SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton

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.     

Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane

Approximately 100 proteins are targeted to the inner nuclear membrane (INM), where they regulate chromatin and nuclear dynamics. The mechanisms underlying trafficking to the INM are poorly understood. The Caenorhabditis elegans SUN protein UNC-84 is an excellent model to investigate such mechanisms. UNC-84 recruits KASH proteins to the outer nuclear membrane to bridge the nuclear envelope (NE), mediating nuclear positioning. UNC-84 has four targeting sequences: two classical nuclear localization signals, an INM sorting motif, and a signal conserved in mammalian Sun1, the SUN--nuclear envelope localization signal. Mutations in some signals disrupt the timing of UNC-84 nuclear envelope localization, showing that diffusion is not sufficient to move all UNC-84 to the NE. Thus targeting UNC-84 requires an initial step that actively transports UNC-84 from the peripheral endoplasmic reticulum to the NE. Only when all four signals are simultaneously disrupted does UNC-84 completely fail to localize and to function in nuclear migration, meaning that at least three signals function, in part, redundantly to ensure proper targeting of UNC-84. Multiple mechanisms might also be used to target other proteins to the INM, thereby ensuring their proper and timely localization for essential cellular and developmental functions.     

UNC-83 IS a KASH protein required for nuclear migration and is recruited to the outer nuclear membrane by a physical interaction with the SUN protein UNC-84

UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane.     

Novel plant SUN-KASH bridges are involved in RanGAP anchoring and nuclear shape determination

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.     

Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function

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.     

A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis

In yeasts and worms, KASH (Klarsicht/ANC-1/Syne/homology) domain and SUN (Sad-1/UNC-84) domain nuclear envelope (NE) proteins play a crucial role in meiotic chromosome movement and homologue pairing. However, although the vertebrate SUN domain protein SUN1 is involved in these processes, its partner has remained identified. Based on subcellular localization screening in mouse spermatocytes, we identified a novel germ cell-specific protein, KASH5, that localized exclusively at telomeres from the leptotene to diplotene stages in both spermatocytes and oocytes. KASH5 possesses hitherto unknown KASH-related sequences that directly interacted with SUN1 and mediated telomere localization. Thus, KASH5 is a mammalian meiosis-specific KASH domain protein. We show that meiotic chromosome movement depended on microtubules and that KASH5 interacted with the microtubule-associated dynein-dynactin complex. These results suggest that KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force-generating mechanism involved in meiotic chromosome movement. Our study strongly suggests that the meiotic homologue-pairing mechanism mediated by the SUN-KASH NE bridge is highly conserved among eukaryotes.     

Sun2 is a novel mammalian inner nuclear membrane protein

Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a sub-population present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein. Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.     

Characterization of SUN-domain proteins at the higher plant nuclear envelope

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.     

Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans

Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase, similar to known inner nuclear membrane proteins. UNC-84 protein is first detected at the 26-cell stage and thereafter is present in most cells during development and in adults. UNC-84 is properly expressed in unc-83 and anc-1 lines, which have phenotypes similar to unc-84, suggesting that neither the expression nor nuclear envelope localization of UNC-84 depends on UNC-83 or ANC-1 proteins. The envelope localization of Ce-lamin, Ce-emerin, Ce-MAN1, and nucleoporins are unaffected by the loss of UNC-84. UNC-84 is not required for centrosome attachment to the nucleus because centrosomes are localized normally in unc-84 hyp7 cells despite a nuclear migration defect. Models for UNC-84 localization are discussed.     

Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants

Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain–interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions.     

Plant SUN domain proteins: Components of putative plant LINC complexes?

We have recently reported the identification and characterization of Sad1/UNC84 (SUN) domain proteins in various plant species. In animals and yeasts, SUN domain proteins are localized at the inner nuclear membrane and form a bridge across the nuclear envelope (NE) by interacting with outer nuclear membrane-localized Klarsicht/Anc-1/Syne-1 homology (KASH) domain proteins. This bridge physically connects cytoskeletal elements with chromatin and nucleoskeletal components. These multiprotein complexes are essential for various cellular and nuclear processes. The identification of SUN domain proteins provides the first evidence of putative NE bridging complexes in plants. Here we speculate on the composition and functions of these in regards to our current understanding of plant SUN domain proteins.Key words: SUN domain protein, LINC complex, plant nuclear envelope, cytoskeleton, KASH domain proteins, Arabidopsis     

Prelamin A-mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle

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.     

Subcellular localization of SUN2 is regulated by lamin A and Rab5

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.     

TRIM-9 functions in the UNC-6/UNC-40 pathway to regulate ventral guidance

TRIpartite Motif (TRIM) family proteins are ring finger domain-containing, multi-domain proteins implicated in many biological processes.Members of the TRIM-9/C-I subfamily of TRIM proteins, including TRIM-9, MIDI and MID2, have neuronal functions and are associated with neurological diseases. To explore whether the functions of C-I TRIM proteins are conserved in invertebrates, we analyzed Caenorhabditis elegans and Drosophila trim-9 mutants. C. elegans trim-9 mutants exhibit defects in the ventral guidance of hermaphrodite specific neuron (HSN) and the touch neuron AVM. Further genetic analyses indicate that TRIM-9 participates in the UNC-6-UNC-40 attraction pathway.Asymmetric distribution of UNC-40 during HSN development is normal in trim-9 mutants. However, the asymmetric localization of MIG-10,a downstream effector of UNC-40, is abolished in trim-9 mutants. These results suggest that TRIM-9 functions upstream of MIG-1 0 in the UNC40 pathway. Moreover, we showed that TRIM-9 exhibits E3 ubiquitin ligase activity in vitro and this activity is important for TRIM-9 function in vivo. Additionally, we found that Drosophila trim-9 is required for the midline attraction of a group of sensory neuron axons. Over-expression of the Netrin/UNC-6 receptor Frazzled suppresses the guidance defects in trim-9 mutants. Our study reveals an evolutionarily conserved function of TRIM-9 in the UNC-40/Frazzled-mediated UNC-6/Netrin attraction pathway.     

Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes

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.     

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane

Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N‐terminal, nucleoplasmic domain of SUN2. A second motif proximal to the cNLS is a cluster of arginines that serves coatomer‐mediated retrieval of SUN2 from the Golgi. Unexpectedly, also the C‐terminal, lumenal SUN domain of SUN2 supports NE localization, showing that targeting elements are not limited to cytoplasmic or transmembrane domains of INM proteins. Together, SUN2 represents the first mammalian INM protein relying on a functional cNLS, a Golgi retrieval signal and a perinuclear domain to mediate targeting to the INM.     

Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.     

unc-119 homolog required for normal development of the zebrafish nervous system

The UNC-119 proteins, found in all metazoans examined, are highly conserved at both the sequence and functional levels. In the invertebrates Caenorhabditis elegans and Drosophila melanogaster, unc-119 genes are expressed pan-neurally. Loss of function of the unc-119 gene in C. elegans results in a disorganized neural architecture and paralysis. The function of UNC-119 proteins has been conserved throughout evolution, as transgenic expression of the human UNC119 gene in C. elegans unc-119 mutants restores a wild-type phenotype. However, the nature of the conserved molecular function of UNC-119 proteins is poorly understood. Although unc-119 genes are expressed throughout the nervous system of the worm and fly, the analysis of these genes in vertebrates has focused on their function in the photoreceptor cells of the retina. Here we report the characterization of an unc-119 homolog in the zebrafish. The Unc119 protein is expressed in various neural tissues in the developing zebrafish embryo and larva. Morpholino oligonucleotide (MO)-mediated knockdown of Unc119 protein results in a "curly tail down" phenotype. Examination of neural patterning demonstrates that these "curly tail down" zebrafish experience a constellation of neuronal defects similar to those seen in C. elegans unc-119 mutants: missing or misplaced cell bodies, process defasciculation, axon pathfinding errors, and aberrant axonal branching. These findings suggest that UNC-119 proteins may play an important role in the development and/or function of the vertebrate nervous system.     

Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)-GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.