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Proc Natl Acad Sci U S A. Jun 23, 2009; 106(25): 10207–10212.
Published online Jun 9, 2009. doi:  10.1073/pnas.0812037106
PMCID: PMC2700906
Developmental Biology

SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice

Abstract

How the nuclei in mammalian skeletal muscle fibers properly position themselves relative to the cell body is an interesting and important cell biology question. In the syncytial skeletal muscle cells, more than 100 nuclei are evenly distributed at the periphery of each cell, with 3–8 nuclei anchored beneath the neuromuscular junction (NMJ). Our previous studies revealed that the KASH domain–containing Syne-1/Nesprin-1 protein plays an essential role in anchoring both synaptic and nonsynaptic myonuclei in mice. SUN domain–containing proteins (SUN proteins) have been shown to interact with KASH domain–containing proteins (KASH proteins) at the nuclear envelope (NE), but their roles in nuclear positioning in mice are unknown. Here we show that the synaptic nuclear anchorage is partially perturbed in Sun1, but not in Sun2, knockout mice. Disruption of 3 or all 4 Sun1/2 wild-type alleles revealed a gene dosage effect on synaptic nuclear anchorage. The organization of nonsynaptic nuclei is disrupted in Sun1/2 double-knockout (DKO) mice as well. We further show that the localization of Syne-1 to the NE of muscle cells is disrupted in Sun1/2 DKO mice. These results clearly indicate that SUN1 and SUN2 function critically in skeletal muscle cells for Syne-1 localization at the NE, which is essential for proper myonuclear positioning.

Keywords: KASH domain, Nesprin, neuromuscular junction, nuclear envelope protein, Syne-1

Proper nuclear positioning relative to the cell body is important for many cellular processes during animal development. Mammalian skeletal muscle fibers are giant syncytial cells, each containing more than 100 nuclei. Each muscle fiber contains a neuromuscular junction (NMJ) that starts to form at approximately embryonic day 14 (E14) in mice (1). Following a multistep process involving the function of the agrin-MuSK-rapsyn-AChR pathway, the motor nerve terminal and the postsynaptic membrane become highly differentiated, ensuring reliable terminal transmission. The nuclei in the muscle fibers are positioned in a nonrandom manner, with 3–8 nuclei (synaptic nuclei) clustered beneath each NMJ and other nuclei (nonsynaptic nuclei) distributed evenly along the cell membrane (1, 2). How synaptic and nonsynaptic nuclei properly position themselves within each muscle fiber and the consequences of disrupting the nuclear positioning are attractive problems to investigate. Recent studies have indicated that KASH (Klarsicht/ANC-1/Syne homologue) domain–containing and SUN domain–containing proteins (KASH/SUN proteins) form a complex at the nuclear envelope (NE) for various cellular functions (36). Whereas a mammalian KASH protein has been found to have a critical function in myonuclear anchorage (79), the roles of SUN proteins in this process remain unclear.

The SUN domain was first defined as a domain of shared homology between Sad1 in Schizosaccharomyces pombe and UNC-84 in Caenorhabditis elegans (10). SUN proteins are conserved inner nuclear membrane proteins containing at least 1 transmembrane domain and a C-terminal SUN domain that is localized inside the lumen of the NE (4, 11). The functions of SUN proteins in nuclear positioning were first revealed by genetic analysis of the unc-84 gene in C. elegans. In C. elegans, UNC-84 has been shown to recruit KASH proteins ANC-1 and UNC-83 to the outer NE for critical functions in both nuclear anchorage and nuclear migration (10, 12, 13). The conserved KASH domain of UNC-83 physically interacts with the SUN domain of UNC-84 in the lumen of NE (14). Similarly, in flies, the SUN protein Klaroid is required for the localization of KASH protein Klarsicht to the NE and for nuclear migration in the eye (15). Genetic studies in the worm and yeast also have indicated roles of SUN proteins in mediating the interactions between the NE and centrosome, centromeres, and telomeres at various stages of mitotic and meiotic cells (1622).

Mammalian SUN1 and SUN2 proteins were first identified as homologues of C. elegans UNC-84 (10). SUN1 and SUN2 are broadly distributed in mouse tissues, whereas 2 other, more recently identified SUN proteins, SUN3 and SPAG4, appear to be expressed in a limited amount of tissues (4, 2325). In tissue culture cells, SUN1 and SUN2 have been shown to be inner NE proteins with their N-terminal domains localized in the nucleoplasm and the C-terminal SUN domains in the lumen of the NE (24, 2628). We have previously shown that SUN1 mediates the telomere–NE interactions during meiosis; loss of Sun1 function disrupts the telomere–NE attachment, homologue pairing, and synapsis formation, leading to the abolishment of gametogenesis (29). Analysis of a recently generated Sun1−/− mouse strain has linked the SUN1 function to piRNA expression in germ cells (30).

The KASH domain is a conserved protein motif of about 60 residues located at the C terminus of KASH proteins (13). Mammalian Syne-1/Nesprin-1 and Syne-2/Nesprin-2 belong to a family of giant KASH proteins (>6,800 residues) that includes ANC-1 in C. elegans and MSP-300 in Drosophila, each of which also contains actin-binding domains at the N terminus and a large central region with mostly spectrin-like repeats (3, 5). Consistent with the studies in C. elegans, SUN1 and SUN2 proteins have been shown to be necessary for the localization of the mammalian KASH protein Syne-2/Nesprin-2 to the NE in tissue culture cells (24, 27). A role for Syne-1/Nesprin-1 in nuclear positioning in skeletal muscle cells was revealed in our previous genetic analysis (7, 8). Overexpression of the KASH domain–containing C-terminal fragment of either Syne-1 or Syne-2 partially damages the anchorage of synaptic nuclei; however, deletion of the KASH domain of Syne-1, but not of Syne-2, severely disrupts both synaptic and nonsynaptic nuclear anchorage. A deletion of the N-terminal actin binding domain of Syne-2/Nesprin-2 also has been generated in mice and shown to cause defects in NE architecture and skin development (31).

In this study, we explored the roles of SUN proteins in myonuclear anchorage in mice carrying single or double mutations in the Sun1 and Sun2 genes. Our results indicate that the 2 SUN proteins play critical roles in recruiting Syne-1/Nesprin-1 to the NE for both synaptic and nonsynaptic nuclear anchorage. These results provide important insights into the mechanism underlying the functions of the KASH–SUN complexes in animal development.

Results

The Anchorage of Synaptic Nuclei Is Partially Perturbed in Sun1 Knockout Mice.

The SUN1 protein is highly expressed and localized on the NE in skeletal muscle cells (29). To elucidate the potential function of SUN1 in myonuclear positioning, we examined whether a Sun1 deletion would disrupt the localization of Syne-1 onto the NE, as well as myonuclear positioning. Syne-1 has been shown to localize at a significantly higher level at the NE of synaptic nuclei than the NE of nonsynaptic nuclei (32). A major component of the NMJ is the acetylcholine receptor (AChR), which can be specifically bound by a chemical compound called alpha-bungrotoxin (α-BTX). NMJ is commonly visualized by staining with α-BTX conjugated to a fluorescent dye (33). Staining with an anti–Syne-1 antibody and tetramethylrhodamine-conjugated α-BTX revealed that Syne-1 remained localized on the NE of myonuclei in Sun1−/− mice (Fig. 1 D and E), although the expected reduction of the level of Syne-1 on synaptic nuclei could not be quantitatively determined (see below). These Sun1−/− mice exhibited a modest but significant decrease in the average number of synaptic nuclei beneath the NMJ compared with control mice of the same age (Fig. 1 B, D, and F); for example, in the mutant mice, 83% of the muscle fibers harbored 2 or more nuclei under the NMJ, compared with 98.2% of the muscle fibers in wild-type (WT) controls (Fig. 1F). Meanwhile, our quantitative analysis indicated that the distribution of nonsynaptic nuclei in Sun1−/− muscle cells was similar to that in WT controls (Fig. 1 C and E; data not shown). These results indicate that although SUN1 is not absolutely required for the NE localization of Syne-1 in skeletal muscle cells, it is required for proper synaptic nuclear anchorage. These results may suggest a role for SUN1 in maintaining a higher level of Syne-1 at synaptic nuclei, and also may indicate the involvement of another SUN protein in the recruitment of Syne-1 to the NE and myonuclear positioning.

Fig. 1.
The anchorage of synaptic nuclei is partially perturbed in Sun1−/− mice. (A) A WT muscle fiber stained with anti–Syne-1 antibody (green), α-BTX (red), and DAPI (blue). (B) Enlarged view of the cropped region in (A), showing ...

Myonuclear Anchorage Is Not Significantly Perturbed in Sun2 Knockout Mice.

Antibody staining of SUN2 revealed that SUN2 was highly expressed and localized on the NE in muscle cells [supporting information (SI) Fig. S1D]. We thus generated Sun2−/− mice to investigate whether SUN2 plays a redundant role with SUN1 in myonuclear anchorage. The mutant allele was constructed by replacing the SUN domain–coding region with a neo cassette (Fig. S1A). Targeted ES clones were identified by PCR and Southern blot analysis (data not shown), and mice carrying the mutant allele were identified by genotyping PCR (Fig. S1B). The absence of the Sun2 transcript and the SUN2 protein on the NE in skeletal muscle cells was demonstrated by RT-PCR and staining with an anti-SUN2 antibody (8), respectively (Fig. S1 C–E).

Unlike Sun1−/− mice, which were completely sterile (29), Sun2−/− mice were fertile and displayed no obvious abnormalities in growth and development. We previously found that SUN2 is not expressed in meiotic cells (29) (Fig. S2), suggesting that SUN2 likely does not play a critical role in meiosis. We examined both the number of synaptic nuclei and the positioning of nonsynaptic nuclei in Sun2−/− mice, and found no significant defects (Fig. S1F; data not shown).

The Anchorage of Synaptic Nuclei Is Severely Disrupted in Sun1/2 Double-Knockout Mice.

The overlapping expression patterns of SUN1 and SUN2 in skeletal muscles and their similar protein structures suggest that SUN1 and SUN2 may have redundant functions in myonuclear anchorage. To test this hypothesis, we combined Sun1 and Sun2 mutant alleles and generated Sun1+/−; Sun2−/− mice, which were then intercrossed to produce Sun1/2 double-knockout (DKO) mice. We examined more than 50 litters and found no Sun1/2 DKO survivors. We then examined 20 litters at E18.5 and found that the number of Sun1/2 DKO embryos was consistent with the expected Mendelian ratio (data not shown). Further analysis found that the Sun1/2 DKO pups reached full term but died soon after birth (Fig. S3).

To assess the anchorage of myonuclei in Sun1/2 DKO embryos, we dissected out the triangularis sterni from the E18.5 embryos and stained them with DAPI and α-BTX. More than 100 cells with each genotype were examined. One or 2 nuclei were found to lie beneath each NMJ in the muscle fibers of WT and Sun1+/−; Sun2+/− mice (Fig. 2 A and B). In contrast, 63% of the muscle cells in Sun1−/−; Sun2+/− mice and 49% of those in Sun1+/−; Sun2−/− mice harbored no nuclei beneath the NMJ (Fig. 2 A and B; P < .001). The anchorage defect was exacerbated in the muscle fibers of Sun1/2 DKO mice, where only 19% of the muscle cells contained 1 nucleus beneath the NMJ (Fig. 2 A and B; P < .001). Next, we examined the anchorage of synaptic nuclei in adult mice, and found significant defects in Sun1−/−; Sun2+/− mice and Sun1+/−; Sun2−/− mice compared with WT and Sun1+/−; Sun2+/− mice (Figs. 1F and and22 C and D; P < .001). These gene dosage–dependent effects indicate that SUN1 and SUN2 have partially redundant functions on synaptic nuclear anchorage.

Fig. 2.
SUN1 and SUN2 play partially redundant roles in anchoring synaptic nuclei. (A) Fluorescent images showing skeletal muscle cells of triangularis sterni in the E18.5 embryos stained with DAPI (blue) and α-BTX (red). Genotypes of the embryos are ...

The Organization of Nonsynaptic Nuclei Is Disrupted in Sun1/2 DKO Mice.

Because the Sun1/2 DKO mice died shortly after birth, when the muscle fibers were not yet completely developed, examining the distribution of the nonsynaptic nuclei was not feasible. We examined adult mice with the genotype of WT, Sun1+/−; Sun2+/−, Sun1−/−; Sun2+/−, and Sun1+/−; Sun2−/− and found a normal distribution of nonsynaptic nuclei in these mice (Fig. 3 A–D and G). Speculating that the lethality of Sun1/2 DKO mice might be due to unknown defects in the central nervous system, we generated transgenic mice that expressed the Sun1 gene driven by the neuron-specific enolase (NSE) promoter. The NSE promoter, derived from the rat NSE gene, has been shown to drive transgene expression in a broad range of neurons in mice (34). The Sun1−/−; Sun2−/−; NES::Sun1 (RDKO) mice were alive at birth, and some mice with this genotype survived to adulthood (Fig. 4). The nuclear positioning defects in skeletal muscles were not rescued in these mice (not shown), consistent with the lack of transgene expression in muscle cells (Fig. 4). This gave us the opportunity to quantitatively examine the positioning of nonsynaptic nuclei in adult mice in the absence of any of the WT Sun1/2 alleles in skeletal muscle cells. We found that on average, more than 3 nuclear clusters were present in each muscle cell of the RDKO mice (Fig. 3 F and G); a nuclear cluster is defined as 3 or more nuclei grouped together, with the distance between adjacent nuclei less than their diameter. This clustering phenotype was not caused by a novel effect of the transgene itself, because the distribution of nonsynaptic nuclei in the control Sun1+/−; Sun2−/−;NSE::Sun1 mice was normal (Fig. 3 E and G).

Fig. 3.
The organization of nonsynaptic nuclei is disrupted in −/−−/−RDKO mice. (A–F) The distribution of nonsynaptic nuclei in skeletal muscle fibers of adult mice stained with DAPI (blue). The arrow indicates a typical ...
Fig. 4.
Expression of SUN1 in neurons rescues the lethality of Sun1/2 DKO mice. (A) The transgenic construct of the NSE-myc-Sun1 fusion gene. (B) Fluorescent images showing expression of the transgenic construct in the cortex of transgenic mice. The brain sections ...

The NE Localization of Syne-1, But Not Lamin A/C, Is Disrupted in Sun1/2 DKO Mice.

Given the similar myonuclear anchorage defects of Sun1/2 DKO mice and Syne-1 KASH deletion (Syne-1−/−) mice, as well as the relationship between KASH proteins and SUN proteins (13, 27), we examined the expression of Syne-1 in skeletal muscle cells of mice harboring the Sun1 and Sun2 mutant alleles. The NE localization of Syne-1 appeared to be normal in Sun1 and Sun2 double-heterozygous mice (Fig. 5 D–F); however, the NE-localized Syne-1 signal in skeletal muscle cells appeared to be reduced in Sun1+/−; Sun2−/− and Sun1−/−; Sun2+/− mice and was completely absent in Sun1/2 DKO mice (Fig. 5 G–O). These results indicate that SUN1 and SUN2 play a critical but redundant role in recruiting Syne-1 to the NE of skeletal muscle cells.

Fig. 5.
The NE localization of Syne-1 but not Lamin A/C is disrupted in Sun1/2 DKO mice. (A–O) Representative images of intercostal muscle sections of the E18.5 embryos stained with the anti–Syne-1 antibody (green) and DAPI (blue). Genotypes of ...

We also examined the NE localization of SUN1 and SUN2 in the muscle fibers of Syne-1/2 double KASH deletion (Syne-1−/−; Syne-2−/−) mice and found no abnormalities (Fig. S4). This finding indicates that these 2 KASH proteins are not required for NE localization of the 2 SUN proteins.

Previous studies have shown that the SUN proteins can interact with nuclear lamina proteins and that the loss of lamin A/C disrupts myonuclear anchorage in mice (25, 28, 35). We examined the localization of Lamin A/C in skeletal muscle cells of Sun1/2 DKO mice. Compared with heterozygotes, the NE localization of Lamin A/C was not disturbed in Sun1/2 DKO mice (Fig. 5 P–T). This suggests that SUN1 and SUN2 are not essential for NE localization of Lamin A/C, in agreement with findings from studies in other organisms and tissue culture cells (15, 24, 25, 28, 36).

Discussion

Previous studies on KASH and SUN proteins have provided evidence of their interaction at the NE in several cellular processes (10, 1215, 17, 20). Syne-1 has been shown to be essential for proper myonuclear anchorage. Our findings provide conclusive evidence to support the idea that SUN1 and SUN2 function redundantly in the anchorage of myonuclei, including synaptic and nonsynaptic nuclei. They also show that SUN1 and SUN2 act redundantly to recruit Syne-1 to the NE.

SUN1 and SUN2 have been shown to be inner NE proteins, with their conserved C terminus SUN domain at the lumen of the NE and the N terminus in the nucleus (26, 28). They can form homodimers and heterodimers in tissue culture cells (28, 37). Our data indicate that both proteins are highly expressed and localized on the NE of muscle cells. These features underlie the redundant roles of SUN1 and SUN2 in skeletal muscle cells.

Investigation of the physiological function of myonuclear anchorage has been an interesting and difficult problem. Previously, it was speculated that synaptic nuclei have specialized roles in maintaining the high expression levels of postsynaptic components and thus the proper formation of the NMJ (1, 38); however, our previous study of the Syne-1/Nesprin-1 protein indicated that neither the formation of the NMJ nor the expression of several synaptic components (synaptophysin, AchR, rapsyn, MuSK, and utrophin) was obviously disrupted (7, 8). In the present study, we also examined the expression of rapsyn, synaptophysin, and utrophin in E18.5 skeletal muscles and found no obvious decrease in these protein levels at the NMJ in the Sun1/2 DKO mice (Fig. S5). Moreover, the lack of prominent developmental defects in the Syne-1/Nesprin-1 KASH deletion mice also suggests that myonuclear anchorage is not essential for mouse development. We did find disturbed formation of proper muscle–nerve connections due to the altered positions of the NMJ within individual mutant myofibers, however (ref. 8 and data not shown). The effects of this abnormal myonuclear positioning on animal development and behavior, as well as other important physiological effects of disrupted myonuclear anchorage, remain to be determined. It is important to note that Syne-1/Nesprin-1 does play essential roles (although redundantly with Syne-2/Nesprin-2) in developmental aspects other than myonuclear anchorage, as clearly indicated by the dramatic lethal phenotype of Syne-1 and Syne-2 double KASH deletion mutants (8).

KASH–SUN protein complexes also have been implicated in roles related to muscular dystrophies (9, 39). The degenerative phenotype may be related to the defects in myonuclear anchorage, including nonsynaptic anchorage. It is interesting that in a recent report, independently generated mice with deletions in the C-terminal KASH domain exhibited more severe developmental and behavior defects than the mice that we generated previously (refs. 8 and 9; L.K. and X.Z., unpublished data). Such a difference might be linked to the different genetic backgrounds of the mice or to mutations induced in ES cells during the targeting process (40), possibly suggesting that mice become highly dependent on the Syne-1/Nesprin-1 function when certain other cellular functions are compromised. Alternatively, the variation in phenotypes could be due to the small difference in the deletion alleles generated in the 2 Syne-1/Nesprin-1 strains. For example, 1 of the alleles might disrupt a KASH domain–independent function. In humans, mutations in Syne-1/Nesprin-1 have been associated with a certain type of recessive cerebellar ataxia (41). Although the precise mechanism of this disease is not clear, it is unlikely to be caused primarily by the defect in nuclear anchorage in muscle cells, based on our analysis of the Syne-1/Nesprin-1 KASH deletion mice (data not shown).

Our observations that the neonatal lethality of Sun1/2 DKO mice was rescued by expressing the Sun1 gene driven by a neuron-specific promoter (RDKO mice) indicates that SUN1 and SUN2 likely play important but redundant roles in the development of the central nervous system. However, the RDKO mice had a smaller body size (Fig. 4) and exhibited defects on a neuronal behavior assay (e.g., feet clasping; data not shown), suggesting that the NSE::Sun1 transgene may not have rescued all neuronal defects in the Sun1/2 DKO mice. The roles of SUN1 and SUN2 in brain development are currently under investigation using Sun1/2 DKO as well as conditional KO mice.

Materials and Methods

Generation of Sun2 Knockout Mice and Sun1/2 DKO Mice.

To construct the Sun2 gene targeting vector, the 11.5-kb BamH I DNA fragment containing exons 5–10 of Sun2 and the 1.4-kb EcoR I fragment containing part of exon 17 were obtained from a BAC clone of the Sv129 strain (no. 49709; Invitrogen). These 2 fragments were cloned into a pPNT vector, flanking the pgk-Neo cassette as the targeting arms. The resulting targeting vector was linearized and electroporated into W4/129S6 ES cells (Taconic Transgenics). After 2 rounds of selection, the ES clones were screened for successful targeting by PCR analysis using primers prDX016 and prDX081. The positive ones were further identified by Southern blot analysis using a 778-bp genomic fragment as the probe (PCR-amplified with primers prDX086 and prDX087). The targeted ES clones were injected into C57BL/6J blastocysts. The genotype of the targeted Sun2 allele in mice was determined by PCR analysis using primers prDX016, prDX081, and prDX115. Sun1/2 DKO mice were generated by intercrossing Sun1−/− and Sun2−/− mice. The primer sequences used are listed in SI Materials and Methods.

Mouse breeding and experimental manipulations were carried out following the general guidelines published by the Association for Assessment and Accreditation of Laboratory Animal Care. All animal-related procedures were reviewed and approved by the Institute of Developmental Biology and Molecular Medicine Institutional Animal Care and Use Committee.

Immunofluorescent Staining and Microscopic Analysis.

Immunofluorescent staining of frozen sections was carried out following standard protocols (42). Whole-mount staining of skeletal muscle cells was carried out as described previously (7, 8). To isolate individual myofibers from E18.5 embryos, the thoraxes were fixed and triangularis sterni were extracted. After staining with tetramethylrhodamine-conjugated α-BTX (Molecular Probes) and DAPI, individual fibers were teased out and mounted in the mounting medium (Sigma). At the E18.5 stage, the motor neuron has not yet invaginated into the muscle fiber. When triangularis stermi were dissected out to obtain the single muscle fiber, a clean separation between the fiber and surrounding cell/nuclei was usually achieved. Thus, the distraction of nonmuscle nuclei in these experiments was minimal and did not present a significant problem for quantitative scoring of the mutant phenotype. For scoring the nonsynaptic nuclei clusters in adult mice, we chose only fibers with minimal contamination of nonmuscle cells. The reliability of the phenotype scoring is supported by the fact that we did not detect a single prominent nuclear cluster in the WT fibers that we examined, and that the clustering phenotype was obvious and highly penetrant in the RDKO mice. The antibodies used in this study were mouse anti-Lamin A/C (Chemicon), goat anti-mouse IgG-FITC (Chemicon), goat anti-rabbit IgG-FITC (Sigma), mouse anti-rapsyn (Sigma), rabbit anti-synaptophysin (Zymed), and mouse anti-utrophin (Novocastra). The production of antibodies against SUN1, SUN2, and Syne-1 has been described previously (8, 29).

Photographs were taken with a Leica DMRXA2 system. Images were manipulated with Leica FW4000, Adobe Photoshop software, and Adobe Illustrator software.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Xiaohui Wu, Yanling Yang, and Yanfeng Tan for their help in producing the chimeras and transgenic mice and Beibei Ying, Xiaohui Wu, Kejing Deng, Lin Sun, Wufan Tao, Aileen Sewell, and other members of the IDM for their assistance and discussions. This work was supported by grants from the National Natural Science Foundation of China, the Chinese Ministry of Science and Technology (973 Program) and Ministry of Education (211 Program), and the Shanghai Municipal Government.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812037106/DCSupplemental.

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