SUN-Domain Proteins 4572–4573 (2006)

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51. Rudd, P. M. & Dwek, R. A. Glycosylation: heterogeneity 63. Wang, L., Xie, J., Deniz, A. A. & Schultz, P. G. Unnatural protein complexes support a broad range of and the 3D structure of proteins. Crit. Rev. Biochem. amino acid mutagenesis of green fluorescent protein. 4 Mol. Biol. 32, 1–100 (1997). J. Org. Chem. 68, 174–176 (2003). functions (BOX 1). At least 80 unique integral 52. Zhang, Z. et al. A new strategy for the synthesis of 64. Wang, L., Brock, A. & Schultz, P. G. Adding L-3-(2- membrane proteins were found to localize glycoproteins. Science 303, 371–373 (2004). naphthyl)alanine to the genetic code of E. coli. J. Am. 5 53. Xu, R. et al. Site-specific incorporation of the mucin- Chem. Soc. 124, 1836–1837 (2002). at the nuclear envelope in mammalian cells . type N-acetylgalactosamine-α-O-threonine into protein 65. Turner, J. M., Graziano, J., Spraggon, G. & Schultz, P. G. It is assumed that most of these proteins in Escherichia coli. J. Am. Chem. Soc. 126, Structural characterization of a p-acetylphenylalanyl 4 15654–15655 (2004). aminoacyl-tRNA synthetase. J. Am. Chem. Soc. 127, interact directly or indirectly with lamins . 54. Lu, W., Gong, D., Bar-Sagi, D. & Cole, P. A. Site-specific 14976–14977 (2005). Among these nuclear membrane proteins are incorporation of a phosphotyrosine mimetic reveals a 66. Turner, J. M., Graziano, J., Spraggon, G. & Schultz, P. G. role for tyrosine phosphorylation of SHP-2 in cell Structural plasticity of an aminoacyl-tRNA synthetase three families, each of which is characterized signaling. Mol. Cell 8, 759–769 (2001). active site. Proc. Natl Acad. Sci. USA 103, 6483–6488 by a distinct motif (specifically, LEM, SUN or 55. Xie, J. Adding Unnatural Amino Acids to the Genetic (2006). Repertoire. Thesis, Scripps Research Institute, La Jolla 67. Tian, F., Tsao, M. L. & Schultz, P. G. A phage display KASH). LEM-domain proteins (named after (2006). system with unnatural amino acids. J. Am. Chem. Soc. LAP2, emerin and MAN1) are reviewed else- 56. Wang, J., Xie, J. & Schultz, P. G. A genetically encoded 126, 15962–15963 (2004). 4 fluorescent amino acid. J. Am. Chem. Soc. 128, 68. Castelli, D. D., Lovera, E., Ascenzi, P. & Fasano, M. where . We will discuss proteins that contain 8738–8739 (2006). Unfolding of the loggerhead sea turtle (Caretta caretta) the SUN domain and their partners, many 57. Summerer, D. et al. A genetically encoded fluorescent myoglobin: a 1H-NMR and electronic absorbance study. amino acid. Proc. Natl Acad. Sci. USA 103, Protein Sci. 11, 2273–2278 (2002). of which contain the KASH (named after 9785–9789 (2006). Klarsicht, ANC-1 and SYNE1 homology) 58. Tsao, M. L., Summerer, D., Ryu, Y. & Schultz, P. G. The Acknowledgements 6–8 genetic incorporation of a distance probe into proteins Work in the laboratory of P.G.S. is supported by the National domain . in Escherichia coli. J. Am. Chem. Soc. 128, Institutes of Health, the United States Department of Energy Most (but not all) SUN-domain proteins 4572–4573 (2006). and the Skaggs Institute for Chemical Biology. 59. Xie, J. et al. The site-specific incorporation of p-iodo-L- are localized at the INM. The situation is phenylalanine into proteins for structure determination. Competing interests statement more complicated for KASH-domain pro- Nature Biotechnol. 22, 1297–1301 (2004). The authors declare no competing financial interests. 60. Deiters, A., Geierstanger, B. H. & Schultz, P. G. Site- teins, most individual isoforms of which are 8,9 specific in vivo labeling of proteins for NMR studies. DATABASES localized on either the ONM or the INM . Chembiochem 6, 55–58 (2005). The following terms in this article are linked online to: 61. Suydam, I. T. & Boxer, S. G. Vibrational Stark effects However, some isoforms lack the KASH UniProtKB: http://ca.expasy.org/sprot calibrate the sensitivity of vibrational probes for electric ClpB | Gal4 | GRB2 | LamB | STAT1 domain, do not localize to the nuclear fields in proteins. Biochemistry 42, 12050–12055 (2003). FURTHER INFORMATION envelope and are instead proposed to 62. Alfonta, L., Zhang, Z., Uryu, S., Loo, J. A. & Schultz, P. G. Peter G. Schultz’s homepage: tether other organelles to the cytoskeleton8. Site-specific incorporation of a redox-active amino acid http://schultz.scripps.edu Several INM-localized SUN-domain and into proteins. J. Am. Chem. Soc. 125, 14662–14663 Access to this links box is available online. (2003). KASH-domain proteins can interact with lamins, and in certain cases this interaction is required for their nuclear envelope localiz ation (see below). OPINION This perspective will focus primarily on SUN-domain proteins: we will depict their structural organization (known and hypo- SUN-domain proteins: ‘Velcro’ that thetical) in complexes that traverse the nuclear envelope, and discuss their roles links the nucleoskeleton to the in nuclear positioning, centrosome attachment to the ONM, links to the cytoskeleton, cytoskeleton and telomere positioning during meiosis. We will propose a model in which SUN- domain proteins serve as mechanical ‘Velcro’ Yonatan B. Tzur, Katherine L. Wilson and Yosef Gruenbaum to interconnect the cytoskeleton and nucle- Abstract | The novel SUN-domain family of nuclear envelope proteins interacts with oskeleton, and suggest that SUN-domain proteins have further, non-mechanical roles various KASH-domain partners to form SUN-domain-dependent ‘bridges’ across as specialized nuclear envelope receptors. the inner and outer nuclear membranes. These bridges physically connect the nucleus to every major component of the cytoskeleton. SUN-domain proteins have SUN-domain and KASH-domain proteins diverse roles in nuclear positioning, centrosome localization, germ-cell Both the SUN domain and regions that are development, telomere positioning and apoptosis. By serving both as mechanical upstream of the SUN domain interact directly with the KASH domain of KASH-domain adaptors and nuclear envelope receptors, we propose that SUN-domain proteins proteins10,11. This interaction is required for connect cytoplasmic and nucleoplasmic activities. the cellular functions of both types of protein.

For a discussion of the SUN-domain (Sad1 pore complexes, which control the traffic of Domain organization of SUN-domain and UNC-84 homology domain) family macromolecules between the nucleoplasm proteins. Malone and colleagues12 coined the of proteins, we must first introduce the and the cytoplasm. In metazoans, the nuclear term SUN domain when they discovered a nuclear envelope. In all eukaryotic cells, pore complexes and the INM are anchored motif of ~120 residues in the C terminus of the nuclear envelope separates the nucleus to a structural network of lamin filaments1 the Caenorhabditis elegans UNC-84 protein. from the cytoplasm. The nuclear envelope (BOX 1). Lamin polymers confer mechanical This protein has significant homology to a is composed of an outer nuclear membrane strength to the nucleus2,3. Many nuclear region in the Schizosaccharomyces pombe (ONM) and an inner nuclear membrane membrane and nucleoplasmic proteins Sad1 protein and several uncharacterized (INM). The two membranes join at nuclear interact with lamins4, and lamin-associated mammalian proteins. Genome-database

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searches indicated that UNC-84 is conserved Box 1 | The lamins and that the number of SUN-domain proteins has increased over the course of Lamins are type-V intermediate filament proteins. They are the main structural constituents of the metazoan nuclear lamina, a protein meshwork that lies between the inner nuclear membrane and evolution (FIG. 1). For example, the S. pombe chromatin. Lamins are also present in the nucleoplasm1,48. Lamins are divided into A and B types on genome contains a single SUN-domain the basis of their expression patterns and protein structure. B-type lamins are essential proteins gene, whereas C. elegans and Drosophila that are encoded by two genes in mammals (LMNB1 and LMNB2) and are present in all metazoan melanogaster each have two, and mammals cells. By contrast, A-type lamins (for example, lamin A and C) are derived by alternative splicing of a have at least four SUN-domain genes, SUN1, single gene (LMNA in human) that is found only in more complex metazoans; this gene is expressed SUN2, SUN3 and SPAG4 (REF. 8). mainly in differentiated cells and is not essential for cell viability49. B-type lamins and lamin A (but Besides the SUN domain, members of not lamin C) are translated as precursors with a C-terminal CAAX motif. The cysteine residue in the this family share other structural features CAAX motif is farnesylated, the last three residues (AAX) are subsequently removed by a zinc- to varying extents (FIG. 2). Most SUN- metalloprotease and the cysteine is then methyl-esterified. To produce mature lamin A, the last 15 domain proteins have at least one predicted residues are then cleaved by the zinc-metalloprotease Zmste24 (REF. 50). 2,3 transmembrane domain. Several span Lamin polymers confer mechanical strength to the nucleus . Lamins also interact with many different nuclear proteins51 and are involved in chromatin organization, DNA replication, the membrane multiple times, which is a transcription, assembly and disassembly of the nuclear envelope, spacing of nuclear pore potentially useful property for proteins that complexes and apoptosis52. Mutations in the human LMNA gene affect muscle, adipose, bone, are proposed to anchor mechanical-load- nerve and skin cells and are responsible for at least 12 heritable diseases, collectively termed bearing structures and transmit mechanical laminopathies, that include muscular dystrophies and premature ageing49,53,54. force. Human SUN1 spans the membrane three times13. SUN1 has an additional hydrophobic region that is conserved in SUN2, but is not membrane inserted in either protein; Enaptin), Nesprin-2 (also known as syne-2 cells, most tested endogenous SUN-domain the function of this hydrophobic region and NUANCE) and Nesprin-3 genes in mam- proteins localize at the nuclear envelope is unknown13,20. Caenorhabditis elegans mals8. Many KASH-domain proteins are during interphase (see below). Their exact UNC-84, which is localized at the INM, is enormous (>1 MDa)9. They are also diverse; topology within the envelope is the focus of predicted to have as many as nine hydro- for example, the Nesprin-1 and Nesprin-2 much interest, as therein lies the key to their phobic domains (FIG. 2), but how many actu- genes each produce more than 12 protein proposed nuclear envelope ‘bridging’ activi- ally span the INM is unknown. Strangely, isoforms (of which some are small or lack ties14,17. Immunogold electron microscopy both of the SUN-domain proteins in the KASH domain) through alternative (EM) analysis of the C. elegans matefin D. melanogaster, which are uncharacterized, transcription initiation and transcription (also known as SUN-1) protein localized its seem to lack transmembrane domains. Last, termination, or alternative pre-mRNA N terminus to the INM, but its exact topol- most SUN-domain proteins have at least one splicing9. Nesprin proteins also have numer- ogy was not determined18. The N-terminal coiled-coil domain, which in human SUN1 ous spectrin-repeat domains, which are domain of mouse and human SUN1 is is proposed to mediate homodimerization13. thought to confer an extended configuration; sufficient to target SUN1 to the nuclear There is also the unexplored possibility that the largest isoforms at the nuclear envelope envelope11,15,19 and also confers direct bind- SUN-domain proteins might form hetero- are estimated to extend up to 500 nm ing to A-type lamins13,19, which hints at a dimers, as depicted speculatively in FIG. 3. into the cytoplasm8. Nesprins and other potential localization mechanism. However, The dimerization idea is important, KASH-domain proteins have recently been SUN1 localization seems to be lamin A because each SUN-domain dimer could reviewed in detail8,9. With a few exceptions, independent in vivo11,13,15,19 (see below). The directly anchor two KASH-domain part- our discussion is limited to Nesprin isoforms N-terminal domain of human SUN2 also ners and thereby significantly enhance for which SUN-domain interactions have binds A-type lamins13, and its C-terminal the mechanical stability of protein com- been characterized. SUN domain has been localized to the lume- plexes that bridge the nuclear envelope. nal space between the INM and the ONM in Furthermore, assuming one-to-one binding Subcellular localization human HeLa cells20. These findings indicate of the SUN and KASH domains, SUN Current evidence indicates that INM- that SUN1 and SUN2 have similar topo- dimers could interact with two KASH positioned SUN-domain proteins interact logies, with their N-terminal domains in the proteins in the same membrane (for exam- with ONM-positioned KASH-domain nucleoplasm and their SUN domains in ple, the ONM), or on opposite membranes proteins, and in this manner create protein the lumen of the nuclear envelope (FIG. 3). (the ONM and the INM). ‘bridges’ that span both nuclear mem- Whether this topology applies to other branes8,14. Other SUN-domain and KASH- SUN-domain proteins is not known. Domain organization of KASH-domain domain proteins might be associated with All studied metazoan SUN-domain pro- proteins. Caenorhabditis elegans encode other organelles8,13,15. So, a precise knowledge teins colocalize with lamins, as determined three known KASH-domain proteins of the localization and topology for each by indirect immunofluorescence11,13,14,18,20. (ANC-1, UNC-83 and ZYG-12), all of which protein is needed to understand their roles. Whereas SUN1 and SUN2 can bind lamins have a C-terminal transmembrane domain directly13,19, others cannot (for example, followed by the ~35-residue KASH domain. SUN-domain proteins. The endogenous C. elegans UNC-84 (REF. 14)). Paradoxically, The KASH domain also is present in several S. pombe Sad1 protein localizes in vivo the second C. elegans SUN-domain protein, vertebrate and invertebrate proteins, includ- to the spindle pole body (SPB; the yeast matefin/SUN-1, can bind Ce-lamin ing those encoded by MSP300 and Klarsicht microtubule-organizing centre)16. However, (a B-type lamin) directly in vitro but does in D. melanogaster and the Nesprin-1 when ectopically expressed, Sad1 also local- not seem to depend on Ce-lamin for its (also known as CPG2, syne-1, myne-1 and izes at the nuclear envelope16. In metazoan nuclear envelope localization in vivo18.

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Hs SUN2 are downregulated for both A-type lamins ONM in an UNC-84-dependent manner, 97 and lamin B1 (REF. 15), but this might be because mutations in UNC-84 displaced 26 41 Hs SUN1 explained by the incomplete knockdown of ANC-1 from the ONM . Likewise, the lamin expression, compensatory binding to localization of the Nesprin-2 giant isoform Dm Q9V996 lamin B2 encoded by a different gene (BOX 1), at the nuclear envelope requires both human or a putative lamin-independent retention SUN1 and SUN2 (REFS 11,13). The localiza- 57 Hs SPAG4 mechanism. SUN2 also remained localized in tion of Nesprin-2 giant and Klarsicht at HeLa cells that are downregulated for A-type the ONM also depends, at least indirectly, 63 Hs SUN3 lamins. However, it was frequently lost from on A-type lamins, as these KASH-domain the nuclear envelope of Lmna-knockout proteins each mislocalize in cells in which 90 13 13,22,27,28 Sp Sad1 fibroblasts . SUN2 was also identified as a A-type lamins are downregulated . predicted stable component of the nuclear The KASH domain is both essential to target Ce UNC-84 lamina network in a large-scale proteomic large KASH-domain proteins to the nuclear analysis of mammalian nuclear envelope envelope, and sufficient to localize to the 5 10,21 Dm Q9VKG2 proteins , and biochemical extraction experi- nuclear envelope when expressed alone . ments show that SUN2 is not extracted by The Nesprin-1α isoform localizes to the 29 Ce mateﬁn/SUN-1 detergent or 8 M urea alone, but can be INM . Its nucleoplasmic domain binds 0.05 extracted by detergent and 8 M urea. These lamin A in vitro, and its nuclear envelope Figure 1 | Phylogenetic relationships among results are consistent with binding to lamins localization requires A-type lamins30. SUN-domain proteins. Phylogenetic analysis but do not rule out other anchoring mecha- Nesprin-1α also has very high (4 nM) affin- of SUN-domain amino-acid sequences from nisms20. The retention mechanisms for SUN- ity for the nuclear membrane protein emerin human (Hs), Drosophila melanogaster (Dm), domain proteins might hold some surprises, in vitro29, the localization of which also Caenorhabditis elegans (Ce) and Schizo- as ‘rescue’ experiments indicate that lamin requires A-type lamins31. This INM- saccharomyces pombe (Sp) shows several statisti- interactions alone are insufficient13, and cells localized KASH-domain protein adds cally significant branching events. Human SUN- domain proteins fall into two subgroups. One might therefore use a novel mechanism(s) to another dimension to the nucleoskeletal side includes the inner-nuclear-membrane-localized retain SUN-domain proteins at the nuclear of nuclear-envelope-bridging complexes, SUN1 and SUN2 (which probably originated from envelope. The human SUN3 (REF. 13) and as it binds the nuclear lamina network both a close common ancestor) and the other more SPAG4 (also known as SUN4)15 proteins, directly (by binding lamin A) and indirectly ancient group includes SPAG4 and SUN3, which when expressed from tagged transgenes, (by binding emerin). However, we do not localize in the endoplasmic reticulum and the did not localize specifically at the nuclear know how Nesprin-1α at the INM connects outer nuclear membrane. Drosophila mela- envelope but instead localized throughout to SUN-domain proteins. Indeed, current nogaster Q9V996 is related to both sets of human the endoplasmic reticulum (ER)13,15. The evidence does not exclude a potential dual proteins and might represent the shared ancestor functions of ER-localized SUN-domain pro- localization of Nesprin-1α also on the ONM. of human SUN-domain proteins. Caenorhabditis teins are unknown. However, we speculate In cardiomyocytes, Nesprin-1α associates elegans UNC-84 and S. pombe Sad1 are significantly closer to the human proteins than to that ER/ONM-localized (the ONM is part with a nuclear-envelope-localized signalling D. melanogaster Q9VKG2 or C. elegans matefin/ of the ER network) SUN proteins might also complex that includes both the muscle- SUN-1. This analysis was done by aligning the form nuclear-envelope-bridging complexes, specific A-kinase anchoring protein amino-acid sequences of each SUN domain using in combination with INM-localized (mAKAP) and the ryanodine receptor, ClustalW55 with default alignment parameters. KASH-domain proteins (see below). an ER/ONM-localized Ca2+ channel that Neighbour-joining phylogenetic analysis was is essential for muscle-cell contraction. applied to the conserved core of these align- KASH-domain proteins. Different Nesprin Whether Nesprin-1α is emerin associated, ments (corresponding to residues 675–810 of isoforms localize to different parts of or localizes to the INM versus the ONM, human SUN1) using default parameters of the nuclear envelope9. In immunogold was not determined in cardiomyocytes32. Bootstrap tree analysis (1,000 trials). The tree was EM experiments, the KASH domain of Endogenous Nesprin-2 also requires A-type plotted using ‘njplot’ and processed with Adobe Illustrator 11.0.0. The bootstrap values for 1,000 Nesprin-1 isoforms localized to the nuclear lamins to localize at the nuclear envelope, as trials are shown. Numbers at the branch points membranes, but this analysis did not dis- it mislocalizes in human SW-13 cells, which 23 indicate the percentage bootstrap support criminate between INM-localized versus naturally lack A-type lamins . calculated from 1,000 trees. The scale bar indi- ONM-localized isoforms21. In a similar cates genetic distance and is equivalent to 5% analysis, the KASH domain of Nesprin-2 Linking the nucleus and the cytoskeleton amino-acid diversity. isoforms localized to both the INM and the SUN-domain proteins regulate the posi- ONM22,23, which is consistent with Nesprin-2 tion of the nucleus in the cell10,26,33,34. The isoforms that extend into either the cyto- hypodermis tissue of C. elegans arises from Are lamins sufficient to localize SUN1 plasm or nucleoplasm24. Immunogold local- the fusion of precursor cells to form a at the nuclear envelope? The answer to ization of green fluorescent protein (GFP) multinucleated syncytium. In this syncy- this question has been surprisingly elusive. in keratinocytes that expressed GFP fused tium, nuclei are evenly spaced and remain Mammalian SUN1 remained localized at to the N-terminal domain of Nesprin-3 separated even when the worm is motion- the nuclear envelope in mouse fibroblasts localized this protein to the ONM25. less. Mutations in either UNC-84 or its that were devoid of A-type lamins (Lmna- The localization of KASH-domain binding partner, the KASH-domain protein knockout cells), but this could have been due proteins at the ONM requires their SUN- ANC-1, disrupt nuclear attachment to the to weak interactions with B-type lamins11. domain partners on the INM. For example, actin cytoskeleton, and these untethered SUN1 also remained localized in cells that the KASH domain of ANC-1 localizes to the nuclei move freely in the shared cytoplasm

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until they clump together26. UNC-84 and Subcellular another KASH-domain partner, UNC-83, localization are also required for developmentally Hs SUN1 INM regulated nuclear migrations in hyp7 cells, gut primordial cells and P-cell precursors12. Hs SUN2 INM However, in these cases, the mutations probably disrupt an interaction between the Hs SUN3 ER/ONM nucleus and microtubule-dependent motors, which are required for directed movement Hs SPAG4 ER/ONM of the nucleus. Nuclear migration failure in P-cells eventually leads to P-cell death and Ce UNC-84 INM uncoordinated movement35. So, a single SUN-domain protein in C. elegans, UNC-84, Ce mateﬁn/SUN–1 INM links the nuclear envelope to two different elements of the cytoskeleton. The simple Dm Q9V996 ? explanation for this dual function is that UNC-84 can bind independently to either Dm Q9VKG2 ? of two KASH-domain partners, ANC-1 and UNC-83, that in turn link to actin and Sp Sad1 SPB, NE microtubules, respectively. The other C. elegans SUN-domain pro- Transmembrane Coiled-coil SUN Lamin-binding domain domain domain region tein, matefin/SUN-1, links the nuclear envelope to a third cytoskeletal element — the Figure 2 | Known functional domains in SUN-domain proteins. Each protein and its predicted centrosome or microtubule-organizing domains are depicted to scale. SUN domains and the coiled-coil domains were predicted using the SMART database. The lamin-binding regions in SUN1 and SUN2 (REF. 13) are indicated as well as centre — through ZYG-12, a nuclear- the subcellular localizations11,13,15,16,18–20. Transmembrane domains were predicted using TMPRED envelope-localized KASH-domain protein. with the lowest threshold, which therefore probably includes hydrophobic regions that do not act ZYG-12 also binds centrosomes, and its as transmembrane domains in vivo. The number of actual transmembrane domains and the roles of interaction with matefin/SUN-1 is required non-embedded hydrophobic domains are important areas of current research on SUN-domain to attach centrosomes to the nuclear proteins. Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; ER, endoplasmic reticulum; envelope during embryogenesis36. Matefin/ Hs, human; INM, inner nuclear membrane; NE, nuclear envelope; ONM, outer nuclear membrane; SUN-1 is expressed only in the germ line; Sp, Schizosaccharomyces pombe; SPB, spindle pole body. the gene product is deposited maternally and the protein persists in the nuclear envelope through to late embryogenesis18. interact with all three major elements of the this model would allow one SUN-protein Matefin/SUN-1 is probably not required for cytoskeleton. The SUN-domain partner for dimer to link KASH proteins on opposite centrosome attachment later in development, Nesprin-3 is not yet known. However, based membranes (for example, see Nesprin-1α as centrosomes localize normally in the on the overlapping roles of SUN1 and SUN2 and Nesprin-1 giant in FIG. 3). germ cells of worms that are homozygous for in anchoring Nesprin-2 giant13, we speculate We propose an additional possibility a mtf-1/sun-1 deletion (A. Fridkin and Y.G., that even if SUN-domain proteins have ‘pre- that does not require a flexible hinge: unpublished observations). ferred’ KASH-domain partners, they might INM-localized KASH-domain proteins recognize other KASH-domain proteins. might be anchored by SUN3 or SPAG4, the KASH-domain proteins bind the cytoskeleton. relatively uncharacterized SUN-domain In addition to their KASH and spectrin- Bridging the nuclear envelope proteins that are proposed to localize in repeat domains, large KASH-domain We are still far from understanding SUN- the ER and the ONM13. In this model, proteins also have a functional domain that domain and KASH-domain proteins at the ONM-localized SUN-domain proteins confers direct binding to a specific element molecular level. Crisp et al.13 propose that could serve as ‘reverse anchors’ for INM- of the cytoskeleton. ANC-1, MSP-300, SUN1 and SUN2 are embedded in the INM localized KASH-domain proteins (FIG. 3), in Nesprin-1 giant and Nesprin-2 giant each as homodimers (and possibly as heterodim- addition to their unknown roles in the ER. have two calponin-homology domains at ers), with each dimer binding two KASH- Although highly speculative, these models their N terminus, which bind actin fila- domain proteins. We further propose that are worth considering because they suggest ments in the cytoplasm8 (FIG. 3). Most small SUN-domains might be flexibly hinged, such mechanisms by which SUN-protein dimers isoforms of Nesprin-1 and Nesprin-2 lack that an INM-localized SUN-domain protein might collectively distribute mechanical this domain and presumably do not bind might be free to interact with KASH-domain force bidirectionally at the nuclear enve- actin. Interestingly, Nesprin-3 also lacks proteins that are located either on the oppo- lope via attachments to KASH-domain the actin-binding domain but instead binds site membrane (the ONM) or, potentially, proteins on both the INM and the ONM. plectin, a protein that links cytoplasmic the same membrane (the INM) (see the Also worth considering is the possibility intermediate filaments to filamentous ‘flexible hinge’ in FIG. 3). This possibility that some KASH-domain proteins, such (F)-actin25. Klarsicht, a D. melanogaster provides a mechanism for INM-localized as Nesprin-1α, might have SUN-domain- Nesprin protein, also lacks an actin-binding SUN-domain proteins to anchor INM- independent anchoring mechanisms, for domain, but instead binds microtubules27. localized KASH-domain proteins (for exam- example, through direct binding to lamins So, KASH-domain proteins collectively ple, Nesprin-1α)37. In its most extreme form, and emerin37.

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Actin

NES2g NES2g IF ONM NES2g NES1g INM Plectin NES3 SUN1 SUN1 NES1g SUN1 SUN2

NES3 SUN? ? NES1α ? ? Emerin

Nuclear actin? NPC Nucleoskeleton

Actin ?

UNC-84 ? NES1α dimer

ANC-1 UNC-84 Mateﬁn/ ER/ONM-localized SUN-1 SUN dimer? NPC

Motor UNC-83 protein ZYG-12 + MTOC – Microtubule Figure 3 | Models for mechanical bridging of the nuclear envelope by (NES2g) at the ONM. NES2g and the giant isoform of Nesprin-1 (NES1g) SUN-domain proteins. Schematic depiction (not to scale) of the interac- each bind actin. Nesprin 3 (NES3) binds plectin, which links cytoplasmic tions between SUN-domain proteins (yellow) and KASH-domain proteins intermediate filaments (IFs) to actin. SUN-domain proteins with at least (green) at the inner and outer nuclear membranes (INM and ONM, respec- one predicted coiled-coil domain (see FIG. 2) are depicted as homodimers tively) of the nuclear envelope, and their interactions with specific ele- or heterodimers. This diagram includes two blatant speculations. First, ments of the cytoskeleton, a generic nucleoskeleton network (light blue), relatively uncharacterized endoplasmic reticulum (ER)/ONM-localized and the INM protein emerin (dark blue). Caenorhabditis elegans SUN- SUN proteins (for example, SUN3), might provide ‘reverse anchors’ for domain proteins (UNC-84, matefin/SUN-1) and KASH-domain proteins INM-localized KASH-domain proteins (for example, NES1α). Second (the (ANC-1, UNC-83 and ZYG-12) are depicted on the lower left. UNC-84 can ‘flexible hinge’ model), INM-localized SUN dimers might interact with bind either ANC-1, which binds actin, or UNC-83, which binds microtu- KASH-domain proteins that are located either on the ONM (for example, bules via an unidentified microtubule-dependent motor protein. Matefin/ NES1g) or the INM (for example, NES1α), or on both simultaneously. SUN-1 binds ZYG-12 dimers, which bind the microtubule-organizing The question marks indicate that the actual structure of the nucleo- centre (MTOC). Human proteins SUN1 and SUN2 anchor Nesprin-2 giant skeleton is unknown. NPC, nuclear pore complex.

It is easy to comprehend how SUN- made by KASH-domain proteins embedded probably serve various roles in the nucleus, domain proteins in the INM could link the in the INM. Might these KASH-domain some of which are structural38,39. Actin and nuclear envelope to specific elements of the proteins link to a correspondingly diverse nuclear spectrin are proposed to form an cytoskeleton via their binding to KASH- group of mechanical elements inside the INM cortical network that is anchored by domain proteins in the ONM, given the nucleus, and if so, what are these elements emerin40, the same protein to which both binding of KASH-domain proteins to actin, (besides lamins and emerin)? Nesprin-1α and the intranuclear Nesprin-2 microtubules, plectin and centrosomes. Nesprin-1α and several small Nesprin-2 isoform can bind directly23,29 (FIG. 3). In The picture of nucleoskeleton organization isoforms interact with the nuclear mem- theory, an INM-localized KASH-domain is less clear, and indeed begs the question of brane protein emerin and A-type lamins23,29, protein that can bind actin would be appeal- whether nuclear-envelope-bridging com- whereas other Nesprin-2 isoforms colocalize ingly symmetric if it could link directly to plexes are symmetric in their mechanisms with heterochromatin and have calponin- nucleoskeletal actin, but such an isoform of attachment to the cytoskeleton and to homology domains that presumably remains hypothetical. One can also consider the nucleoskeleton. Given that mechanical bind actin polymers in the nucleus23. potential symmetry in the mechanisms networks are only as strong as their weakest Nuclear actin polymers are conformation- by which INM-localized compared to link, we must consider the contacts that are ally distinct from cytoplasmic F-actin, and ER/ONM-localized SUN-domain proteins

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themselves are anchored. In human cells, So, UNC-84 might have interesting novel Yonatan B. Tzur and Yosef Gruenbaum are at the INM-localized SUN-domain proteins bind partners. How or why a nuclear-envelope- Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, directly to lamins. Perhaps ER/ONM- associated SUN-domain protein influences Jerusalem 91904, Israel. localized ones bind cytoplasmic intermedi- fat levels is unknown. However, we note that Katherine L. Wilson is at the Department of Cell ate filaments. certain mutations in A-type lamins cause Biology, Johns Hopkins University School of Medicine, 47 Why do cells use SUN-domain proteins partial lipodystrophy in humans . 725 N. Wolfe Street, Baltimore, Maryland 21205, rather than nuclear pore complexes to Novel functions are also emerging for USA. mechanically bridge the nuclear envelope? the second C. elegans SUN-domain Correspondence to Y.G. and K.L.W. Pore complexes link the INM and the protein, matefin/SUN1. Most embryos in e-mails: [email protected]; [email protected] ONM, are highly stable and are strongly which matefin/SUN-1 was downregulated doi:10.1038/nrm2003 anchored to the nuclear lamina network41. died at the ~300-cell stage with phenotypes Published online 23 August 2006 Although pore complexes cannot currently that cannot be attributed to centrosome 1. Stuurman, N., Heins, S. & Aebi, U. Nuclear lamins: be excluded as load-bearing elements of detachment18. Worms that are homozygous their structure, assembly, and interactions. J. Struct. Biol. 122, 42–66 (1998). the nuclear envelope, indirect evidence for a mtf-1/sun-1-deletion allele show that 2. Dahl, K. N., Kahn, S. M., Wilson, K. L. & Discher, D. E. indicates that their core activity — nucleo- this gene is essential for germline matura- The nuclear envelope lamina network has elasticity 18 and a compressibility limit suggestive of a molecular cytoplasmic transport — is vulnerable to tion and survival , with late larval pheno- shock absorber. J. Cell Sci. 117, 4779–4786 (2004). mechanical deformation42. We speculate types that hint at it having fundamental 3. Lammerding, J. et al. Lamin A/C deficiency causes defective nuclear mechanics and that the evolution of a specific bridg- roles in germline cell proliferation or germ- mechanotransduction. J. Clin. Invest. 113, 370–378 ing mechanism, based on SUN-domain cell maintenance, or both18. Matefin/SUN-1 (2004). 4. Gruenbaum, Y., Margalit, A., Shumaker, D. K. & and KASH-domain proteins, provided also binds the apoptotic activator CED-4 Wilson, K. L. The nuclear lamina comes of age. Nature a versatile mechanism to anchor many and is required for apoptosis (Y.Z. and Y.G., Rev. Mol. Cell Biol. 6, 21–31 (2005). 5. Schirmer, E. C., Florens, L., Guan, T., Yates, J. R. & cytoskeletal and nucleoskeletal structures unpublished observations). Gerace, L. Nuclear membrane proteins with potential to one integrative ‘mechanical syncytium’ disease links found by subtractive proteomics. Science 531, 1380–1382 (2003). at the nuclear envelope. Consistent with Concluding remarks and outlook 6. Gruenbaum, Y. et al. The nuclear lamina and its this idea, the links between SUN-domain On the basis of current evidence, we functions in the nucleus. Int. Rev. Cyt. 226, 1–62 (2003). and KASH-domain proteins are needed propose that SUN-domain proteins are 7. Worman, H. J. Inner nuclear membrane and signal to maintain the uniform spacing between the ‘Velcro’ in the nuclear envelope that transduction. J. Cell Biochem. 96, 1185–1192 (2005). 13 8. Starr, D. A. & Fischer, J. A. KASH ‘n Karry: the KASH the INM and the ONM . The importance mechanically links the cytoskeleton to the domain family of cargo-specific cytoskeletal adaptor of integrating mechanical connections is nucleoskeleton. SUN-domain proteins that proteins. Bioessays 27, 1136–1146 (2005). 9. Warren, D. T., Zhang, Q., Weissberg, P. L. & Shanahan, intuitively obvious when one considers localize in the ER and the ONM might have C. M. Nesprins: intracellular scaffolds that maintain that chromosomes are dense and must be similar roles in spacing and reinforcing the cell architecture and coordinate cell function? Expert. Rev. Mol. Med. 7, 1–15 (2005). hauled about without damaging either the ER-membrane network, while also poten- 10. McGee, M. D., Rillo, R., Anderson, A. S. & Starr, D. A. nuclear envelope or the cell. tially ‘reverse-anchoring’ INM-localized UNC-83 Is a KASH protein required for nuclear migration and is recruited to the outer nuclear KASH-domain proteins (FIG. 3). We are membrane by a physical interaction with the SUN New roles for SUN-domain proteins also beginning to appreciate potential non- protein UNC-84. Mol. Biol. Cell 17, 1790–1801 (2006). Other, non-mechanical roles for SUN- mechanical roles for SUN-domain proteins 11. Padmakumar, V. C. et al. The inner nuclear membrane domain proteins are also emerging. In during apoptosis, meiosis and germ-cell protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope. J. Cell Sci. 118, 3419–3430 S. pombe, telomeres become clustered at maintenance. (2005). the nuclear envelope near the SPB during The proposed mechanical roles of SUN- 12. Malone, C. J., Fixsen, W. D., Horvitz, H. R. & Han, M. UNC-84 localizes to the nuclear envelope and is meiosis. This clustering facilitates the align- domain proteins in bridging the nuclear required for nuclear migration and anchoring during ment of homologous chromosomes and envelope raise exciting new questions about C. elegans development. Development 126, 43 3171–3181 (1999). promotes their pairing and recombination . the nature and purpose of these attach- 13. Crisp, M. et al. Coupling of the nucleus and cytoplasm: Telomere clustering is mediated by the ments, not only during nuclear migration role of the LINC complex. J. Cell Biol. 172, 41–53 (2006). SUN-domain protein Sad1, which binds a in specialized cell types, but in everyday 14. Lee, K. K. et al. Lamin-dependent localization of protein named Bqt1, which joins with Bqt2 interphase nuclei. SUN-domain proteins UNC-84, a protein required for nuclear migration in C. elegans. Mol. Biol. Cell 13, 892–901 (2002). to bind the telomere-associated protein Rap1 have the potential to directly transduce 15. Hasan, S. et al. Nuclear envelope localization of (REF. 44). Sad1 also interacts with the KASH- mechanical signals from the cytoplasm to human UNC84A does not require nuclear lamins. FEBS Lett. 580, 1263–1268 (2006). domain protein Kms1 (REF. 45); interestingly, the nucleus and back, indicating that they 16. Hagan, I. & Yanagida, M. The product of the spindle this interaction is required to maintain might serve as the ‘integrins’ of the nuclear formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. Sad1 at the SPB. So, the yeast SUN-domain envelope. Many open questions remain, J. Cell Biol. 129, 1033–1047 (1995). and KASH-domain proteins have roles in however, including whether human cells 17. Starr, D. A. & Han, M. ANChors away: an actin based mechanism of nuclear positioning. J. Cell Sci. 116, meiosis that involve chromosome (telomere) use KASH-domain proteins to anchor 211–216 (2003). tethering to the nuclear envelope. centrosomes (that is, are they equivalent 18. Fridkin, A. et al. Matefin, a C. elegans germ-line specific SUN-domain nuclear membrane protein, is In C. elegans, besides disrupting nuclear to ZYG-12 in C. elegans), and why both essential for early embryonic and germ cell migration during development, mutations predicted SUN-domain proteins in development. Proc. Natl Acad. Sci. USA 101, 6987–6992 (2004). in the SUN-domain protein UNC-84 cause D. melanogaster seem to lack transmem- 19. Haque, F. et al. SUN1 interacts with nuclear lamin A additional phenotypes (such as improper brane domains. As more binding partners and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the migration of gonad distal tip cells, egg- are discovered, a better understanding of cytoskeleton. Mol. Cell. Biol. 26, 3738–3851 (2006). laying defects and reduced fat levels12,35,46) their connections will be achieved, and we 20. Hodzic, D. M., Yeater, D. B., Bengtsson, L., Otto, H. & Stahl, P. D. Sun2 is a novel mammalian inner nuclear that are not reported for its known KASH- will better comprehend the importance of membrane protein. J. Biol. Chem. 279, domain partners UNC-83 or ANC-1. these bridges. 25805–25812 (2004).

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Genetics 168, nuclear pore complex glycoprotein. J. Cell Biol. 116, (BSF) and the European Union’s FP6, Life Science, Genomics 1385–1393 (2004). 15–30 (1992). and Biotechnology for Health to Y.G., and the National 29. Mislow, M. K. J. et al. Nesprin-1a self-associates and 43. Ding, D. Q., Chikashige, Y., Haraguchi, T. & Hiraoka, Y. Institutes of Health to K.L.W. binds directly to emerin and lamin A in vitro. FEBS Oscillatory nuclear movement in fission yeast meiotic Lett. 525, 135–140 (2002). prophase is driven by astral microtubules, as revealed Competing interests statement 30. Muchir, A. et al. Nuclear envelope alterations in by continuous observation of chromosomes and The authors declare no competing financial interests. fibroblasts from LGMD1B patients carrying nonsense microtubules in living cells. J. Cell Sci. 111, 701–712 Y259X heterozygous or homozygous mutation in (1998). lamin A/C gene. Exp. Cell Res. 291, 352–362 44. Chikashige, Y. et al. Meiotic proteins Bqt1 and Bqt2 DATABASES (2003). tether telomeres to form the bouquet arrangement of The following terms in this article are linked online to: 31. Sullivan, T. et al. Loss of A-type lamin expression chromosomes. Cell 125, 59–69 (2006). Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. compromises nuclear envelope integrity leading to 45. Niwa, O., Shimanuki, M. & Miki, F. Telomere-led fcgi?db=gene muscular dystrophy. J. Cell Biol. 147, 913–920 bouquet formation facilitates homologous Nesprin-1 | Nesprin-3 | SUN1 | SUN2 (1999). chromosome pairing and restricts ectopic interaction UniProtKB: http://ca.expasy.org/sprot 32. Pare, G. C., Easlick, J. L., Mislow, J. M., McNally, E. M. in fission yeast meiosis. EMBO J. 19, 3831–3840 ANC-1 | Sad1 | UNC-84 | ZYG-12 & Kapiloff, M. S. Nesprin-1α contributes to the (2000). targeting of mAKAP to the cardiac myocyte nuclear 46. Greer, E. L. & Brunet, A. FOXO transcription factors FURTHER INFORMATION envelope. Exp. Cell Res. 303, 388–399 (2005). at the interface between longevity and tumor Kathy Wilson’s homepage: http://biolchem.bs.jhmi.edu/ 33. Yu, J. et al. The KASH domain protein MSP-300 plays suppression. Oncogene 24, 7410–7425 (2005). bcmb/Faculty_person.asp?PersonID=688 an essential role in nuclear anchoring during 47. Worman, H. J. & Courvalin, J. C. Nuclear envelope, Yosef Gruenbaum’s homepage: Drosophila oogenesis. Dev. Biol. 289, 336–345 nuclear lamina, and inherited disease. Int. Rev. Cytol. http://www.ls.huji.ac.il/grulabs (2006). 246, 231–279 (2005). SMART database: http://smart.embl-heidelberg.de 34. Grady, R. M., Starr, D. A., Ackerman, G. L., Sanes, J. R. 48. Cohen, M., Lee, K. K., Wilson, K. L. & Gruenbaum, Y. TMPRED: http://www.ch.embnet.org/software/TMPRED_ & Han, M. Syne proteins anchor muscle nuclei at the Transcriptional repression, apoptosis, human disease form.html neuromuscular junction. Proc. Natl Acad. Sci. USA and the functional evolution of the nuclear lamina. 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