Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

Abstract

The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.

Structural insights into SUN-KASH complexes across the nuclear envelope

“…The addition of amino acids at the Cterminus of the PPPT anchor leads to steric hindrance of PPPT docking into the BI-pocket. These observations are consistent with a previous report showing that an extension of the C-terminal KASH domain sequence prevented the binding to the SUN domain [22][23][24].…”

“…In fact, we have repeatedly observed that both the SUN domain homotrimer alone and the luminal region of SUN2 containing two coiled-coils and the SUN domain (CC1-CC2-SUN) are sufficient for KASH binding ( Figure 4). However, a truncated form of SUN2 that only contains the second coiled-coil and the SUN domain (CC2-SUN) was not able to bind the KASH peptide, consistent with previous studies [1,10,22,24]. Of note, we previously found that the truncated form containing only CC2 and the SUN domain also showed reduced oligomerization [10].…”

“…Our current structure revealed intimate hydrophobic docking of four C-terminal residues of the KASH domain (PPPT motif) into the BI-pocket of the SUN domain, which is consistent with earlier studies showing that the conserved PPPT motif of the KASH domain was involved in association with the SUN proteins [22][23][24]. Based on structural analysis, we tested the contribution of this interaction by GST pull-down assay using a KASH peptide with a deletion of C-terminal PPPT motif (GST-KASH∆).…”

“…Our result clearly showed that removal of the PPPT motif abolished KASH association with the SUN domain, indicating an essential role of this motif in LINC complex formation ( Figure 4A). A previous study by Stewart-Hutchinson et al [22] indicated that addition- npg al residues added at the C-terminus of the KASH domain prevented KASH from binding with SUN proteins. This observation can be explained by the structural feature of the BI-pocket of the SUN domain, which can only sterically accommodate a ligand of the size of the PPPT motif.…”

A perinuclear actin cap regulates nuclear shape

“…Traditional fluorescence microscopy focusing on the bottom of the cell suggests that the displacement of these NE proteins caused by lamin depletion does not affect actin filament organization (14). Because the NE distribution of LINC complex proteins is altered in embryonic fibroblasts derived from mice lacking A-type lamins, a model of dilated cardiomyopathy and muscular dystrophy (15,16), we hypothesized that the depletion of lamin A/C from the nucleus would disrupt the organization of the actin cap and the regulation of nuclear shape by cell shape.…”