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1.  A dynamic meiotic SUN belt includes the zygotene-stage telomere bouquet and is disrupted in chromosome segregation mutants of maize (Zea mays L.) 
The nuclear envelope (NE) plays an essential role in meiotic telomere behavior and links the cytoplasm and nucleoplasm during homologous chromosome pairing and recombination in many eukaryotic species. Resident NE proteins including SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne-homology) domain proteins are known to interact forming the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that connects chromatin to the cytoskeleton. To investigate the possible cross-kingdom conservation of SUN protein functions in plant meiosis, we immunolocalized maize SUN2 using 3D microscopy of pollen mother cells from maize (Zea mays L.), a large-genome plant model with a canonical NE zygotene-stage telomere bouquet. We detected SUN2 at the nuclear periphery and found that it exhibited a distinct belt-like structure that transitioned to a half-belt during the zygotene stage and back to a full belt during and beyond the pachytene stage. The zygotene-stage half-belt SUN structure was shown by 3D immuno-FISH to include the NE-associated telomere cluster that defines the bouquet stage and coincides with homologous chromosome synapsis. Microtubule and filamentous actin staining patterns did not show any obvious belt or a retracted-like structure other than a general enrichment of tubulin staining distributed widely around the nucleus and throughout the cytoplasm. Genetic disruption of the meiotic SUN belt staining patterns with three different meiosis-specific mutants, desynaptic (dy1), asynaptic1 (as1), and divergent spindle1 (dv1) provides additional evidence for the role of the nuclear envelope in meiotic chromosome behavior. Taking into account all of the observations from this study, we propose that the maize SUN belt is directly or indirectly involved in meiotic telomere dynamics, chromosome synapsis, and possibly integration of signals and forces across the meiotic prophase nuclear envelope.
doi:10.3389/fpls.2014.00314
PMCID: PMC4093829  PMID: 25071797
telomere; SUN; nuclear envelope; bouquet; meiosis
2.  Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function 
PLoS Genetics  2014;10(2):e1004099.
LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1−/− meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1−/− mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.
Author Summary
Correct genome haploidization during meiosis requires tightly regulated chromosome movements that follow a highly conserved choreography during prophase I. Errors in these movements cause subsequent meiotic defects, which typically lead to infertility. At the beginning of meiotic prophase, chromosome ends are tethered to the nuclear envelope (NE). This attachment of telomeres appears to be mediated by well-conserved membrane spanning protein complexes within the NE (LINC complexes). In mouse meiosis, the two main LINC components SUN1 and SUN2 were independently described to localize at the sites of telomere attachment. While SUN1 has been demonstrated to be critical for meiotic telomere attachment, the precise role of SUN2 in this context, however, has been discussed controversially in the field. Our current study was targeted to determine the factual capacity of SUN2 in telomere attachment and chromosome movements in SUN1 deficient mice. Remarkably, although telomere attachment is impaired in the absence of SUN1, we could find a yet undescribed SUN1-independent telomere attachment, which presumably is mediated by SUN2 and KASH5. This SUN2 mediated telomere attachment is stable throughout prophase I and functional in moving telomeres within the NE. Thus, our results clearly indicate that SUN1 and SUN2, at least partially, fulfill redundant meiotic functions.
doi:10.1371/journal.pgen.1004099
PMCID: PMC3937131  PMID: 24586178
3.  Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and Myonuclear Organization 
PLoS Genetics  2014;10(9):e1004605.
Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.
Author Summary
Emery-Dreifuss muscular dystrophy (EDMD) is an inherited disorder involving muscle wasting and weakness, accompanied by cardiac defects. The disease is variable in its severity and also in its genetic cause. So far, 6 genes have been linked to EDMD, most encoding proteins that form a structural network that supports the nucleus of the cell and connects it to structural elements of the cytoplasm. This network is particularly important in muscle cells, providing resistance to mechanical strain. Weakening of this network is thought to contribute to development of muscle disease in these patients. Despite rigorous screening, at least 50% of patients with EDMD have no detectable mutation in the 6 known genes. We therefore undertook screening and identified mutations in two additional genes that encode other components of the nuclear structural network, SUN1 and SUN2. Our findings add to the genetic complexity of this disease since some individuals carry mutations in more than one gene. We also show that the mutations disrupt connections between the nucleus and the structural elements of cytoplasm, leading to mis-positioning and clustering of nuclei in muscle cells. This nuclear mis-positioning is likely to be another factor contributing to pathogenesis of EDMD.
doi:10.1371/journal.pgen.1004605
PMCID: PMC4161305  PMID: 25210889
4.  Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes* 
The Journal of Biological Chemistry  2009;285(5):3487-3498.
The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.
doi:10.1074/jbc.M109.071910
PMCID: PMC2823409  PMID: 19933576
Diseases/Aging; Diseases/Muscular Dystrophy; Methods/Microscopic Imaging; Protein/Protein-protein interactions; Subcellular Organelles/Cytoskeleton; Subcellular Organelles/Nuclear Membrane; Laminopathies
5.  Structural insights into SUN-KASH complexes across the nuclear envelope 
Cell Research  2012;22(10):1440-1452.
Linker of the nucleoskeleton and the cytoskeleton (LINC) complexes are composed of SUN and KASH domain-containing proteins and bridge the inner and outer membranes of the nuclear envelope. LINC complexes play critical roles in nuclear positioning, cell polarization and cellular stiffness. Previously, we reported the homotrimeric structure of human SUN2. We have now determined the crystal structure of the human SUN2-KASH complex. In the complex structure, the SUN domain homotrimer binds to three independent “hook”-like KASH peptides. The overall conformation of the SUN domain in the complex closely resembles the SUN domain in its apo state. A major conformational change involves the AA'-loop of KASH-bound SUN domain, which rearranges to form a mini β-sheet that interacts with the KASH peptide. The PPPT motif of the KASH domain fits tightly into a hydrophobic pocket on the homotrimeric interface of the SUN domain, which we termed the BI-pocket. Moreover, two adjacent protomers of the SUN domain homotrimer sandwich the KASH domain by hydrophobic interaction and hydrogen bonding. Mutations of these binding sites disrupt or reduce the association between the SUN and KASH domains in vitro. In addition, transfection of wild-type, but not mutant, SUN2 promotes cell migration in Ovcar-3 cells. These results provide a structural model of the LINC complex, which is essential for additional study of the physical and functional coupling between the cytoplasm and the nucleoplasm.
doi:10.1038/cr.2012.126
PMCID: PMC3463262  PMID: 22945352
crystal structure; SUN2; KASH; LINC complexes; mechanical force transduction; nuclear envelope
6.  Novel plant SUN–KASH bridges are involved in RanGAP anchoring and nuclear shape determination 
The Journal of Cell Biology  2012;196(2):203-211.
SUN–KASH nuclear envelope bridges formed by WIP and SUN proteins are present in the plant branch of the tree of life but have functionally diverged from their opisthokont counterparts and are involved in nuclear morphology and RanGAP–nuclear envelope association.
Inner nuclear membrane Sad1/UNC-84 (SUN) proteins interact with outer nuclear membrane (ONM) Klarsicht/ANC-1/Syne homology (KASH) proteins, forming linkers of nucleoskeleton to cytoskeleton conserved from yeast to human and involved in positioning of nuclei and chromosomes. Defects in SUN–KASH bridges are linked to muscular dystrophy, progeria, and cancer. SUN proteins were recently identified in plants, but their ONM KASH partners are unknown. Arabidopsis WPP domain–interacting proteins (AtWIPs) are plant-specific ONM proteins that redundantly anchor Arabidopsis RanGTPase–activating protein 1 (AtRanGAP1) to the nuclear envelope (NE). In this paper, we report that AtWIPs are plant-specific KASH proteins interacting with Arabidopsis SUN proteins (AtSUNs). The interaction is required for both AtWIP1 and AtRanGAP1 NE localization. AtWIPs and AtSUNs are necessary for maintaining the elongated nuclear shape of Arabidopsis epidermal cells. Together, our data identify the first KASH members in the plant kingdom and provide a novel function of SUN–KASH complexes, suggesting that a functionally diverged SUN–KASH bridge is conserved beyond the opisthokonts.
doi:10.1083/jcb.201108098
PMCID: PMC3265956  PMID: 22270916
7.  Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans 
PLoS Genetics  2010;6(11):e1001219.
The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2–dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.
Author Summary
During meiosis, homologous chromosomes from each parent must pair, synapse, and recombine before being assorted to the gametes. In Caenorhabditis elegans, to find the correct pairing partner, telomere-led chromosome movement occurs in a restricted subvolume of the nucleus. This feature is comparable to the widely conserved meiotic bouquet, a configuration where telomeres cluster in a limited area at the nuclear periphery. Chromosomes are moved by cytoskeletal forces transmitted via the SUN/KASH bridge across the nuclear envelope, and abrogation of movement leads to precocious nonhomologous synapsis. Using live cell imaging, we followed the movement of matefin/SUN-1 aggregates, which highlight chromosome ends. Instead of single chromosome ends looking for their homologous partners, we observed that duplets/multiplets and single chromosome ends were brought together into “patches” by the ongoing movement during the leptotene/zygotene stages of meiosis. Chromosome ends then shuffled through these patches in search of the correct partner. This study was a comprehensive analysis of matefin/SUN-1 aggregate dynamics in wild type, known Caenorhabditis elegans pairing mutants, and the recombination mutant spo-11; and it examined the contributions of these genotypes to leptotene/zygotene chromosome movement.
doi:10.1371/journal.pgen.1001219
PMCID: PMC2991264  PMID: 21124819
8.  Subcellular Localization of SUN2 Is Regulated by Lamin A and Rab5 
PLoS ONE  2011;6(5):e20507.
SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis.
doi:10.1371/journal.pone.0020507
PMCID: PMC3105078  PMID: 21655223
9.  Evidence for LINC1-SUN Associations at the Plant Nuclear Periphery 
PLoS ONE  2014;9(3):e93406.
Sad1/UNC84 (SUN) domain proteins are a highly conserved family of inner nuclear membrane localised proteins in eukaryotes. One of their main functions is as key components of nucleo-cytoskeletal bridging complexes, in which SUN proteins associate with nucleoskeletal elements. In metazoans these are the lamins, which form a supportive structural network termed the lamina. Plants lack sequence homologs of lamins but have a similar nucleoplasmic structural network to support the plant NE. Putative components of this plant lamina-like structure are Little Nuclei (LINC) proteins, which bear structural resemblance to lamins and fulfil similar functions. This work explores the associations between AtLINC1, AtSUN1 and AtSUN2. AtLINC1 is recruited to the NE by SUN proteins and is immobilised therein. This recruitment and the immobile properties are likely due to AtSUN1/2-AtLINC1 protein interactions occurring in planta. In addition, the SUN N-terminus appears to play an important role in mediating these interactions. The associations between AtLINC1 and plant SUN proteins are a first indicator of how the nucleoskeleton may be anchored to the nuclear membrane in plants. Building on the previous characterisation of Klarsicht/Anc1/Syne1 homology (KASH) like proteins in plants, this study advances the identification and characterisation of nucleo-cytoskeletal bridging complexes in plants.
doi:10.1371/journal.pone.0093406
PMCID: PMC3965549  PMID: 24667841
10.  SUN-1 and ZYG-12, Mediators of Centrosome–Nucleus Attachment, Are a Functional SUN/KASH Pair in Caenorhabditis elegans 
Molecular Biology of the Cell  2009;20(21):4586-4595.
Klarsicht/ANC-1/Syne/homology (KASH)/Sad-1/UNC-84 (SUN) protein pairs can act as connectors between cytoplasmic organelles and the nucleoskeleton. Caenorhabditis elegans ZYG-12 and SUN-1 are essential for centrosome–nucleus attachment. Although SUN-1 has a canonical SUN domain, ZYG-12 has a divergent KASH domain. Here, we establish that the ZYG-12 mini KASH domain is functional and, in combination with a portion of coiled-coil domain, is sufficient for nuclear envelope localization. ZYG-12 and SUN-1 are hypothesized to be outer and inner nuclear membrane proteins, respectively, and to interact, but neither their topologies nor their physical interaction has been directly investigated. We show that ZYG-12 is a type II outer nuclear membrane (ONM) protein and that SUN-1 is a type II inner nuclear membrane protein. The proteins interact in the luminal space of the nuclear envelope via the ZYG-12 mini KASH domain and a region of SUN-1 that does not include the SUN domain. SUN-1 is hypothesized to restrict ZYG-12 to the ONM, preventing diffusion through the endoplasmic reticulum. We establish that ZYG-12 is indeed immobile at the ONM by using fluorescence recovery after photobleaching and show that SUN-1 is sufficient to localize ZYG-12 in cells. This work supports current models of KASH/SUN pairs and highlights the diversity in sequence elements defining KASH domains.
doi:10.1091/mbc.E08-10-1034
PMCID: PMC2770946  PMID: 19759181
11.  Prelamin A-mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle 
Cell Death and Differentiation  2011;18(8):1305-1315.
Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery–Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.
doi:10.1038/cdd.2010.183
PMCID: PMC3097169  PMID: 21311568
muscle differentiation; nuclear positioning; muscular dystrophy; SUN1; prelamin A
12.  KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development 
Human Molecular Genetics  2010;20(6):1061-1073.
Nuclear movement relative to cell bodies is a fundamental process during certain aspects of mammalian retinal development. During the generation of photoreceptor cells in the cell division cycle, the nuclei of progenitors oscillate between the apical and basal surfaces of the neuroblastic layer (NBL). This process is termed interkinetic nuclear migration (INM). Furthermore, newly formed photoreceptor cells migrate and form the outer nuclear layer (ONL). In the current study, we demonstrated that a KASH domain-containing protein, Syne-2/Nesprin-2, as well as SUN domain-containing proteins, SUN1 and SUN2, play critical roles during INM and photoreceptor cell migration in the mouse retina. A deletion mutation of Syne-2/Nesprin-2 or double mutations of Sun1 and Sun2 caused severe reduction of the thickness of the ONL, mislocalization of photoreceptor nuclei and profound electrophysiological dysfunction of the retina characterized by a reduction of a- and b-wave amplitudes. We also provide evidence that Syne-2/Nesprin-2 forms complexes with either SUN1 or SUN2 at the nuclear envelope to connect the nucleus with dynein/dynactin and kinesin molecular motors during the nuclear migrations in the retina. These key retinal developmental signaling results will advance our understanding of the mechanism of nuclear migration in the mammalian retina.
doi:10.1093/hmg/ddq549
PMCID: PMC3043658  PMID: 21177258
13.  Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1. 
Molecular Biology of the Cell  1997;8(1):171-187.
Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.
Images
PMCID: PMC276068  PMID: 9017604
14.  LINC Complexes Form by Binding of Three KASH Peptides to the Interfaces of Trimeric SUN proteins 
Cell  2012;149(5):1035-1047.
SUMMARY
Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled-coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.
doi:10.1016/j.cell.2012.03.046
PMCID: PMC3383001  PMID: 22632968
nuclear envelope; SUN; Nesprin; KASH; nuclear migration
15.  The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration 
Molecular Biology of the Cell  2014;25(18):2853-2865.
The nucleoplasmic domain of the Caenorhabditis elegans SUN protein UNC-84 interacts with lamin. If this interaction is disrupted, a partial failure in nuclear migration occurs.
Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect—live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus.
doi:10.1091/mbc.E14-05-0971
PMCID: PMC4161519  PMID: 25057012
16.  Chromosome Movements Promoted by the Mitochondrial Protein SPD-3 Are Required for Homology Search during Caenorhabditis elegans Meiosis 
PLoS Genetics  2013;9(5):e1003497.
Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC). These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.
Author Summary
Sexually reproducing organisms carry two copies of each chromosome (homologs), which must be separated during gamete formation to prevent chromosome duplication in each generation. This chromosome halving is achieved during meiosis, a type of cell division in which the homologs recognize and pair with one another before they become intimately glued together by a structure called the synaptonemal complex (SC). Homolog pairing and SC assembly coincide with movement of chromosomes inside the nucleus, but how chromosome mobility impacts these events is not understood. We find that the mitochondrial protein SPD-3 is required to ensure normal levels of motor-driven chromosome movement and that, although pairing-promoting proteins are normally recruited at the start of meiosis in spd-3 mutants, reduced chromosome mobility impairs homolog pairing. In contrast, SC assembly is normally started, leading to the installation of SC between non-homologous chromosomes and demonstrating a failure in the coordination of pairing and SC assembly. Reduced movement also causes a controlled delay in exit from early meiotic stages characterized by chromosome clustering and active homology search. Our findings show how the different events that lead to the correct association of homologous chromosomes during early meiosis are affected by chromosome mobility.
doi:10.1371/journal.pgen.1003497
PMCID: PMC3649994  PMID: 23671424
17.  The LINC complex is essential for hearing 
Hereditary hearing loss is the most common sensory deficit. We determined that progressive high-frequency hearing loss in 2 families of Iraqi Jewish ancestry was due to homozygosity for the protein truncating mutation SYNE4 c.228delAT. SYNE4, a gene not previously associated with hearing loss, encodes nesprin-4 (NESP4), an outer nuclear membrane (ONM) protein expressed in the hair cells of the inner ear. The truncated NESP4 encoded by the families’ mutation did not localize to the ONM. NESP4 and SUN domain–containing protein 1 (SUN1), which localizes to the inner nuclear membrane (INM), are part of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the nuclear envelope. Mice lacking either Nesp4 or Sun1 were evaluated for hair cell defects and hearing loss. In both Nesp4–/– and Sun1–/– mice, OHCs formed normally, but degenerated as hearing matured, leading to progressive hearing loss. The nuclei of OHCs from mutant mice failed to maintain their basal localization, potentially affecting cell motility and hence the response to sound. These results demonstrate that the LINC complex is essential for viability and normal morphology of OHCs and suggest that the position of the nucleus in sensory epithelial cells is critical for maintenance of normal hearing.
doi:10.1172/JCI66911
PMCID: PMC3561815  PMID: 23348741
18.  Coupling of the nucleus and cytoplasm 
The Journal of Cell Biology  2006;172(1):41-53.
The nuclear envelope defines the barrier between the nucleus and cytoplasm and features inner and outer membranes separated by a perinuclear space (PNS). The inner nuclear membrane contains specific integral proteins that include Sun1 and Sun2. Although the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum, it is nevertheless enriched in several integral membrane proteins, including nesprin 2 Giant (nesp2G), an 800-kD protein featuring an NH2-terminal actin-binding domain. A recent study (Padmakumar, V.C., T. Libotte, W. Lu, H. Zaim, S. Abraham, A.A. Noegel, J. Gotzmann, R. Foisner, and I. Karakesisoglou. 2005. J. Cell Sci. 118:3419–3430) has shown that localization of nesp2G to the ONM is dependent upon an interaction with Sun1. In this study, we confirm and extend these results by demonstrating that both Sun1 and Sun2 contribute to nesp2G localization. Codepletion of both of these proteins in HeLa cells leads to the loss of ONM-associated nesp2G, as does overexpression of the Sun1 lumenal domain. Both treatments result in the expansion of the PNS. These data, together with those of Padmakumar et al. (2005), support a model in which Sun proteins tether nesprins in the ONM via interactions spanning the PNS. In this way, Sun proteins and nesprins form a complex that links the nucleoskeleton and cytoskeleton (the LINC complex).
doi:10.1083/jcb.200509124
PMCID: PMC2063530  PMID: 16380439
19.  The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis 
PLoS Genetics  2011;7(11):e1002365.
The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.
Author Summary
Accurate segregation of chromosomes during mitosis is essential to prevent genetic instability and aneuploidy that lead to cancer and other diseases. Centrosomes and spindle pole bodies mediate the assembly of a microtubule-based structure known as the mitotic spindle, which physically separates chromosomes during mitosis so that the two daughter cells contain a complete copy of the genetic material as well as a spindle pole. During every cell cycle, the DNA and the spindle pole must be duplicated exactly once to ensure proper formation of a bipolar mitotic spindle. In yeast cells, the nuclear envelope does not break down, so the spindle pole must be inserted into the nuclear membrane so that it can form both the microtubules involved in the mitotic spindle and those involved in positioning of the nucleus. How a large protein complex such as the spindle pole body is inserted into the lipid layers of the nuclear membrane is not well understood. We show that the evolutionarily conserved SUN protein Mps3 is involved in spindle pole insertion into the nuclear membrane. This likely reflects a function for SUN proteins in controlling nuclear envelope structure by modulating the types of lipids that are present in the nuclear membrane.
doi:10.1371/journal.pgen.1002365
PMCID: PMC3219597  PMID: 22125491
20.  Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice 
Human Molecular Genetics  2008;18(4):607-620.
Mutations in the gene encoding the inner nuclear membrane proteins lamins A and C produce cardiac and skeletal muscle dysfunction referred to as Emery Dreifuss muscular dystrophy. Lamins A and C participate in the LINC complex that, along with the nesprin and SUN proteins, LInk the Nucleoskeleton with the Cytoskeleton. Nesprins 1 and 2 are giant spectrin-repeat containing proteins that have large and small forms. The nesprins contain a transmembrane anchor that tethers to the nuclear membrane followed by a short domain that resides within the lumen between the inner and outer nuclear membrane. Nesprin’s luminal domain binds directly to SUN proteins. We generated mice where the C-terminus of nesprin-1 was deleted. This strategy produced a protein lacking the transmembrane and luminal domains that together are referred to as the KASH domain. Mice homozygous for this mutation exhibit lethality with approximately half dying at or near birth from respiratory failure. Surviving mice display hindlimb weakness and an abnormal gait. With increasing age, kyphoscoliosis, muscle pathology and cardiac conduction defects develop. The protein components of the LINC complex, including mutant nesprin-1α, lamin A/C and SUN2, are localized at the nuclear membrane in this model. However, the LINC components do not normally associate since coimmunoprecipitation experiments with SUN2 and nesprin reveal that mutant nesprin-1 protein no longer interacts with SUN2. These findings demonstrate the role of the LINC complex, and nesprin-1, in neuromuscular and cardiac disease.
doi:10.1093/hmg/ddn386
PMCID: PMC2722216  PMID: 19008300
21.  Protein interactions at the higher plant nuclear envelope: evidence for a linker of nucleoskeleton and cytoskeleton complex 
Following the description of SAD1/UNC84 (SUN) domain proteins in higher plants, evidence has rapidly increased that plants contain a functional linker of nucleoskeleton and cytoskeleton (LINC) complex bridging the nuclear envelope (NE). While the SUN domain proteins appear to be highly conserved across kingdoms, other elements of the complex are not and some key components and interactions remain to be identified. This mini review examines components of the LINC complex, including proteins of the SUN domain family and recently identified plant Klarsicht/Anc/Syne-1 homology (KASH) domain proteins. First of these to be described were WIPs (WPP domain interacting proteins), which act as protein anchors in the outer NE. The plant KASH homologs are C-terminally anchored membrane proteins with the extreme C-terminus located in the nuclear periplasm; AtWIPs contain a highly conserved X-VPT motif at the C-terminus in contrast to PPPX in opisthokonts. The role of the LINC complex in organisms with a cell wall, and description of further LINC complex components will be considered, together with other potential plant-specific functions.
doi:10.3389/fpls.2014.00183
PMCID: PMC4019843  PMID: 24847341
nuclear envelope; SUN domain proteins; KASH domain proteins; nuclear structure; LINC complex
22.  SUN1 Interacts with Nuclear Lamin A and Cytoplasmic Nesprins To Provide a Physical Connection between the Nuclear Lamina and the Cytoskeleton 
Molecular and Cellular Biology  2006;26(10):3738-3751.
Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.
doi:10.1128/MCB.26.10.3738-3751.2006
PMCID: PMC1488999  PMID: 16648470
23.  Expression of individual mammalian Sun1 isoforms depends on the cell type 
Mammalian Sun1 belongs to an evolutionarily conserved family of inner nuclear membrane proteins, which are known as SUN domain proteins. SUN domain proteins interact with KASH domain partners to form bridging complexes, so-called LINC complexes, that physically connect the nuclear interior to the cytoskeleton. LINC complexes are critical for nuclear integrity and play fundamental roles in nuclear positioning, shaping and movement. The mammalian genome codes for at least five different SUN domain proteins used for the formation of a number of different LINC complexes. Recently, we reported on the identification of several Sun1 isoforms, which tremendously enlarges the alternatives to form functional LINC complexes. We now confirmed that Sun1 actually exists in at least seven distinct splice variants. Besides that, we observed that expression of individual Sun1 isoforms remarkably depends on the cell type, suggesting a cell type-specific adaption of Sun1 dependent LINC complexes to specific cellular and physiological requirements.
doi:10.4161/cib.4.4.15369
PMCID: PMC3181515  PMID: 21966565
Sun1; SUN domain protein; LINC complex; mouse; nuclear envelope; isoform
24.  Requirement for Candida albicans Sun41 in Biofilm Formation and Virulence▿  
Eukaryotic Cell  2007;6(11):2046-2055.
The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41, Orf19.5412, Orf19.1277, MSB2, Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41Δ/sun41Δ deletion mutants and sun41Δ/sun41Δ+pSUN41-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41Δ/sun41Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41Δ/sun41Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.
doi:10.1128/EC.00314-07
PMCID: PMC2168420  PMID: 17873081
25.  Genetic Analysis of Mps3 SUN Domain Mutants in Saccharomyces cerevisiae Reveals an Interaction with the SUN-Like Protein Slp1 
G3: Genes|Genomes|Genetics  2012;2(12):1703-1718.
In virtually all eukaryotic cells, protein bridges formed by the conserved inner nuclear membrane SUN (for Sad1-UNC-84) domain-containing proteins and their outer nuclear membrane binding partners span the nuclear envelope (NE) to connect the nucleoplasm and cytoplasm. These linkages are important for chromosome movements within the nucleus during meiotic prophase and are essential for nuclear migration and centrosome attachment to the NE. In Saccharomyces cerevisiae, MPS3 encodes the sole SUN protein. Deletion of MPS3 or the conserved SUN domain is lethal in three different genetic backgrounds. Mutations in the SUN domain result in defects in duplication of the spindle pole body, the yeast centrosome-equivalent organelle. A genome-wide screen for mutants that exhibited synthetic fitness defects in combination with mps3 SUN domain mutants yielded a large number of hits in components of the spindle apparatus and the spindle checkpoint. Mutants in lipid metabolic processes and membrane organization also exacerbated the growth defects of mps3 SUN domain mutants, pointing to a role for Mps3 in nuclear membrane organization. Deletion of SLP1 or YER140W/EMP65 (for ER membrane protein of 65 kDa) aggravated growth of mps3 SUN domain mutants. Slp1 and Emp65 form an ER-membrane associated protein complex that is not required directly for spindle pole body duplication or spindle assembly. Rather, Slp1 is involved in Mps3 localization to the NE.
doi:10.1534/g3.112.004614
PMCID: PMC3516490  PMID: 23275891
Mps3; SUN protein; spindle pole body duplication; nuclear/ER membrane; SGA

Results 1-25 (983086)