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Keywords:

  • nuclear envelope;
  • SUN domain;
  • INM targeting;
  • LINC complex

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sad1/UNC-84 (SUN)-domain proteins are inner nuclear membrane (INM) proteins that are part of bridging complexes linking cytoskeletal elements with the nucleoskeleton, and have been shown to be conserved in non-plant systems. In this paper, we report the presence of members of this family in the plant kingdom, and investigate the two Arabidopsis SUN-domain proteins, AtSUN1 and AtSUN2. Our results indicate they contain the highly conserved C-terminal SUN domain, and share similar structural features with animal and fungal SUN-domain proteins including a functional coiled-coil domain and nuclear localization signal. Both are expressed in various tissues with AtSUN2 expression levels relatively low but upregulated in proliferating tissues. Further, we found AtSUN1 and AtSUN2 expressed as fluorescent protein fusions, to localize to and show low mobility in the nuclear envelope (NE), particularly in the INM. Deletion of various functional domains including the N terminus and coiled-coil domain affect the localization and increase the mobility of AtSUN1 and AtSUN2. Finally, we present evidence that AtSUN1 and AtSUN2 are present as homomers and heteromers in vivo, and that the coiled-coil domains are required for this. The study provides evidence suggesting the existence of cytoskeletal–nucleoskeletal bridging complexes at the plant NE.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The nuclear envelope (NE) is a double-membrane structure in eukaryotic cells, consisting of an outer nuclear membrane (ONM), which is closely associated with perinuclear endoplasmic reticulum (ER), and an inner nuclear membrane (INM) that is connected to the ONM via the nuclear pores. The NE has a variety of functions in addition to separating nuclear material from the rest of the cell contents. These include regulating transport into and out of the nucleus, physical positioning of the nucleus and facilitating processes in cell division and nucleo-cytoplasmic signalling. The protein constituents of the membrane reflect these functions.

The higher plant NE resembles that of other higher eukaryotes in a variety of respects. It is structurally similar, with inner and outer membranes connected by a pore membrane and nuclear pore complexes (NPCs). In addition, plant cells (unlike some fungi, including Saccharomyces cerevisiae) undergo open cell division (Evans et al., 2009). There are, however, significant differences: plants lack homologues of the nuclear lamins (Brandizzi et al., 2004; Meier, 2007; Evans et al., 2009), and, in place of microtubule organizing centres (MTOCs) and spindle pole bodies (SPBs), the whole surface of the NE is able to nucleate microtubules (MT) (Stoppin et al., 1996). Studies also reveal significant differences in the nuclear membrane proteome; for instance, most well-characterized animal INM proteins such as the lamin B receptor (LBR) and Lap/Emerin/Man1 (LEM) domain proteins do not have homologues in plants (Mans et al., 2004; Meier, 2007). Studies using GFP fused to the N terminus of human LBR expressed in plant tissue show that the construct targets the INM, as in animal cells, but that there is an absence of strong binding interactions for the retention of the construct in plant cells (Irons et al., 2003; Graumann et al., 2007; Evans et al., 2009).

So far, only a few functional membrane-integral protein constituents of the plant NE have been characterized. These include a SERCA-type Ca2+ pump, LCA, shown to localize to the NE in tomato cells by immunofluorescent and immunogold labelling (Downie et al., 1998), as well as the Ca2+ channel doesn’t make infection 1 (DMI1), detected at the NE of Medicago truncatula, and shown to be essential for perinuclear calcium spikes during symbiotic interactions between the plant root and nitrogen-fixing bacteria (Riely et al., 2006; Peiter et al., 2007). Both appear significant in nucleoplasmic signalling, specifically in regulating Ca2+ levels; however, it is not known whether they reside specifically in the INM or ONM (Downie et al., 1998; Riely et al., 2006; Peiter et al., 2007).

Two further groups of plant NE proteins have been characterized that appear to be involved in processes related to the NPC and traffic through the pores. The tryptophan-proline-proline (WPP) domain interacting proteins WIP1, WIP2a and WIP3, as well as the WPP domain interacting tail anchored proteins WIT1 and WIT2 are thought to be present in the ONM and pore membrane, and are necessary for the anchorage of RanGAP to the NE in undifferentiated Arabidopsis root tip cells (Xu et al., 2007; Zhao et al., 2008). The WPP-mediated targeting of proteins to the NE has been shown to be specific to plants and is also used by soluble, NE-associated proteins such as Arabidopsis WPP1 and WPP2 and their tomato homologue MFP associating factor 1 (MAF1) (Gindullis and Meier, 1999;Patel et al., 2004; Meier, 2007).

Proteins yet to be characterized in plants with key importance in animal and fungal cells are the membrane intrinsic proteins involved in physical interactions between the nucleoskeleton and the cytoskeleton, which in animal and fungal cells form bridging structures known as the linker of nucleoskeleton and cytoskeleton (LINC) complex (Crisp et al., 2006). These complexes are essential for various cellular and nuclear processes, such as duplication and anchorage of centrosomes and SPB to the NE (Kemp et al., 2007; Starr, 2009), chromosome decondensation (Chi et al., 2007), telomere anchorage and the formation of the meiotic chromosome bouquet (Chikashige et al., 2006; Tomita and Cooper, 2006; Schmitt et al., 2007).

The LINC complex comprises members of two families of membrane intrinsic proteins conserved in animals and fungi. The Sad1/UNC-84 (SUN)-domain proteins (Gruenbaum et al., 2005; Tzur et al., 2006) are located in the INM and associate with the Klarsicht/ANC-1/SYNE1 homology (KASH)-domain proteins located in the ONM. The two membranes are therefore physically bridged with the SUN and KASH domains interacting in the periplasm (Starr and Fischer, 2005;Wilhelmsen et al., 2006). SUN-domain proteins also interact directly with proteins of the nucleoskeleton and with chromatin, whereas the KASH-domain proteins interact with proteins of the cytoskeleton (Crisp et al., 2006; Tzur et al., 2006).

The work described in this paper follows the discovery of homologues of SUN-domain proteins in plants. Expression, localization and binding interactions, as well as characterisation of key domains of the two Arabidopsis SUN-domain proteins are presented, together with discussion of their likely role in the plant NE.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Arabidopsis SUN-domain proteins and homologues

To identify putative SUN-domain homologues in plants, the Caenorhabditis elegans SUN-domain protein UNC-84 sequence was used as the query in a protein BLAST search, in which At3g10730 (30% identical, 46% similar, e-value 2e−12) and At5g04990 (28% identical, 47% similar, e-value 2e−10) were identified as Arabidopsis homologues of the protein. At3g10730 is 455 amino acids long, with a predicted molecular weight of 50 kDa, and At5g04990 is 471 amino acids long, with a predicted molecular weight of 51.5 kDa. Both contain the highly conserved SUN domain in the C terminus (amino acids 310–446 of At3g10730 and amino acids 313–451 of At5g04990) and a putative coiled-coil domain (amino acids 205–225 of At3g10730 and amino acids 191–225 of At5g04990), as well as an N-terminal transmembrane domain (amino acids 107–128 of At3g10730 and amino acids 109–129 of At5g04990) and bipartite nuclear localization signal (NLS) (amino acids 89–106 of At3g10730 and amino acids 89–106 of At5g04990; Figure 1a). In addition, a putative EF-hand Ca2+ binding domain is predicted to be present in At5g04990 (amino acids 253–268) (Figure 1a). The two proteins appear to be closely related as their amino acid sequences are 62% identical and 72% similar (Figure 1b).

image

Figure 1.  (a) Plant and non-plant members of the Sad1/UNC-84 (SUN) domain protein family: yellow, SUN domain; green, putative EF-hand Ca2+ binding domain; purple, coiled-coil domain; blue, transmembrane domain; red, bipartite nuclear localization signal (NLS); pink, lamin binding domain; location and size of domains as determined by Pfam, SMART and MotifScan; all accession numbers given are from UniProt apart from *NCBI. The plant SUN-domain proteins are similar in size, but are generally smaller than animal SUN-domain proteins. All members feature a conserved SUN domain at the C terminus (Pfam, InterPro), and contain putative membrane domains. Most also feature coiled-coil domains. (b) ClustalW aligned protein sequences of At3g10730 (AtSUN2) and At5g04990 (AtSUN1): blue highlights identical amino acids, grey highlights conserved amino acids; AtSUN1 and AtSUN2 share 62% identical and 72% similar sequences.

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Further investigations using Interpro, Pfam and BLAST searches with At3g10730 and At5g04990 as queries revealed the presence of putative SUN-domain proteins in Oryza sativa, Vitus vinifera, Physcomitrella patens and Zea mays. Two SUN-domain proteins appear to be present in each plant species, apart from P. patens, and all identified plant SUN-domain proteins are of similar size, have a transmembrane domain and the conserved C-terminal SUN domain (Figure 1a). A coiled-coil domain, bipartite NLS and the EF-hand Ca2+ binding domain are also present in some of the other plant SUN-domain proteins (Figure 1a). The length and location of the domains within the protein sequences are listed in Table S1.

A comparison with well-characterized animal and yeast SUN-domain proteins revealed that the domain layout appears to be generally conserved across the kingdoms. An N-terminal transmembrane domain is followed by coiled-coil domains and the C-terminal SUN domain (Figure 1a). Size appears to be a major difference, with the animal SUN-domain proteins generally being significantly larger than the plant SUN-domain proteins. Additional homology comparisons of At3g10730 and At5g04990 with animal and yeast SUN-domain proteins revealed that At3g10730 shares greater homology with animal SUN2 proteins (Mus musculus SUN2 e-value 6e−21, Homo sapiens SUN2 e-value 7e−20, Bos taurus SUN2 e-value 6e−20), and that At5g04990 shares greater homology with animal SUN1 proteins (H. sapiens SUN1 e-value 5e−21, M. musculus SUN1 e-value 1e−18, B. taurus SUN1 e-value 2e−15, Rattus norvegicus SUN1 e-value 3e−19). Therefore, At3g10730 was renamed AtSUN2 and At5g04990 was renamed AtSUN1.

Endogenous AtSUN1 and AtSUN2

The mRNAs of AtSUN1 and AtSUN2 were detected in adult Arabidopsis leaf and inflorescence tissue, as well as in Arabidopsis cell suspension by RT-PCR, verifying that both genes are expressed in various tissues and cell types, and are not pseudogenes (Figure 2a). In addition, relative expression values based on microarray data were obtained for AtSUN2 from Genevestigator (https://www.genevestigator.ethz.ch/; Zimmermann et al., 2004). The gene was found to be expressed in 60 different tissue types and developmental stages of Arabidopsis at relatively similar levels, although its expression appears to be upregulated in inflorescence and root tip tissue (Figure 2b,c; Table S2). In comparison with reference genes ubiquitin conjugating enzyme UBC9, yellow leaf specific protein 8 (YLS8) and protein phosphatase 2A subunit (PP2A), which are expressed at relatively high, medium and low levels, respectively, AtSUN2 is expressed at very low levels (Figure 2b,c; Table S2). Upregulation of AtSUN2 expression in roots of 7-day-old seedlings was reported by Spencer et al. (2007) during transcriptional profiling of Arabidopsis embryos and seedlings. No significant difference in AtSUN2 expression levels was found during the globular, heart and torpedo stages of embryonic development.

image

Figure 2.  Endogenous AtSUN1 and AtSUN2. (a) RT-PCR analysis of AtSUN1, AtSUN2 and reference gene PP2A: all three genes are expressed in leaf (L) and inflorescence (I) tissue, as well as in cell culture (CC); AtSUN1 mRNA is 1416 bp, AtSUN2 mRNA is 1368 bp and PP2A mRNA is 1764 bp. (b, c) Relative expression levels: relative expression values for AtSUN2 and reference genes PP2A, YLS8 and UBC9, obtained from Genevestigator; AtSUN1 expression data were not available. AtSUN2 is expressed at very low levels, and mostly evenly through development and in different tissues, but appears upregulated in root tips and inflorescence tissue. (d) Detection of endogenous AtSUN1 and AtSUN2. Anti-AtSUN1 antiserum (AS1) and anti-AtSUN2 antiserum (AS2) was used to detect approximately 43-kDa bands in Arabidopsis leaf extract representative of AtSUN1 and AtSUN2. The bands were not detected with pre-immune serum (PI).

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Polyclonal anti-peptide antisera were raised to amino acids 92–105 of AtSUN1 and AtSUN2, as these domains were found to be unique to each of the two proteins. Bands of approximately 43 kDa were detected with both anti-AtSUN1 and anti-AtSUN2 antisera, but not with the pre-immune sera (Figure 2d) indicating the presence of endogenous AtSUN1 and AtSUN2 in Arabidopsis leaf tissue.

Nuclear envelope localization of fluorescent protein fusions

Animal and yeast SUN-domain proteins have been shown to reside in the NE, in particular the INM. The presence of a predicted transmembrane domain and NLS in both AtSUN1 and AtSUN2 suggest that both proteins might localize similarly in plants. To investigate the subcellular localization of the two proteins, fluorescent protein fusions of AtSUN1 and AtSUN2 were expressed transiently in tobacco leaf tissue and stably in tobacco BY-2 cells. Expression was observed by confocal and electron microscopy.

The plant NE marker LBR-GFP was used as a positive control (Figure 3a). Similar to LBR-GFP, both YFP-AtSUN1 and CFP-AtSUN2 were present at the nuclear periphery around chromatin, indicative of NE localization (Figure 3b,c), where they co-localized (Figure 3d). Additional weak fluorescence was also observed in either cytoplasmic puncta or the ER of some cells (Figure S1). In addition to the transient expression, tobacco BY-2 cells were transformed with AtSUN1-YFP and AtSUN2-YFP. Stable expression of the two fusion proteins also resulted in their localization to the NE of BY-2 cells, and in weak fluorescent labelling of ER and cytoplasmic puncta (Figure 3e,f).

image

Figure 3.  Subcellular localization of AtSUN1 and AtSUN2. (a–d) Confocal micrographs of transiently transformed tobacco leaf lower epidermal cells: green, fluorescent protein fusion; magenta, ethidium bromide-labelled DNA. Scale bars: 10 μm. (a) Plant nuclear envelope (NE) marker LBR-GFP at the NE. (b) YFP-AtSUN1 at the NE. (c) CFP-AtSUN2 at the NE. D) mRFP-AtSUN1 (red) and YFP-AtSUN2 (green) co-localize (yellow) at the NE. (e, f) Confocal micrographs of stable expression in tobacco BY-2 cells: green, fluorescent protein fusion; magenta, DRAQ5-labelled DNA. Scale bars: 10 μm. (e) AtSUN1-YFP at the NE. (f) AtSUN2-YFP at the NE. Scale bars: 10 μm. (g–j) Electron micrographs of transiently transformed tobacco leaf lower epidermal cells: fluorescent protein fusions are marked by 10-nm gold particles; white arrows mark inner nuclear membrane (INM); black arrows mark outer nuclear membrane (ONM); c, cytoplasm; ER, endoplasmic reticulum; n, nucleoplasm. Scale bar: (g), 100 nm; all others, 200 nm. (g) Non-infiltrated control with no gold labelling at the NE. (h) mRFP-AtSUN1 located at the INM, NE and ER. (i) YFP-AtSUN2 located at the INM, ONM and periplasm. (j) YFP-AtSUN2 located in the ER.

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Ultrastructural observations of immunogold-labelled tobacco leaf tissue transiently expressing mRFP-AtSUN1 and YFP-AtSUN2 were carried out to confirm the NE localization. Gold labelling was primarily observed at the INM, ONM and periplasm (Figure 3h,i), with a few gold particles also decorating the ER (Figure 3h,j). The ultrastructural observations verify the localization of AtSUN1 and AtSUN2, expressed as fluorescent fusion proteins, at the plant NE, in particular at the INM.

Effect of domain deletions on subcellular localization

To investigate whether AtSUN1 and AtSUN2 are localized specifically to the INM, and to determine what domains are required for their NE localization, truncation and deletion mutants of the two proteins were created, fused to fluorescent proteins and transiently expressed in tobacco leaf tissue (Figure 4a). The subcellular localization of the mutants was observed by both confocal and electron microscopy.

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Figure 4.  Subcellular localization of AtSUN1 and AtSUN2 deletion mutants. (a) Schematic overview of deletion mutants: all AtSUN1 mutants were fused to YFP, and all AtSUN2 mutants were fused to CFP; domain colours as described in the legend to Figure 1; YFP-AtSUN1ΔN and CFP-AtSUN2ΔN lack the N terminus, CFP-AtSUN2ΔNLS lacks the putative bipartite nuclear localization signal (NLS), YFP-AtSUN1ΔCC and CFP-AtSUN2ΔCC lack the colied-coil domain, YFP-AtSUN1ΔSUN and CFP-AtSUN2ΔSUN lack the SUN domain. (b–l) Confocal micrographs of transiently transformed tobacco leaf lower epidermal cells: green, fluorescent protein fusion; magenta, ethidium bromide-labelled DNA. Scale bars: 10 μm. (b) YFP-AtSUN1ΔN in the nuclear envelope (NE). (c) YFP-AtSUN1ΔN in the endoplasmic reticulum (ER). (d) YFP-AtSUN1ΔCC in NE (weak) and aggregates. (e) YFP-AtSUN1ΔCC in ER (weak) and aggregates. (f) YFP-AtSUN1ΔSUN in NE (weak) and aggregates. (g) CFP-AtSUN2ΔN in NE. (h) CFP-AtSUN2ΔN in ER. (i) CFP-AtSUN2ΔNLS in NE and ER. (j) CFP-AtSUN2ΔCC in NE and aggregates. (k) CFP-AtSUN2ΔCC in aggregates. (l) CFP-AtSUN2ΔSUN in aggregates and absent from NE. (m–q) Electron micrographs of transiently transformed tobacco leaf lower epidermal cells, with fluorescent protein fusions marked by 10-nm gold particles: white arrows mark the inner nuclear membrane (INM), black arrows mark the outer nuclear membrane (ONM); c, cytoplasm; Cm, cytoplasmic membranes; ER, endoplasmic reticulum; n, nucleoplasm. Scale bars: 100 nm. (m) CFP-AtSUN2ΔNLS at INM and ONM. (n) CFP-AtSUN2ΔNLS in ER. (o) CFP-AtSUN2ΔSUN absent from the NE. (p, q) CFP-AtSUN2ΔSUN in cytoplasmic membranes.

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Deletion of the 106 N-terminal amino acids of AtSUN1 and AtSUN2 did not affect NE localization, but resulted in a significant increase of peripheral ER labelling (Figure 4b,c,g,h; Figure S1). This was also observed in cells expressing CFP-AtSUN2ΔNLS, in which only the NLS domain was deleted (Figure 4i). Ultrastructural observations revealed the mutant to be present mainly in the ONM and ER, although INM-associated gold particles were also observed (Figure 4m,n). The affect of the coiled-coil domain on NE targeting was observed in cells expressing either YFP-AtSUN1ΔCC or CFP-AtSUN2ΔCC, in both of which the coiled-coil domain had been deleted (Figure 4a). Although both mutants were present at the NE, NE fluorescence was weak, and the two mutants were mainly localized to bright aggregates (Figure 4d,j). By lowering gain levels and laser transmission, the aggregates were found to be ring-like structures of various sizes likely to be associated with the ER (Figure 4d,e,j,k). These aggregates were also observed in cells expressing YFP-AtSUN1ΔSUN and CFP-AtSUN2ΔSUN. In these mutants, the C-terminal 159 amino acids and 146 amino acids of AtSUN1 and AtSUN2, respectively, including the SUN domain, were deleted (Figure 4a). Although a small quantity of YFP-AtSUN1ΔSUN was still present at the NE, as indicated by the low NE fluorescence in Figure 4f, CFP-AtSUN2ΔSUN was absent from the NE and completely localized in aggregates (Figure 4l). Ultrastructural observations of CFP-AtSUN2ΔSUN-expressing cells support the confocal observations, as gold particles were not detected at the INM or ONM, but were detected instead at cytoplasmic membranous structures, likely to be the cytoplasmic aggregates (Figure 4o–q).

Mobile behaviour of SUN proteins and mutants

Animal and yeast SUN-domain proteins associate with nuclear components such as lamins and chromatin, as well as other NE components such as KASH domain and other SUN-domain proteins. Binding interactions affect the mobility of the protein in the membrane with strong binding interactions immobilizing the protein or slowing down its rate of movement. To gain an understanding of AtSUN1 and AtSUN2 binding at the NE, their mobility was investigated using fluorescence recovery after photobleaching (FRAP). In addition, the mobility of mutants lacking the N terminus and coiled-coil domain were also investigated, as these two domains are essential in animal and yeast SUN-domain proteins for associations with nuclear components and other SUN-domain proteins, respectively.

Approximately half of YFP-AtSUN1 and CFP-AtSUN2 molecules are immobile at the NE, with the mobile fraction moving at 0.03 ± 0.01 and 0.04 ± 0.02 μm2 s−1, respectively, making these two proteins the least mobile in the NE (Table 1). Interestingly, when the YFP is fused to the C terminus of AtSUN1 and AtSUN2, their mobility significantly increases (< 0.05), with a higher mobile fraction and diffusion rate (Table 1). Deletion of the N terminus and coiled-coil domain also significantly increase the mobility of AtSUN1 and AtSUN2 (< 0.05). YFP-AtSUN1ΔN is more mobile than YFP-AtSUN1ΔCC (< 0.05; Table 1). Overall, CFP-AtSUN2ΔN has the highest mobile fraction with 79.61 ± 9.87% mobile, and CFP-AtSUN2ΔCC moves the fastest, at a rate of 0.24 ± 0.11 μm2 s−1 (Table 1).

Table 1.   Quantification of AtSUN1 and AtSUN2 mobility measured by fluorescence recovery after photobleaching (FRAP). (A) Mobility of AtSUN1: YFP-AtSUN1 is the least mobile of the four AtSUN1-based fusion proteins. (B) Mobility of AtSUN2: YFP-AtSUN2 is the least mobile of the four AtSUN2-based fusion proteins. Deletion of the N terminus and coiled-coil domain increase the mobility of both AtSUN1 and AtSUN2
 Mobile fraction (%)Diffusion rate (μm2 s−1)
A
 YFP-AtSUN1(710 aa)49.04 ± 6.620.03 ± 0.01
 AtSUN1-YFP(710 aa)65.04 ± 6.030.07 ± 0.02
 YFP-AtSUN1ΔN(602 aa)68.31 ± 5.860.21 ± 0.05
 YFP-AtSUN1ΔCC(604 aa)59.85 ± 9.120.13 ± 0.04
B
 CFP-AtSUN2(694 aa)54.55 ± 8.550.04 ± 0.02
 AtSUN2-YFP(694 aa)64.02 ± 7.930.08 ± 0.02
 CFP-AtSUN2ΔN(588 aa)79.61 ± 9.870.16 ± 0.05
 CFP-AtSUN2ΔCC(630 aa)70.49 ± 9.710.24 ± 0.11

Interactions between AtSUN1 and AtSUN2

The observation that deletion of the coiled-coil domain increases the mobility of YFP-AtSUN1 and CFP-AtSUN2 (Table 1) suggests that this domain is involved in binding interactions that affect the mobile behaviour of the two proteins. In animal and yeast SUN-domain proteins, the coiled-coil domain facilitates oligomerization for the formation of dimers, trimers and tetramers. Co-expression of CFP-AtSUN2ΔSUN and mRFP-AtSUN2 abolished the NE localization of the non-mutated protein and caused it to co-localize with the mutant in aggregates (data not shown), raising the possibility that the two Arabidopsis SUN-domain proteins may also interact with each other to form oligomeric complexes. To investigate this and the putative involvement of the coiled-coil domain, acceptor photobleaching fluorescence resonance energy transfer (apFRET) of fluorescent protein fusions of AtSUN1 and AtSUN2 at the NE was employed. The FRET efficiency EF was calculated according to the method described by Karpova et al. (2003), and denotes the increase of CFP fluorescence after bleaching YFP. As a control, the change of CFP fluorescence was observed when YFP was not bleached. The fluorescence of CFP-AtSUN1 increased significantly (< 0.05) when it was co-expressed with either YFP-AtSUN2 (EF = 7.84 ± 3.47) or YFP-AtSUN1 (EF = 11.35 ± 3.39), and the YFP moiety was bleached (Table 2). Similarly, the CFP-AtSUN2 fluorescence increased significantly (EF = 19.78 ± 5.39; < 0.05) after the co-expressed YFP-AtSUN2 was bleached (Table 2). This demonstrates the ability of AtSUN1 and AtSUN2 to form homomers and heteromers in vivo. However, the CFP fluorescence did not significantly alter in comparison with the control CFP levels when CFP-AtSUN1 was co-expressed with YFP-AtSUN1ΔCC, implying that deletion of the coiled-coil domain abolishes the ability of AtSUN1 to form homomers (Table 2).

Table 2.   Fluorescence resonance energy transfer (FRET) efficiencies as indicators of SUN domain protein interactions. An increase of CFP fluorescence after YFP photobleaching of co-expressed non-mutated AtSUN1 and AtSUN2 indicates that they are able to form homomers and heteromers. No significant increase of CFP-AtSUN1 fluorescence when co-expressed with YFP-AtSUN1ΔCC shows that the coiled-coil domain is necessary for homomerization of AtSUN1
Co-expressed proteinsEF (%)Control EF (%)
CFP-AtSUN1+ YFP-AtSUN27.84 ± 3.470.56 ± 3.85
CFP-AtSUN1+ YFP-AtSUN111.35 ± 3.39−1.34 ± 4.24
CFP-AtSUN2+ YFP-AtSUN219.78 ± 5.391.81 ± 4.44
CFP-AtSUN1+ YFP-AtSUN1ΔCC2.28 ± 7.631.17 ± 7.16

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This paper gives a detailed description of two membrane intrinsic proteins of the plant INM, and provides evidence for the presence of members of the SUN domain family in plants. In this study, At5g04990 and At3g10730 were identified as structural homologues of the C. elegans SUN domain protein UNC-84. Previously, the two proteins were found to be homologous with Schizosaccharomyces pombe Sad1 (Van Damme et al., 2004). Although these authors showed that expressed as fluorescent protein fusions, the two localize to the plant NE, phragmoplast and spindle, they did not associate them with SUN-domain proteins, and did not investigate them further. Here, we have shown that At5g04990 and At3g10730 are not only structurally similar to UNC-84 and Sad1, but are also similar to other SUN-domain proteins, and we have therefore named them AtSUN1 and AtSUN2, respectively, to reflect their association with this protein family. In addition, we have presented evidence for the presence of SUN-domain proteins in other plant species, and have investigated the domain structure, expression, binding properties, and NE targeting mechanisms of AtSUN1 and AtSUN2.

Endogenous AtSUN1 and AtSUN2 were detected in Arabidopsis leaf extract, and their expression was detected in leaf, inflorescence and root tissue, as well as suspension cells, indicating that the two proteins are present in various tissue types. In support of this, relative expression data for AtSUN2 showed the protein to be expressed in 60 different tissue types at low levels (Figure 2b; Table S2) indicating that AtSUN2 may fulfil a ‘housekeeping’ function. As animal SUN-domain proteins are involved in nuclear positioning and anchorage of chromatin to the NE, similar roles for AtSUN1 and AtSUN2 would be worth investigating. Interestingly, although the expression of AtSUN2 is comparably low, it appears to be upregulated in root tip tissue, inflorescence and 7-day-old seedling roots (Figure 2b,c). These tissues contain actively dividing cells, suggesting that AtSUN2 may have an additional function in cell division. Some animal and yeast SUN-domain proteins are known to be involved in mitotic and meiotic processes. For instance, human SUN1 is required for chromosome decondensation at the end of mitosis (Chi et al., 2007), and both SUN1 and SUN2 homologues have been found to anchor meiotic telomeres to the NE of C. elegans, mammals and yeast, and are thus essential for homologous chromosome pairing in gametogenesis (Tomita and Cooper, 2006; Bupp et al., 2007; Conrad et al., 2007; Penkner et al., 2007; Schmitt et al., 2007). Whether AtSUN1 and AtSUN2 are implied in similar roles in plant cell divisions remains to be established. Also to be further examined are the expression patterns of AtSUN1, and whether or how they differ from AtSUN2 expression.

Western blots of antisera raised against AtSUN1 and AtSUN2 detected bands of approximately 43 kDa in Arabidopsis leaf extract, smaller than the predicted 50 kDa based on the sequence. In general, the plant SUN-domain proteins appear to be similar in size but smaller than the animal and yeast homologues (Figure 1a). Their domain layout, however, is very similar, with the prominent approximately 124 amino acid long SUN domain at the C terminus (Figure 1a). For human SUN1 and SUN2 it has been established that the C terminus and the SUN domain reside in the periplasm, and that the N terminus resides in the nucleoplasm (Hodzic et al., 2004). A similar membrane orientation is predicted for other SUN-domain proteins (Padmakumar et al., 2005; Haque et al., 2006). In the periplasm, the SUN domain has been found to interact with the KASH domain of ONM-localized KASH-domain proteins, which in turn associate with cytoskeletal elements such as actin or MTOC/SPB, thereby forming a bridge across the NE that links the NE to the cytoskeleton (Crisp et al., 2006; Worman and Gundersen, 2006; Starr, 2009). Also at the C terminus, SUN-domain proteins feature coiled-coil domains. These have been implicated in the formation of dimers, trimers and tetramers of mammalian SUN1 and SUN2, which in turn assemble into highly immobile oligomeric complexes ideal for the anchorage of large cytoskeletal elements such as the MTOC (Wang et al., 2006; Lu et al., 2008). The N terminus of SUN-domain proteins is involved in interactions with nucleoplasmic components such as chromatin and lamin A (Tzur et al., 2006; Worman and Gundersen, 2006). It also contains targeting information, for example NLS, and is essential for the correct localization of SUN-domain proteins in the INM (Tzur et al., 2006; Wang et al., 2006). Binding to lamins in animal cells allows SUN-domain proteins to link nucleoskeletal elements to cytoskeletal elements via KASH interactions. The functionality of some of the domains of AtSUN1 and AtSUN2 were tested by expression of fluorescent protein fusions of deletion mutants, and examining their localization, mobility and binding abilities.

Non-mutated AtSUN1 and AtSUN2 expressed as fluorescent protein fusions both transiently and stably were found to localize to the NE, and ultrastructural observation revealed the fusion proteins present in the INM (Figure 3). Taken together with the presence of a putative NLS and the homology with animal and yeast INM-localized SUN-domain proteins, this suggests that AtSUN1 and AtSUN2 are INM-intrinsic proteins. In support of this we found that the deletion of the NLS or the entire N terminus increased ER localization (Figure 4; Figure S1). Fluorescent protein fusions of AtSUN1 and AtSUN2 N termini, with and without NLS localized to the nucleus (data not shown). This proves the functionality of the NLS, and indicates that the N termini contain NE-targeting information. Deletion of the N termini and NLS appear to affect the efficiency of AtSUN1/AtSUN2 targeting to the plant NE, which is in accordance with current NE-targeting models for animal INM proteins (Lusk et al., 2007). Interestingly, deletion of the C-terminal coiled-coil and SUN domains reduced or abolished NE localization of AtSUN1 and AtSUN2 (Figure 4). This is contrary to observations in animal and yeast cells, where deletion of the SUN domain does not affect NE localization (Padmakumar et al., 2005; Crisp et al., 2006). Instead of localization to the NE, the coiled-coil and SUN-domain deletion mutants of AtSUN1 and AtSUN2 were found in cytoplasmic membrane aggregates, with some also found in the ER, suggesting that the origins of the aggregates are ER membranes (Figure 4). Formation of these aggregates may have been caused by the mistargeting of the mutants or sequestration of misfolded protein to the aggregates. In animal cells, sequestration of misfolded membrane proteins in ER aggregates is thought to protect the ER and maintain its functionality (Granell et al., 2008).

Previously, we have used FRAP to examine the mobile behaviour of the mammalian-based LBR-GFP, and found it to be highly mobile in the plant NE, with a mobile fraction of approximately 87% (Graumann et al., 2007). Here, we show that approximately half of AtSUN1 and AtSUN2 expressed as fluorescent protein fusions are immobile at the plant NE (Table 1), indicating that in contrast to LBR-GFP the two SUN-domain proteins are involved in binding interactions strong enough to partly immobilize them. Deletion of either the N terminus or coiled-coil domain significantly increased the mobility of AtSUN1 and AtSUN2 (Table 1). The N terminus may be hypothesized to be nucleoplasmic, as shown for other SUN-domain proteins (Hodzic et al., 2004), and is thus likely to interact with nucleoplasmic components. Animal SUN-domain proteins interact with chromatin and nuclear lamins, which immobilize animal INM proteins such as LBR (Ellenberg et al., 1997). In the absence of lamins in plants, plant-specific nuclear filamentous proteins recently shown to underly the plant INM (Dittmer et al., 2007; Fiserova et al., 2009) may be involved in immobilizing AtSUN1 and AtSUN2. This, as well as putative interactions with chromatin, awaits investigation. The increased mobile fraction and diffusion rate of AtSUN1 and AtSUN2 mutants lacking the coiled-coil domain could reflect a size change. In common with mammalian SUN-domain proteins, which exist in multimeric protein complexes consisting of between two and four molecules (Wang et al., 2006; Lu et al., 2008), AtSUN1 and AtSUN2 appear to be able to form homomers and heteromers in vivo (Table 2). Deletion of the coiled-coil domain, which facilitates oligomerization of mammalian SUN-domain proteins (Wang et al., 2006; Lu et al., 2008), abolishes the ability of AtSUN1 to oligomerize, and increases mobility. The observation that YFP fused to the C terminus of AtSUN1 and AtSUN2 also increases the mobility of the two proteins may suggest that YFP hinders the C-terminal binding interactions. This suggests that the coiled-coil domain in AtSUN1, and potentially in AtSUN2, is functionally involved in oligomerization. It also indicates that AtSUN1 and AtSUN2, like their mammalian homologues, appear to be functional in multimeric protein complexes at the plant INM. In animal and yeast cells these include the KASH-domain proteins, which together with the SUN-domain proteins form the NE bridging complexes that connect the cytoskeleton and the nucleoskeleton (Starr, 2009). Whether complexes like these exist in plants remains to be established. It is well documented that cytoskeletal elements associate with the plant NE. For instance, AtGCP2 and AtGCP3 are two components of the γ-tubulin ring complexes that mediate microtubule nucleation at the NE (Seltzer et al., 2007). Both proteins contain NE-targeting domains, and, as they are soluble, it has been speculated that these domains facilitate the anchorage to ONM-intrinsic proteins (Seltzer et al., 2007). This membrane anchor, however, has not yet been identified. In addition, it has been shown recently that nuclear histone H1 is associated with MT nucleation on the outer surface of the NE in BY-2 cells (Nakayama et al., 2008). This implies that in plants, as in animal cells, nuclear components are linked to cytoskeletal elements (Nakayama et al., 2008). Our identification of plant SUN-domain homologues is a further indicator for the existence of LINC complex-like structures in plants. However, the lack of KASH-domain homologues and lamin homologues in plants suggests that such complexes may vary significantly in composition and properties compared with those in animals and yeast. The identification of AtSUN1 and AtSUN2 interaction partners will be the next step in elucidating the functions of the two proteins and the nature of a putative plant LINC complex.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Bioinformatics

The expasy and tair blast facilities (http://us.expasy.org/tools/blast/; http://arabidopsis.org/Blast/) were used to identify At3g10730 and At5g04990 as putative homologues of C. elegans UNC-84, and to find putative homologues of the two Arabidopsis SUN-domain proteins in other species. The amino acid sequences of At3g10730 and At5g04990 were compared with amino acid sequences in Pfam, Sanger, Aramemnon and Interpro predictive protein and pattern databases (http://pfam.sanger.ac.uk/search?tab=searchSequenceBlock; http://smart.embl-heidelberg.de; http://aramemnon.botanik.uni-koeln.de/index.ep; http://www.ebi.ac.uk/interpro) to investigate putative protein motifs. Nuclear localization signals were determined with MotifScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Relative expression data for AtSUN2 and the three reference genes were obtained from the Genevestigator database (https://www.genevestigator.ethz.ch/; Zimmermann et al., 2004). Relative expression data for AtSUN1 were not available from Genevestigator or other expression databases.

RT-PCR

RNA was extracted from adult Arabidopsis leaf and inflorescence tissue and suspension culture cells using the Nucleospin II RNA purification kit (Macherey-Nagel, http://www.macherey-nagel.com). The Superscript III one-step RT-PCR system with Platinum®Taq high fidelity (Invitrogen, http://www.invitrogen.com) was used with the following primer pairs to synthesize and amplify AtSUN1, AtSUN2 and protein phosphatase 2A subunit (PP2A) cDNA. AtSUN1 forward, 3′-ATGTCGGCATCAACGGTGTCG-5′, AtSUN1 reverse, 3′-TTATTCACTTTCAGGTGAAGAGTCCTG-5′; AtSUN2 forward, 3′-ATGTCGGCGTCAACGGTGTC-5′, AtSUN2 reverse, 3′-TCAAGCATGAGCAACAGAGAC-5′; PP2A forward, 3′-ATGTCTATGGTTGATGAGCC-5′, PP2A reverse, 3′-GCTAGACATCATCACATTGTC-5′.

Antibody production

Amino acids 92–105 of AtSUN1 and AtSUN2 were chosen as epitopes for polyclonal antipeptide antisera, as these sequences were found to be unique to AtSUN1 and AtSUN2 (determined by BLAST), and had high predicted antigenicity (Alpha Diagnostics, http://www.4adi.com). A cysteine was added to the C terminus of each peptide for keyhole limpet hemocyanin conjugation. The antibody production including peptide synthesis and purification was carried out by Genosphere (http://www.genosphere-biotech.com).

Immunoblotting

Total protein was extracted from adult Arabidopsis rosette leaf tissue, as described by Sparkes et al. (2005). Protein samples were electrophoretically separated on a 12% SDS gel alongside a broad-range, pre-stained protein marker (New England Biolabs, http://www.neb.uk.com), and were subsequently transferred onto nitrocellulose membrane. The membrane was blocked with 5% milk phosphate-buffered saline plus Tween 20 (PBST) before incubation with either rabbit anti-AtSUN1 pre-immune and antiserum (IgG fraction) or rabbit anti-AtSUN2 pre-immune and antiserum. Primary antibodies were detected using a goat anti-rabbit antibody conjugated to Cy5 diluted to 1:4000. Fluorescence was detected with a Typhoon scanner.

Cloning and fluorescent protein fusions

ABRC-supplied clones U14318 and U50949 were the source of AtSUN1 and AtSUN2 CDS, respectively, for the cloning and production of mutants. PCR was used to create deletion and truncation mutants. Gateway attB sequences were added to either end of the PCR products to recombine the amplicon into the Gateway entry vector pDONR207. The destination vectors used include the following binary expression vectors: pB7WGR2 (for the N-terminal mRFP fusion pB7WGR2AtSUN1), pB7WGC2 (for the N-terminal CFP fusions pB7WGC2AtSUN1, pB7WGC2AtSUN2, pB7WGC2AtSUN2ΔN, pB7WGC2AtSUN2ΔSUN, pB7WGC2AtSUN2ΔNLS and pB7WGC2AtSUN2ΔCC), 35S-eYFP-CasB-NOS::pCambia1300 (for the N-terminal YFP fusions pCambia1300YFP-AtSUN1, pCambia1300YFP-AtSUN1ΔN, pCambia1300YFP-AtSUN1ΔSUN, pCambia1300YFP-AtSUN1ΔCC and pCambia1300YFP-AtSUN2) (Steve Slocombe, Leeds University; Plant Systems Biology, Ghent University; Karimi et al., 2002). Agrobacterium tumefaciens strain GV3101::pMP90 was transformed with expression vectors and used for the transient and stable expression of plant material.

Transient and stable expression

Nicotiana tabaccum leaves were infiltrated with agrobacteria carrying expression vectors for transient expression, as described by Sparkes et al. (2006). Stable expression of fluorescent fusion constructs in BY-2 cells was achieved using the protocol of Irons et al. (2003).

Confocal microscopy, FRAP and apFRET

Confocal imaging was performed as described by Brandizzi et al. (2002) and Irons et al. (2003). Chromatin of BY-2 cells was stained with DRAQ5 (Biostatus, http://www.biostatus.com). Fluorescence recovery after photobleaching was carried out as described by Graumann et al. (2007). For apFRET, performed as described by Karpova et al. (2003), a Zeiss LSM 510 META confocal microscope was used (Zeiss, http://www.zeiss.co.uk). In this case, CFP was used as the FRET donor and YFP was used as the FRET acceptor. FRET between donor and acceptor was confirmed by bleaching of YFP and monitoring the concomitant increase in CFP fluorescence. Laser transmission was kept at 5–10% to avoid photobleaching during scanning, apart from during the YFP bleaching step, when the 514-nm laser was set to 100%. Leaf segments expressing either CFP or YFP alone were imaged with the apFRET settings to confirm that fluorophore crosstalk was minimized, and that the bleaching step did not reduce CFP fluorescence. Four pre-bleach scans and 10 post-bleach scans were taken of the YFP and CFP fluorescence. For data analysis the background fluorescence was subtracted and the CFP fluorescence intensity was normalized onto a percentage scale, as described by Graumann et al. (2007). To calculate the FRET efficiency EF, the following equation was used:

  • image

where CFPpost is the CFP fluorescence intensity after the photobleaching and CFPpre is the CFP fluorescence intensity before the photobleaching (Karpova et al., 2003). Statistical analysis was performed using F-tests and Student’s t-tests. For each sample pair and each control, 30–40 NEs were used.

Electron microscopy

Tobacco leaf tissue was preserved by high-pressure freezing (HPF) with a Bal-Tec HPM010 HPF machine (http://www.bal-tec.com), followed by freeze substitution and embedding in LR white resin. Ultrathin sections were cut and transferred onto formvar-covered nickel grids for immunogold labelling. Grids were blocked in goat serum diluted 1:30 in Tris-BSA for 30 min, followed by a 15-min incubation in Tris-BSA-Tween, and a 15-min incubation in Tris-BSA-glycine. Grids were than incubated overnight in either anti-GFP antibody (AbCam, http://www.abcam.com) or anti-DsRed antibody (BD Pharmingen, http://www.bdbiosciences.com) at 4°C. A goat anti-rabbit antibody (BBInternational, http://www.britishbiocell.co.uk) conjugated to 10-nm gold particles was incubated for 1 h at room temperature (21°C), followed by 15-min incubations in uranyl acetate and lead citrate for contrast staining. Samples were imaged with a JEOL 1200EXII transmission electron microscope (http://www.jeol.com) at 120 kV, and images were captured with a Gatan camera (http://www.gatan.com).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by a grant from the Leverhulme Trust: grant number F/00382/H. A studentship for Katja Graumann was provided by Oxford Brookes University. We would like to thank Coralie Lefevre for technical assistance.

References

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Accession numbers: At5g04990, At3g10730. Gene annotation data for TAIR has been included with this submission.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Quantification of ER localisation. Cells transiently expressing fluorescent fusion proteins, which localise to the NE, were examined for additional ER fluorescence. For each fusion protein, 20 field of view images were examined by confocal microscope. The total number of expressing cells with NE fluorescence as well as the number of cells with additional ER fluorescence was quantified.

Table S1. Overview of domain layout of plant, animal and fungal SUN domain proteins and their homologies to AtSUN1 and AtSUN2. This table summarises the position of the various domains in plant, animal and fungal SUN domain proteins as depicted in figure 1 and their homologies to AtSUN1 and AtSUN2.

Table S2. Complete list of the relative expression values for AtSUN2 and reference genes PP2A, YLS8 and UBC9 obtained from Genevestigator. This table lists the relative expression values for AtSUN2, PP2A, YLS8 and UBC9 obtained from Genevestigator.

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