A molecular mechanism for LINC complex branching by structurally diverse SUN-KASH 6:6 assemblies

Abstract

The LINC complex mechanically couples cytoskeletal and nuclear components across the nuclear envelope to fulfil a myriad of cellular functions, including nuclear shape and positioning, hearing and meiotic chromosome movements. The canonical model of the LINC complex is of individual linear nucleocytoskeletal linkages provided by 3:3 interactions between SUN and KASH proteins. Here, we provide crystallographic and biophysical evidence that SUN-KASH is a constitutive 6:6 complex in which two SUN trimers interact back-to-back. A common SUN-KASH topology is achieved through structurally diverse 6:6 interaction mechanisms by distinct KASH proteins, including zinc-coordination by Nesprin-4. The SUN-KASH 6:6 complex is incompatible with the current model of a linear LINC complex and instead suggests the formation of a branched LINC complex network. In this model, SUN-KASH 6:6 complexes act as nodes for force distribution and integration between adjacent SUN and KASH molecules, enabling the coordinated transduction of large forces across the nuclear envelope. KeywordsLINC complex, nuclear envelope, SUN1, Nesprin-4, KASH5, Nesprin-1, X-ray crystallography, smallangle X-ray scattering, biophysics 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 Ntermini 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; Starr and Fridolfsson, 2010). Nesprin-3 shares a similar KASH domain but its cytoplasmic region binds to plectin, mediating interactions with intermediate filaments (Ketema and Sonnenberg, 2011). 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). KA...

The Role of the LINC Complex in Sperm Development and Function

“…Surprisingly, this 6:6 assembly was also observed with SUN1:KASH1 complex. Thus, it can be concluded that SUN-KASH complexes are rather forming a network of interacting oligomers instead of the current linear model [41]. KASH1 is a large KASH domain protein with a molecular weight of approximately 1000 kDa.…”

“…This was proposed for LINC complexes consisting of SUN1, KASH1, and KASH2 based on the X-ray crystallographic analysis [28,31]. However, the assembly of LINC complexes containing KASH4 or KASH5 interacting with SUN1, turned out to form a complex net of 6:6 hetero-oligomeric structures that are in fact 3:3 hetero-hexamers that interact "back-to-back" with one another in the PNS [41]. Surprisingly, this 6:6 assembly was also observed with SUN1:KASH1 complex.…”