Mechanical Response of Nesprin-2G to Cytoskeletal Forces Regulates Linc Complex-Dependent Mechanotransduction

Abstract

Nuclear geometry and positioning are affected by cytoskeletal forces, which plays an important role in cellular mechanotrasnduction. The LINC (LInkers of the Nucleoskeleton and Cytoskeleton) complex has been shown to bear mechanical load and mediate force transmission from the cytoskeleton into the nucleus, however, molecular mechanisms of this phenomenon are not yet understood. Giant nesprin-2 (nesprin-2G) forms a direct linkage between the actin cytoskeleton and SUN proteins inside the perinuclear space. These linkers are shown to be under constant tension especially in the apical and equatorial planes of the nucleus and have 56 spectrin repeats along their rod domain. A functional construct of nespring-2G containing only four spectrin repeats, referred to as mini-nesprin-2G, was shown to rescue the nuclear movement defects observed in nesprin-2G-depleted fibroblasts. Since it is intrinsically difficult to measure forces and track residue-level interactions across individual proteins using current experimental techniques, we used molecular dynamics simulations to study mechanical response of nesprin molecules to cytoskeletal forces. Mini-nesprins are excellent candidates for this purpose as their small size allows for long simulation periods necessary for capturing important molecular events. Our results provides a detailed understanding of how mechanical modes of mini-nesprin-2G are excited and contribute to its response to various cytoskeletal forces around the nucleus. Also, we simulated force-induced conformational changes of the Actin Binding Domain (ABD) of mini-nesprin-2G, which are most likely induced by the axial tension along the actin bundles at the apical plane of the nucleus. Furthermore, we examined the effect of ABD conformation on its actin binding affinity by comparing the strength of interactions between distinct conformations of the ABD domain of mini-nesprin-2G and actin. Lastly, we used force distribution analysis to identify force-sensitive residues along the mini-nesprin-2G structure and tested the effect of mutating them to alanine on transferring forces to the nucleus.