Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin

Abstract

The outer nuclear membrane proteins nesprin-1 and nesprin-2 are retained at the nuclear envelope through an interaction of their klarsicht/ANC-1/syne homology (KASH) domain with Sun proteins present at the inner nuclear membrane. We investigated the requirements for the localization of nesprin-3α at the outer nuclear membrane and show that the mechanism by which its localization is mediated is similar to that reported for the localization of nesprin-1 and nesprin-2: the last four amino acids of the nesprin-3α KASH domain are essential for its interaction with Sun1 and Sun2. Moreover, deletion of these amino acids or knockdown of the Sun proteins results in a redistribution of nesprin-3α away from the nuclear envelope and into the endoplasmic reticulum (ER), where it becomes colocalized with the cytoskeletal crosslinker protein plectin. Both nesprin-3α and plectin can form dimers, and dimerization of plectin is required for its interaction with nesprin-3α at the nuclear envelope, which is mediated by its N-terminal actin-binding domain. Additionally, overexpression of the plectin actin-binding domain stabilizes the actin cytoskeleton and prevents the recruitment of endogenous plectin to the nuclear envelope. Our studies support a model in which the actin cytoskeleton influences the binding of plectin dimers to dimers of nesprin-3α, which in turn are retained at the nuclear envelope through an interaction with Sun proteins.

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].…”

“…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∆).…”

“…However, it was recently found that both SUN proteins and KASH proteins can form high-order oligomers or clusters during specific cellular events [23,[34][35][36][37][38][39]. Given that mechanical transduction is one of the salient features of LINC complex function, we have previously proposed and would like to reiterate that regulated networking could create a "machine" for the SUN-KASH-mediated force (de)coupling between the nucleus and the cytoskeleton ( Figure 6B).…”

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

“…Sun2-GFP was from Hodzic et al (2004). Nesprin-3␣-GFP was described in Ketema et al (2007) and was obtained from Arnoud Sonnenberg (the Netherlands Cancer Institute). The sequences of all constructs were verified by nucleotide sequencing.…”

“…Finally, the outer nuclear membrane protein nesprin-3 (Wilhelmsen et al, 2005) is abnormally distributed in fibroblasts from TorA knockout mice, and these cells move slower than controls in a polarized cell migration assay (Nery et al, 2008). Because nesprin-3 participates in linker of the nucleoskeleton and cytoskeleton (LINC) complexes (Wilhelmsen et al, 2005;Ketema et al, 2007;Crisp and Burke, 2008;StewartHutchinson et al, 2008;Starr, 2009), these data suggest that TorA activity may help regulate NE structure and connections between nucleus and cytoskeleton.…”