderately (f) and severely affected regions (g) of TRV:Rae1 lines. Scale bars = 50 μm.
(h) Reduced cyclin-dependent kinase (CDK) activities. Total CDKAPSTAIRE from the shoot apex of TRV and TRV:Rae1 plants was bound to the p13Suc1-conjugated agarose beads. The CDK activities of the bound material were measured by kinase assay using histone H1 as a substrate. The reaction mixture was then resolved by SDS–PAGE. An autoradiogram (32P-histone H1) and Coomassie-stained histone H1 band (input) are shown.
(i) Real-time quantitative (q)RT-PCR analyses for expression of cell cycle-related genes. The transcript levels of Nicotiana benthamiana CycB1, CDKB1-1, and histone H3 and H4 genes were examined by real-time qRT-PCR with gene-specific PCR primers relative to a reference gene of β-actin. Complementary DNA was prepared from 1 μg of the total RNA from the shoot apex of the virus-induced gene silencing (VIGS) plants. Each value represents the mean ± SE of three replicates per experiment. Asterisks denote the statistical significance of the differences between TRV and TRV:Rae1: * 0.01; **P ≤ 0.005.
(j–l) Scanning electron micrographs of adult vegetative meristem with leaf primordia from TRV (j) and TRV:Rae1 lines (k, l). m, meristem; p, leaf primordium. Scale bars = 50 μm in (j), 100 μm in (k) and 200 μm in (l).
(m, n) Increased ploidy levels in the leaf cells of TRV:Rae1 lines. Nuclei isolated from the fourth leaf above the infiltrated leaf were stained with propidium iodide and analyzed by flow cytometry. Ploidy distribution of leaf nuclei from the representative TRV and TRV:Rae1 lines (m) and quantification of the ploidy levels (n) are shown. TRV:Nup85 and TRV:NUA VIGS plants were analyzed as controls.

To confirm the correlation between reduced cell division and NbRae1 deficiency, we examined the cyclin-dependent kinase (CDK) activity in the shoot apex. The CDK activity in TRV:Rae1 lines decreased to about 34% of that in the TRV control (Figure 5h). Furthermore, the transcript levels of the histones H3 and H4, Cyclin B1 (CycB1), and CDKB1-1, all of which are expressed in actively dividing cells (Boudolf et al., 2004), were significantly reduced in the shoot apex of the NbRae1 VIGS plants based on quantitative real-time RT-PCR analyses (Figure 5i). Semi-quantitative RT-PCR analyses showed that the CDKB1-2 and CDKA3 transcript levels were also reduced in the shoot apex of TRV:Rae1 plants (Figure S3). Reduced CDK activities and downregulation of these cell cycle-related genes indicate decreased mitotic activity.

We examined if the reduced cell division affects the initiation of the leaf primordia in the shoot apical meristem (SAM) of the NbRae1 VIGS lines (Figure 5k,l; cf. control in j). The adult vegetative SAM of the TRV control exhibited multiple emerging leaf primordia (Figure 5j). However, the SAM in the TRV:Rae1 plants was associated with significantly reduced numbers of leaf primordia, and furthermore their morphology was abnormal and the size difference between the primordia was less distinct (Figure 5k,l). These results suggest that NbRae1 deficiency resulted in a decreased rate of leaf initiation as well as defective leaf development, probably caused by cell division defects in a peripheral organogenic zone of the SAM.

Increased ploidy levels in the TRV:Rae1 leaves

Using flow cytometry, we measured and quantified the ploidy levels in nuclei from the mature leaves of the TRV and TRV:Rae1 lines (Figure 5m,n). In TRV leaves, almost all nuclei had a ploidy level of 2C, consistent with previous reports in Nicotiana tabacum and N. benthamiana (Kosugi and Ohashi, 2003; Park et al., 2005). However, TRV:Rae1 lines exhibited increased numbers of nuclei with a ploidy level of 4C. Flow cytometry of the TRV:Nup85 and TRV:NUA lines used as controls resulted in a ploidy pattern of mostly 2C, similar to TRV (Figure 5n).

Reduced cambial cell division in the stem of the TRV:Rae1 lines

Based on the light and fluorescence microscopy of transverse stem sections, TRV:Rae1 lines had much narrower vascular bundles than TRV cells with a decrease in xylem cells and the external phloem cells, indicating that the secondary growth of the vasculature is mainly affected in the stem of the NbRae1 VIGS lines (Figure S4a–f, Text S2). Transmission electron microscopy of the thin sections of stem suggested that the secondary-wall formation of the tracheary element (TE), a hallmark of xylem differentiation, was also defective or delayed in the NbRae1 VIGS lines (Figure S4g–n, Text S2). These results indicate that NbRae1 depletion impairs division and differentiation of cambial cells in N. benthamiana stem vasculature.

NbRae1 closely associates with mitotic microtubules

To characterize the cell cycle-related function of NbRae1 in more detail, we assessed the localization of NbRae1 during mitosis (Figure 6a). Transgenic BY-2 cells expressing HA:NbRae1 (Figure 2b) were synchronized with aphidicolin, then fixed and double-labeled with anti-HA and anti-α-tubulin antibodies. Cell lines 4 and 5 were both analyzed, and yielded the same results. After release from aphidicolin-mediated cell cycle arrest, NbRae1 protein was distributed along the full length of the mitotic microtubules throughout mitosis, including pre-prophase bands, spindles, and phragmoplasts, but not with interphase arrays (Figure 6a). In particular, NbRae1 was localized in the cell plate-forming midzone and around the newly formed NE in telophase (Figure 6a). Localization of NbRae1 along the entire length of microtubules and in the cell plate-forming region significantly differed from the preferential localization of mammalian Rae1 at the spindle poles (Blower et al., 2005; Wong et al., 2006).

Figure 6.

 NbRae1 localized to mitotic microtubules of tobacco BY-2 cells throughout mitosis.
(a) Confocal laser scanning micrographs showing subcellular localization of NbRae1 during mitosis. BY-2 cells expressing hemagglutinin (HA):NbRae1 were immunolabeled at various phases of the cell cycle with anti-α-tubulin (green) and anti-HA (red) antibodies, and stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The arrows in interphase and late telophase indicate the nuclear envelope (NE). The arrow in prophase indicates the pre-prophase band. A three dimensional image of cytokinesis is shown (3D; the NE staining is not visible due to weakened fluorescence to see the ring in the cell plate-forming zone). Scale bars = 10 μm.
(b) In vitro binding of recombinant NbRae1 to microtubules. Purified maltose-binding protein (MBP):NbRae1 protein and control MBP were incubated with increasing concentrations of taxol-stabilized microtubules in a pelleting assay. The presence of microtubules and MBP:NbRae1 or MBP in the pellet was analyzed by Western blotting using anti-α-tubulin and anti-MBP antibodies, respectively.

The immunolabeling experiments suggested that NbRae1 binds to microtubules. To examine whether NbRae1 directly binds to microtubules, full-length NbRae1 protein was expressed and purified as a maltose-binding protein (MBP) fusion protein in Escherichia coli. The MBP:NbRae1 or control MBP protein was incubated with taxol-stabilized microtubules, then the microtubules were pelleted by centrifugation, and the presence of MBP:NbRae1 or MBP in the pellet was analyzed by western blotting using anti-MBP antibody (Figure 6b). To detect microtubules in the pellet, the membrane was stripped and reprobed with anti-α-tubulin antibody. MBP:NbRae1 was first detected in the pellet at a concentration of 0.2 μm tubulin, and proportionally increasing amounts of the protein were precipitated in the pellet fraction with increasing amounts of polymerized microtubules, whereas control MBP was only detected in the pellet at a concentration of 3.2 μm tubulin (Figure 6b). These results demonstrate that NbRae1 directly binds to microtubules in vitro.

To compare the NbRae1 localization pattern with that of γ-tubulin during mitosis, the transgenic BY-2 cells were double-labeled with anti-HA and anti-γ-tubulin antibodies (Figure S5). γ-Tubulin is essential for microtubule nucleation and microtubule organization in acentrosomal plant cells as well as in other eukaryotes (Binarováet al., 2006; Pastuglia et al., 2006; Lüders and Stearns, 2007). Consistent with the previous observation (Drykováet al., 2003), punctuate γ-tubulin staining was associated with the mitotic microtubules along their entire length, and localized in the pole regions, in the vicinity of the nucleus, and near the plasma membrane (Figure S5). NbRae1 fluorescence faintly overlapped with that of γ-tubulin along the mitotic microtubules and in the polar regions (Figure S5).

NbRae1 is required for spindle assembly in BY-2 cells

To better understand the function of NbRae1 during mitosis, we transformed BY-2 cells with NbRae1 RNAi constructs containing either an inverted repeat of a 300-bp C-terminal coding region, RNAi(C), or a 336-bp DNA fragment corresponding to a 236-bp C-terminal coding region and a 100-bp 3′-untranslated (UTR) region, RNAi(U), under control of a β-estradiol-inducible transcription system. In actively growing NbRae1 RNAi BY-2 cells, β-estradiol treatments significantly reduced NbRae1 transcript levels based on RNA gel blot analyses (Figure 7a; data not shown).

Figure 7.

 Northern blot analysis of NbRae1 transcript levels and cell cycle progression in NbRae1 RNAi BY-2 cells.
(a) Northern blot analysis was carried out with total RNA from untreated (0 h) and treated RNAi(U) cells with β-estradiol (+ER) or with DMSO only as a control (−ER) for 12, 24 and 48 h (top). The β-estradiol treatment resulted in reduced levels of NbRae1 mRNA in RNAi(U) cells. The rRNA levels served as a control. Quantification of the relative transcript levels compared with the untreated (0 h) sample is shown (bottom).
(b) The RNAi(U) and RNAi(C) cells were treated with β-estradiol (+ER) or with DMSO (−ER). Cells were synchronized by addition of aphidicolin 24 h after the start of the β-estradiol treatment. After incubation for a further 24 h, cells were transferred to fresh medium containing β-estradiol or DMSO, and mitotic indices were plotted at the indicated times after removal of aphidicolin. A total of 300 cells were counted per sample.

The RNAi(U) and RNAi(C) BY-2 cells were treated with β-estradiol for 24 h, and aphidicolin was added to the cells for synchronization. After 24 h the cells were washed and transferred to fresh medium containing β-estradiol (+ER), and mitotic indices were plotted (= 300) at indicated times after the removal of aphidicolin (Figure 7b). As a control, the same experiments were carried out with DMSO treatment only (−ER). In response to β-estradiol, cell cycle progression of synchronized cells was significantly delayed in both RNAi(U) and RNAi(C) cell lines: the maximum mitotic index occurred ∼10 h after the removal of aphidicolin in β-estradiol-treated cells (+ER), compared with ∼8 h in DMSO-treated cells (−ER). This delay appeared to be caused by retarded progression through M phase rather than delayed entry into M phase, because entry into M phase occurred ∼4 h after the removal of aphidicolin with either DMSO or β-estradiol treatment (Figure 7b). Both DMSO- (−ER) and β-estradiol-treated (+ER) wild-type BY-2 cells exhibited normal progression of the cell cycle, reaching the maximum mitotic index at ∼8 h. (Figure S6a).

We examined the cortical microtubule arrangement in RNAi(U) and RNAi(C) BY-2 cells using confocal laser scanning microscopy (Figure 8a). Following treatment with β-estradiol or DMSO for 48 h, microtubules and DNA in interphase cells were visualized by immunolabeling using anti-α-tubulin antibody and DAPI staining, respectively. There was no significant difference in the arrangement of cortical microtubules between DMSO-treated (−ER) and β-estradiol-treated (+ER) RNAi(U) and RNAi(C) BY-2 cells: in both cases the microtubules were organized in parallel arrays, perpendicular to the main axis of the cell (Figure 8a). Thus, silencing of NbRae1 did not significantly affect cortical arrays.

Figure 8.

 NbRae1 is required for spindle assembly and function in BY-2 cells.
(a) Confocal laser scanning micrographs of immunofluorescent staining showing cortical microtubule arrangement in NbRae1 RNAi(U) BY-2 cells. BY-2 cells in interphase were stained with anti-α-tubulin antibodies (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) following 48 h treatment with DMSO (−ER) or β-estradiol (+ER). Merged images are shown. Scale bars = 10 μm.
(b) Confocal laser scanning micrographs of immunofluorescent staining for mitotic microtubule arrangement in NbRae1 RNAi(U) BY-2 cells. Mitotic BY-2 cells were observed after staining as in (a). (A) Bipolar spindles in control metaphase cells (−ER); after induction (+ER), NbRae1-silenced cells show disorganized (B–D) or multipolar spindles with independent spindle arrays (E, F), and abnormal telophase with mislocalized phragmoplast (G, H); (I) localization of γ-tubulin around the multipolar spindles. Scale bars = 10 μm in (A–H); 5 μm in (I).
(c) Quantification of the mitotic spindle defects observed in NbRae1 RNAi(U) BY-2 cells, showing a significant increase in disorganized and multipolar spindles. Data points represent mean ± SD of three independent experiments each analyzing 300 mitotic cells.

We next monitored mitotic and cytokinetic arrays in RNAi(U) and RNAi(C) BY-2 cells (Figure 8b). RNAi inhibition of NbRae1 led to complete randomization of spindle assembly, which was associated with disorganized chromosome congression at the metaphase plate. The spindles were disorganized, unfocussed, and fragmented (Figure 8b, B–D; cf. control in 8b, A). Multipolar spindles with three or four spindle clusters were common, whereas monopolar spindles were not observed (Figure 8b, E,F). Chromosomes failed to align properly in prometaphase/metaphase, forming a central mass or spreading around the spindle clusters, possibly leading to anaphase segregation defects (Figure 8b, B–F; cf. control in 8b, A). Infrequently, telophase cells that exhibited aberrant patterns of phragmoplast formation were observed (Figure 8b, G,H). This unusual morphology was rarely seen in DMSO-treated RNAi cells. In the multipolar spindles, γ-tubulin was co-localized with the abnormal spindles and accumulated around the aberrantly localized spindle poles (Figure 8b, I). Next, we quantified the mitotic spindle defects of NbRae1 RNAi(U) cells after β-estradiol treatment (Figure 8c). Defects in spindle assembly and chromosome alignment, including disorganized or multipolar spindles, were observed in ∼18% of the mitotic cells (n = 300) after β-estradiol treatment, compared with ∼5% after DMSO treatment (Figure 8c). In wild-type BY-2 cells, both DMSO- (–ER) and β-estradiol-treated (+ER) samples exhibited the mitotic spindle defects in <5% of the mitotic cells, similar to the DMSO-treated RNAi samples (Figure S6b). Thus, NbRae1 plays a critical role in spindle organization and chromosome segregation during mitosis in plant cells.

Discussion

In plants, relatively few studies have been carried out on the mechanism of nucleocytoplasmic transport through the NPC (Gong et al., 2004; Dong et al., 2006; Parry et al., 2006; Jacob et al., 2007). However, Arabidopsis databases contain many proteins that are similar to the protein components involved in nucleocytoplasmic transport in mammals and yeast, suggesting a conserved transport mechanism among diverse eukaryotes. For example, a DEAD box RNA helicase is enriched in the nuclear rim, and the mutation of the gene cryophyte/los4 blocks poly(A) RNA export in Arabidopsis (Gong et al., 2004). MOS3/SAR3 and SAR1, which exhibit homology to the human nucleoporins Nup96 and Nup160, respectively, and TRANSLOCATED PROMOTER REGION (AtTPR), a nuclear pore protein, were shown to localize to the NE and play a role in nucleocytoplasmic transport of RNA in Arabidopsis (Dong et al., 2006; Parry et al., 2006; Jacob et al., 2007; ). Interestingly, in addition to the defects in mRNA export, inactivation of these components resulted in a variety of developmental phenotypes. The sar1 (suppressor of auxin resistance1)/atnup160 mutants exhibited early flowering, retarded seedling growth, and reduced tolerance to cold stress (Dong et al., 2006). The mos3 (modifier of snc1)/sar3 (suppressor of auxin resistance3) mutation caused pleiotropic growth defects as well as altered disease resistance response and auxin signaling (Zhang and Li, 2005; Parry et al., 2006). The cryophyte/los4 mutants exhibited a reduced stature and early flowering (Gong et al., 2004). It is not clear how the NPC and the nucleocytoplasmic transport machinery affect these various physiological processes. These pleiotropic phenotypes of the mutants may be an indirect consequence of the defect in RNA export, since certain regulators may require normal RNA export more critically than the others. Alternatively, the mutations may reduce or alter the activity in other functions of the NPC and the transport machinery.

Recent studies in mammals and yeast documented that Nups perform functions in addition to their role as structural components of NPC, particularly during mitosis (Rabut et al., 2002; Griffis et al., 2004). Nup358 relocates to kinetochores after nuclear envelope breakdown, and in its absence the assembly of other kinetochore components is inhibited, leading to severe perturbation of chromosome alignment and segregation (Salina et al., 2003). Multiple members of the Nup106–160 complex are associated with kinetochores, spindle poles, and spindle fibers during prometaphase, and depletion of the complex severely disturbs spindle assembly in Xenopus egg extracts (Orjalo et al., 2006). The Rae1–Nup98 complex also illustrates the various roles of Nups in mitotic mechanisms. Rae1 localizes to sperm spindles and Ran asters in Xenopus egg extracts, and binds directly to microtubules with a low affinity in vitro; depletion of Rae1 disrupts assembly of sperm spindles and Ran asters (Blower et al., 2005). The Rae1–Nup98 complex was independently identified as an inhibiter of the anaphase-promoting complex (APC) in a mouse model system (Jeganathan et al., 2005); however, these authors did not observe any defects in spindle assembly in mice lacking both Rae1 and Nup98. Thus, the precise role of Rae1 in mitosis requires clarification.

In this study, we observed that NbRae1 mainly associated along the entire length of mitotic microtubules throughout mitosis and localized in the cell plate-forming midzone, and only a minor portion was present in the spindle pole area. This is different from the localization of animal Rae1, which is enriched at spindle poles with a minor fraction present along the length of microtubules and on chromosomes (Blower et al., 2005; Wong et al., 2006). Nonetheless, RNAi inhibition of NbRae1 in BY-2 cells resulted in formation of disorganized or multipolar spindles and defects in chromosome segregation similar to the phenotype of Rae1-silenced animal cells, suggesting a conserved function of the Rae1 proteins in spindle organization that is distinct from their roles at the interphase NE. In particular, the reduced pole focusing and weakened bipolarity of the spindles observed in NbRae1 RNAi lines indicate that NbRae1 contributes to spindle pole formation. In animal systems, Rae1 transiently interacts with the Nuclear Mitotic Apparatus Protein (NuMA) during mitosis, which cross-links the microtubule bundles (Wong et al., 2006). Rae1 might increase the microtubule cross-linking valency of NuMA and stabilize microtubules at the spindle pole, or alternatively the Rae1–NuMA complex might be involved in centrosome duplication or stabilization (Wong et al., 2006). Plant cells lack mitotic centrosomes, and there is no Arabidopsis gene with high sequence homology to NuMA. The dynamic relocation of NbRae1 during interphase and mitosis and the mechanism of its spindle-organizing function remain to be determined.

Interestingly, it has been proposed that the Arabidopsis homolog of NbRae1 (At1g80670) may function as a substrate receptor for Damaged DNA-Binding 1–Cullin 4–Regulator of Cullins 1 (DDB1–CUL4–ROC1)-based E3 ubiquitin ligases, based on the presence of a DWD motif (for DDB1-binding WD40) (Lee et al., 2008). Yeast two-hybrid and in vivo co-immunoprecipitation suggested the interaction of the Rae1 homolog with DDB1 (Lee et al., 2008). The DWD motif is also conserved in the NbRae1 sequences. It would be important to determine whether NbRae1 indeed acts as part of a CUL4-based E3 ligase, and, if so, to identify the substrates of the NbRae1-mediated E3 ligase in mitosis and/or in interphase. Possessing multiple WD40 repeats with a β-propeller structure, NbRae1 may also function as a scaffolding platform that recruits important protein factors to the mitotic microtubule network. So far, there has been no report suggesting that Rae1 or nucleoporins are components of E3 ubiquitin ligase in other eukaryotes. In yeast and mammals, Nup358/RanBP2 is unique among nucleoporins due to its small ubiquitin-related modifier (SUMO) E3 ligase activities (Dawlaty et al., 2008). Nup358 functions in both nucleocytoplasmic transport and mitosis. In interphase, Nup358 sumoylates heterogenous nuclear ribonucleoprotein (hnRNP) C and M proteins to facilitate nucleocytoplasmic transport of mRNAs (Vassileva and Matunis, 2004). In mitosis, Nup358 sumoylates topoisomerase IIα to regulate its localization to inner centromeres, and Nup358 deficiency induces severe aneuploidity and tumorigenesis in mice (Dawlaty et al., 2008). Deciphering the molecular networks of NbRae1 activity and interaction is essential for understanding the mechanisms of its multiple functions.

Rae1 is essential for early embryogenesis in mammals (Babu et al., 2003). Rae1-null mouse embryos degenerate at 6.5–8.5 embryonic days, while the loss of a single Rae1 allele causes a mitotic checkpoint defect and chromosome missegregation (Babu et al., 2003). When we examined the silencing phenotypes of NbRae1 at the whole plant level, we observed growth arrest of the plants, reduced cell division activities in the shoot apex and the vascular cambium, and increased ploidy levels in mature leaves. The reduced mitotic activity is probably caused by retarded cell division in the meristem tissues, supported by the finding that NbRae1-silencing resulted in delayed progression of the mitotic phase in BY-2 cells. The latter observation indicates that the abnormal RNAi cells were able to progress through the cell cycle regardless of the spindle anomaly and chromosome missegregation, instead of being arrested at metaphase. It is also consistent with the elevated ploidy observed in differentiated leaves of NbRae1 VIGS plants, which was probably caused by aberrant mitotic exit without proper chromosome segregation, followed by differentiation. Similar findings have been observed in other spindle mutants: Arabidopsis γ-tubulin double mutants demonstrate significantly reduced mitotic activity in the shoot and root apices, associated with increased duration of the cell cycle in the mutant root cells (Pastuglia et al., 2006). Furthermore, inhibition of nuclear division and abnormally large nuclei indicative of polyploidy were observed in these mutants (Pastuglia et al., 2006). Increased ploidy has also been reported in γ-tubulin mutants of Aspergillus nidulans (Prigozhina et al., 2004), S. pombe (Paluh et al., 2000), and Drosophila (Sunkel et al., 1995), with re-entry of cells into interphase without division suggesting defective mitotic checkpoint regulation (Prigozhina et al., 2004). Similarly, an Arabidopsis temperature-sensitive rsw7 mutant with defective kinesin-5 motor protein (AtKRP125c) demonstrated disrupted mitotic spindles; however, the cells were not arrested at metaphase but enlarged or infrequently multinucleate cells were present in interphase, indicating that the disrupted spindles do not cause metaphase arrest as they do in animal cells (Bannigan et al., 2007).

Experimental procedures

Synchronization of BY-2 cell culture

For synchronization at the G1/S boundary of the cell cycle, 5 ml of 7-day-old RNAi BY-2 cell culture was transferred to 45 ml modified Linsmaier and Skoog medium containing DMSO or 10 μmβ-estradiol (Sigma, http://www.sigmaaldrich.com/), and incubated for 24 h before addition of 5 mg L−1 aphidicolin (Sigma). After 24 h the cells were washed, resuspended in 45 ml fresh medium containing DMSO or β-estradiol, and further incubated as indicated.

Determination of the mitotic index

BY-2 cells were stained with DAPI and observed under a fluorescence microscope to calculate the number of cells in the mitotic phase. A total of 300 cells were counted in each of three independent experiments.

Immunofluorescence

Preparation of BY-2 cells and immunofluorescence were carried out as described (Sasabe et al., 2006). For double-labeling of HA and α-tubulin, fixed and permeabilized BY-2 cells expressing HA:NbRae1 fusion proteins were immunolabeled with 1:500 dilution of anti-HA (IgG3) antibodies (Applied Biological Materials Inc., http://www.abmgood.com/) and 1:1000 dilution of anti-α-tubulin (IgG2a) antibodies (DM1A, Sigma). Then the cells were incubated with a 1:1000 dilution of IgG3-specific Alexa Fluor 593-conjugated anti-mouse IgG antibodies (Molecular Probes, http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes.html) and a 1:1000 dilution of IgG2a-specific Alexa Fluor 488-conjugated anti-mouse IgG antibodies (Molecular Probes). For double-labeling of HA and γ-tubulin, cells were immunolabeled with a 1:500 dilution of anti-HA antibodies (Applied Biological Materials Inc.) and a 1:500 dilution of anti-γ-tubulin antibodies (Erhardt et al., 2002). Then the cells were incubated with a 1:1000 dilution of Alexa Fluor 488-conjugated anti-mouse IgG antibodies (Molecular Probes) and a 1:1000 dilution of Alexa Fluor 593-conjugated anti-rabbit IgG antibodies (Molecular Probes) before staining with 0.2 μg ml−1 DAPI. The cells were observed under a confocal laser scanning microscope (Carl Zeiss LSM 510, http://www.zeiss.com/) with optical filters LP 560 (excitation 543 nm, emission 560–615 nm), BP505-530 (excitation 488 nm, emission 525 nm), and BP420-480 (excitation 405 nm, emission 461 nm) for Alexa Fluor 593, Alexa Fluor 488, and DAPI, respectively.

Microtubule binding assay

Increasing concentrations (0–3.2 μm) of purified bovine brain tubulin subunits (Cytoskeleton, http://www.cytoskeleton.com/) were incubated in tubulin resuspension buffer [1× BRB80 buffer (80 mm K-piperazine-N,N′-bis(ethanesulfonic acid) (PIPES), pH 6.8; 1 mm MgCl2; 1 mm EGTA), 20% glycerol, 1 mm guanosine-5′-triphosphate (GTP)] at 30°C for 30 min in the presence of 20 μm taxol to induce in vitro microtubule polymerization. Then MBP:NbRae1 and MBP were added to the reaction mix at a final concentration of 10 nm before further incubation for 30 min at 30°C. Taxol-stabilized microtubules were pelleted by centrifugation at 25 000g for 20 min at 30°C and washed three times in 1× BRB80 buffer. Proteins in the pellet were analyzed by SDS–PAGE and Western blotting using anti-MBP and anti-α-tubulin antibodies.

Other procedures

Detailed descriptions of the following procedures are included in the Text S2: virus-induced gene silencing, plant treatment, subcellular localization of NbRae1 in leaf protoplasts, semi-quantitative RT-PCR, histochemical analyses, whole-mount in situ hybridization of mRNA, localization of lignin, analysis of starch content, quantitative real-time RT-PCR, statistical analyses, flow cytometry, measurement of CDK activity, DNA constructs and transformation of BY-2 cells, protein extraction and immunoblotting, northern blot analysis, and purification of the recombinant NbRae1 protein.

Acknowledgements

This research was supported by grants to HSP from the Plant Diversity Research Center of the 21st Century Frontier Research Program, a KOSEF grant (M10749000002-07N4900-00210), and the Plant Signaling Network Research Center (at Korea University) of the Science Research Center Program, all of which are funded by the Ministry of Science and Technology of Korea.

EMBL accession numbers: ABG29731.

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