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

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.