The nuclear envelope partitions nuclear components from the cytoskeleton, thereby necessitating their mechanical coupling across the nuclear envelope to enable cytoskeletal function in the structure and positioning of nuclear contents. This is achieved by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which traverses the nuclear envelope and binds to cytoskeletal and nuclear structures to mediate force transduction between these partitioned components (Haque et al., 2006; Crisp et al., 2006; Lee and Burke, 2018; Meinke and Schirmer, 2015; Figure 1a). In this capacity, the LINC complex is essential for cellular life, performing critical functions in nuclear structure, shape, and positioning (Alam et al., 2015; Luxton et al., 2010; Crisp et al., 2006), in addition to tissue-specific functions including sound perception in the inner ear and chromosome movements during meiosis (Horn et al., 2013a; Roux et al., 2009; Horn et al., 2013b; Lee et al., 2015). Further, mutations of the LINC complex and its interacting partners are associated with human laminopathies, including Hutchison-Gilford progeria syndrome and Emery-Dreifuss muscular dystrophy (Meinke et al., 2011; Chen et al., 2012; Taranum et al., 2012; Zhou et al., 2018b; Chang et al., 2019).

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The LINC complex is formed of SUN (Sad1 and UNC84 homology) domain and KASH (Klarsicht, ANC-1, and Syne homology) domain proteins (Padmakumar et al., 2005; Haque et al., 2006; Crisp et al., 2006), which interact immediately below the outer nuclear membrane, through complex formation between their C-terminal eponymous SUN and KASH domains (Sosa et al., 2012; Wang et al., 2012; Zhou et al., 2012). SUN proteins then traverse the approximately 50 nm peri-nuclear space and cross the inner nuclear membrane, enabling their N-termini to bind to nuclear contents, including reported interactions with the nuclear lamina (Crisp et al., 2006; Haque et al., 2006; Haque et al., 2010), chromatin (Chi et al., 2007), and the telomeric ends of meiotic chromosomes (Shibuya et al., 2014). Similarly, KASH domain proteins cross the outer nuclear membrane and have large cytoplasmic extensions to enable their N-termini to bind to the cytoskeleton (Spindler et al., 2019; Starr and Fridolfsson, 2010). Thus, the LINC complex axis is established by a peri-nuclear SUN-KASH core interaction and mechanically couples the cytoskeleton and nuclear contents (Figure 1a).

In mammals, there are five SUN proteins, of which SUN1 and SUN2 are widely expressed and perform partially redundant functions (Lei et al., 2009; Zhang et al., 2009). There are similarly multiple KASH proteins, four of which are Nesprins (Nuclear Envelope Spectrin Repeat proteins). Nesprin-1 and Nesprin-2 are widely expressed, perform overlapping functions and contain large cytoplasmic spectrin-repeat domains with N-termini that bind to actin (Banerjee et al., 2014; Sakamoto et al., 2017; Zhou et al., 2018a). Nesprin-3 shares a similar KASH domain but its cytoplasmic region binds to plectin, mediating interactions with intermediate filaments (Wilhelmsen et al., 2005). The two most divergent KASH proteins, Nesprin-4 and KASH5, exhibit substantial sequence diversity within their KASH domains (Figure 1b). Nesprin-4 functions in the outer hair cells of the inner ear and is essential for hearing (Horn et al., 2013a). Its N-terminus interacts with kinesin, which mediates microtubule binding and plus-end directed movements that achieve the basal positioning of nuclei (Horn et al., 2013a; Roux et al., 2009). KASH5 functions in meiosis and is essential for fertility (Horn et al., 2013b; Morimoto et al., 2012). Its N-terminus interacts with dynein-dynactin (Morimoto et al., 2012; Horn et al., 2013b), which mediates microtubule binding and minus-end directed motility that drives rapid chromosomal movements to facilitate homologous chromosome pairing (Lee et al., 2015; Zetka et al., 2020). Thus, KASH proteins execute a range of LINC complex functions in transmitting actin forces, plus-/minus-end directed microtubule movements and the tensile strength of intermediate filaments into the nucleus.

The canonical model of the LINC complex is based on crystal structures of the SUN-KASH domain complexes formed between SUN2 and Nesprin-1/2 (Sosa et al., 2012; Wang et al., 2012). The SUN domain adopts a ‘three-leaf clover’-like structure, in which a globular trimer extends from a short N-terminal trimeric coiled-coil (Sosa et al., 2013). KASH domains are intertwined between SUN protomers and their path is defined by three distinct regions. The KASH C-terminus contains a triple proline motif that packs between the globular cores of SUN protomers. The KASH mid-region winds around the trimeric arc and is wedged between the globular core of one SUN protomer and a β-turn-β loop, known as the KASH-lid, of the adjacent protomer. The KASH N-terminus then turns by >90° to radiate out from the trimer axis and forms a disulphide bond with a SUN protomer (between SUN2 and KASH1 amino acids C563 and C8774, respectively), which is predicted to enhance the tensile strength of SUN-KASH (Jahed et al., 2015; Sosa et al., 2012). The extensive 3:3 complex of three KASH domains bound to a single SUN trimer was interpreted as the biological unit of the crystal lattice (Sosa et al., 2012; Wang et al., 2012). On this basis, it was proposed that the LINC complex consists of a SUN-KASH 3:3 complex that is orientated vertically to allow KASH proteins to cross the outer nuclear membrane and SUN to form an extended trimeric coiled-coil that spans the peri-nuclear space (Sosa et al., 2012; Sosa et al., 2013; Figure 1a). In support of this model, the luminal region of SUN2 was shown to be trimeric in vitro by analytical ultracentrifugation, SEC-MALS, and gel filtration (Sosa et al., 2012; Nie et al., 2016; Zhou et al., 2012; Jahed et al., 2018b) and upon targeting to the nuclear envelope in vivo, the luminal regions of SUN2 and SUN1 were shown to form trimers and larger structures by fluorescence fluctuation spectroscopy (Hennen et al., 2017; Hennen et al., 2018). However, the stoichiometry of SUN-KASH complexes has not yet been tested in solution. Further, whilst it has been widely recognised that branching or higher order assembly of LINC complexes may be advantageous in distributing large forces and achieving coordinated motions (Zhou et al., 2012; Lu et al., 2008; Jahed et al., 2018a; Wang et al., 2012; Sosa et al., 2013; Lu et al., 2012), we have hitherto lacked structural evidence and a molecular basis for higher order assembly of the LINC complex.

Here, we provide crystallographic and biophysical evidence in support of the LINC complex forming a branched network. We find that SUN-KASH complexes between SUN proteins and Nesprin-4, KASH5 and Nesprin-1 are 6:6 structures formed of constitutive interactions between two 3:3 complexes. The three distinct KASH domains provide structurally diverse but related 6:6 interfaces that achieve the same topology with potential hinge-like motion between SUN trimers. The SUN-KASH 6:6 interface consists of a ‘head-to-head’ interaction between SUN’s trimeric C-terminal ‘heads’, thereby providing a mechanistic basis for formation of a branched LINC complex network. Thus, we propose that SUN-KASH domain complexes act as nodes for branching and integration between LINC complexes to achieve the coordinated transduction of large forces across the nuclear envelope.

How does our finding of a constitutive SUN-KASH 6:6 assembly integrate with previous biochemical studies of the LINC complex? It was previously shown by analytical ultracentrifugation, SEC-MALS, and gel filtration that luminal SUN2 is trimeric, and its isolated SUN domain is a trimer or monomer, depending on biochemical conditions (Zhou et al., 2012; Wang et al., 2012; Sosa et al., 2013; Jahed et al., 2018b). These findings agree with our observations that the isolated SUN domain of SUN1 becomes monomeric upon cleavage of its N-terminal GCN4 expression tag (which mimics the trimeric luminal coiled-coil), so is entirely dependent on KASH-binding to stabilise its trimeric structure and head-to-head assembly. The only previous analysis of SUN-KASH in solution involved demonstrating complex formation by analytical gel filtration, without means for oligomer determination (Esra Demircioglu et al., 2016). Thus, the 3:3 SUN-KASH model was the natural conclusion of combining SUN’s luminal trimer with the extensive 3:3 complexes within SUN2-KASH1/2 crystal lattices (Sosa et al., 2012; Wang et al., 2012). Our SEC-MALS and SEC-SAXS analyses provide the first reported evidence of solution structure, revealing that SUN-KASH complexes formed by SUN1 and SUN2 are 6:6 hetero-oligomers in which 3:3 structures are locked in head-to-head interactions, as observed in our SUN1-KASH crystal structures and in previous SUN2-KASH crystal lattices (Sosa et al., 2012; Wang et al., 2012). Further, mutational analysis confirmed that SUN1/2-KASH1 complexes depend on interactions across the 6:6 interface for their stability. Hence, our conclusion that SUN-KASH complexes are 6:6 hetero-oligomers in vitro is consistent with all existing crystallographic, biochemical, and biophysical data.

How does the SUN-KASH 6:6 assembly relate to previous observations of LINC complex structure and function within the cell? The oligomeric states of luminal regions of SUN1 and SUN2, upon expression and targeting to the nuclear envelope, were determined by fluorescence fluctuation spectroscopy as trimers with additional higher order SUN1 structures (Hennen et al., 2017; Hennen et al., 2018). In these studies, expressed KASH domains and isolated SUN domains remained mostly monomeric, suggesting that expressed constructs did not form SUN-KASH complexes with endogenous partners. Hence, these studies provided important evidence that the coiled-coils of SUN’s luminal regions form trimers and larger oligomers but did not determine the stoichiometry of SUN-KASH complexes. The assembly of higher order LINC structures has also been suggested by numerous other cellular findings, including immobility within the nuclear envelope (Lu et al., 2008), foci formation within the meiotic nuclear envelope (Ding et al., 2007; Morimoto et al., 2012; Horn et al., 2013b), and the formation of transmembrane actin-associated nuclear (TAN) lines (Luxton et al., 2010). Our model of LINC complex branching by SUN-KASH 6:6 assembly is consistent with the observed oligomeric state of SUN’s luminal region and higher order LINC assembly, but its molecular details are not directly tested by any existing cellular data. Thus, our molecular model of a branched LINC complex, and similarly the role of zinc-binding in the SUN1-KASH4 complex, must be tested in future studies of the consequence of separation of function mutations (such as targeting the 6:6 interfaces of SUN1/2-KASH1 complexes by I673E and I579E mutations) on the cellular structure and function of the LINC complex.

The advantages of a branched LINC complex network include its ability to transmit large forces, being impervious to the breakage of individual linkages, and in mediating communication and coordination between adjacent molecules. The SUN-KASH 6:6 assembly provides an attractive structural means for branching, which may combine with a series of episodic instances of oligomer variation along the SUN-KASH axis to generate a highly branched LINC complex network. Firstly, oligomer variation could occur through higher order assembly of SUN-KASH 6:6 complexes, as indicated by our observation of higher order SUN2-KASH structures and the formation of SUN1-KASH4 12:12 complexes upon disruption of zinc-binding. Secondly, oligomeric variation within SUN’s luminal regions may mediate branching, such as indicated by the formation of trimers and larger oligomer by luminal SUN1 (Hennen et al., 2018) and disulphide bond formation by SUN1 amino acid C526 (Lu et al., 2008). Finally, oligomer variation between SUN and KASH proteins could mediate branching across the outer nuclear membrane. Indeed, KASH5 is dimeric (Gurusaran and Davies, unpublished findings), raising the question of how SUN trimers and KASH dimers are organised into discrete 6:6 complexes? We suggest that each KASH dimer likely spans both SUN trimers, thereby establishing a symmetrical array of SUN1-KASH interfaces within each 6:6 structure, which constitute branching events between SUN-KASH5 6:6 complexes and their dimeric cytoskeletal attachments. Thus, we propose that coordinated force transduction is achieved by a highly branched LINC complex network in which SUN-KASH 6:6 hetero-oligomers contribute to branching by mediating force distribution and integration between three KASH dimers and two SUN trimers (Figure 8).

The head-to-head nature of SUN-KASH 6:6 complexes suggests their orientation parallel to the outer nuclear membrane, with SUN trimers organised obliquely within the peri-nuclear space (Figure 8). Our SAXS analysis of SUN1-KASH1 indicated that it adopts a hinged conformation in solution, stabilised by two KASH-lid tip-to-tip interactions and laterally associated central KASH-lids. Whilst hinged motions were not required to explain SAXS data of SUN1-KASH4/5, normal mode analysis predicted that hinged structures of up to approximately 60° angulation are conformationally accessible states for all three SUN1-KASH complexes. Thus, we suggest that all SUN-KASH head-to-head structures can undergo hinge-like motion at their 6:6 interface, with a large proportion of highly angled conformations accounting for their dominance in the SAXS data of SUN1-KASH1 but not other complexes. This hinge-like motion would result in SUN-KASH complexes becoming angled in response to the magnitude and direction of tension forces carried by SUN and KASH molecules, whilst providing the conformational flexibility necessary for constituent KASH proteins to adopt orientations that allow upstream transmembrane sequences to cross the outer nuclear membrane. Further, all three SUN-KASH 6:6 interfaces are largely hydrophobic, so could be stabilised by interactions with phospholipids, possibly as part of integrated membrane-bound complexes that include KASH’s transmembrane regions. Thus, hinge-like flexibility of SUN-KASH may result in a diverse range of angled conformations owing to distinct tension forces, steric constraints and membrane structures of particular spatiotemporal environments.

What are the roles of distinct SUN and KASH proteins in LINC complex structure and function? Whilst SUN1 and SUN2 form similar 6:6 hetero-oligomers, we observed notable differences in the stability and higher assembly of their SUN-KASH complexes. The reduced stability of SUN2-KASH complexes could facilitate a faster turnover of SUN2-containing LINC complexes, whilst higher order assembly of SUN2-KASH may combine with differential SUN1/2 luminal assemblies (Hennen et al., 2017; Hennen et al., 2018) to achieve distinct LINC complex architectures. These findings may underlie some of the observed asymmetries between SUN1 and SUN2 LINC complexes, such as their differential preference for cytoskeletal components and their non-redundant functions (Link et al., 2014; Zhu et al., 2017; Thakar et al., 2017; May and Carroll, 2018). It is important to note that SUN2-KASH4, in which the 6:6 interface is mediated solely by KASH4 zinc sites, is the only SUN2 complex that retains the high affinity observed for SUN1 complexes. In contrast, SUN amino acids contribute to the 6:6 interfaces of KASH1/5 complexes, explaining how SUN protein sequence diversity can account for the substantially reduced affinity of SUN2-KASH1/5 in comparison with their SUN1 complexes. The variation of KASH proteins seemingly provides even greater functional diversity given their entirely non-redundant roles. An intriguing observation is that Nesprin-4 and KASH5, which transduce microtubule forces (Horn et al., 2013a; Roux et al., 2009; Morimoto et al., 2012; Horn et al., 2013b), demonstrate extensive interactions at their 6:6 interfaces. In contrast, a far less extensive 6:6 interface is found in classical Nesprins, which transduce actin forces and the tensile strength of intermediate filaments (Banerjee et al., 2014; Starr and Fridolfsson, 2010; Ketema and Sonnenberg, 2011). Thus, cytoskeletal components may have differential requirements for the strength, structure and stability of SUN-KASH 6:6 hetero-oligomers. Further, differences in regulatory mechanisms, such as zinc-binding in SUN1-KASH4 assembly, may contribute towards specialisation. The expression levels and relative availability of SUN and KASH proteins will determine their incorporation into LINC complexes, and specialised functionalities may be achieved by combining distinct isoforms within the same LINC complex network or within separate networks of the same cell.

How is LINC complex assembly regulated within the cell? An intriguing finding is that SUN proteins undergo autoinhibition, in which SUN domains become bound by upstream sequences in monomeric conformations that are incapable of binding to KASH domains (Nie et al., 2016; Xu et al., 2018; Jahed et al., 2018b; Jahed et al., 2018a). These autoinhibitory conformations likely represent unassembled states that may be crucial intermediates in the dynamic process of LINC complex expression, localisation, and assembly within the cell. They may also represent a ‘storage form’ of SUN proteins that form when quantities of available KASH proteins are limiting. This would establish discrete pools of assembled and unassembled SUN proteins, which could play an important role in preventing unbound SUN molecules from weakening established LINC structures by continually competing for KASH-binding. Further, given the myriad of LINC complex functions in almost all eukaryotic cells (Lee and Burke, 2018; Meinke and Schirmer, 2015; Starr and Fridolfsson, 2010), assembly is likely directed along specific pathways to achieve distinct LINC complex structures for the fulfilment of specialised functions. Thus, regulatory processes must overcome autoinhibition, enable KASH-binding, and direct LINC assembly in a timely manner. These may involve chaperones, enzymatic modification, protein interactions, and/or chemical conditions of the nuclear envelope environment. In specific, these may include regulation by luminal ion concentration and pH (Jahed et al., 2018b), local regulation of SUN-KASH angulation, control of SUN1-KASH4 assembly by zinc availability, and determining the nature of LINC complexes through relative availability of SUN and KASH protein isoforms. We have hitherto considered variations within SUN-KASH 6:6 complexes, but also recognise the potential for regulatory mechanisms of the nuclear envelope to induce more substantial structural changes. Thus, whilst our model of LINC complex branching through SUN-KASH 6:6 assembly is consistent with all existing data, it remains possible that alternative LINC complex conformations may form within the spatial and temporal contexts of disparate cell types.

Crystallographic structure factors and atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession numbers 6R15, 6R16 and 6R2I, and raw diffraction data have been uploaded to https://proteindiffraction.org/. SEC-SAXS data have been deposited in the Small Angle Scattering Biological Data Bank (https://www.sasbdb.org/) under accession numbers SASDJC5, SASDJD5, SASDJE5 and SASDJF5. Uncropped gel images relating to Figures 4b and 5a are available in source data files.

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   (2020) RCSB Protein Data Bank

   ID 6R15. Crystal structure of the SUN1-KASH1 6:6 complex.

   https://www.rcsb.org/structure/6R15
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   ID 6R16. Crystal structure of the SUN1-KASH4 6:6 complex.

   https://www.rcsb.org/structure/6R16
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   ID 6R2I. Crystal structure of the SUN1-KASH5 6:6 complex.

   https://www.rcsb.org/structure/6R2I
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   ID SASDJC5. LINC complex between the SUN domain of SUN1 and KASH domain of Nesprin-4 - SUN1-KASH4 6:6 complex.

   https://www.sasbdb.org/data/SASDJC5/
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Author details

1. Manickam Gurusaran

   1. Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
   2. Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, United Kingdom

   Contribution

   Data curation, Validation, Investigation, Methodology, Project administration

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6603-3118

2. Owen Richard Davies

   1. Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
   2. Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   owen.davies@ed.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3806-5403

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