Characterization of Unfolding Mechanism of Human Lamin A Ig Fold by Single-Molecule Force Spectroscopy—Implications in EDMD

Abstract

A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath the nuclear envelope thereby conferring proper shape and mechanical rigidity to the nucleus. Lamin proteins are also shown to be related diversely to basic nuclear processes. More than 400 mutations in human lamin A protein alone have been reported to produce at least 11 different disease conditions jointly termed as laminopathies. These mutations in lamin A are scattered throughout its helical rod domain, as well as the C-terminal domain containing the conserved Ig-fold region. The commonality of phenotypes in all these diseases is characterized by misshapen nuclei of the affected tissues which might stem from altered rigidity of the supporting lamina hence lamins. Here we have focused on autosomal dominant Emery-Dreifuss Muscular Dystrophy, one such laminopathy where R453W is the causative mutation located in the Ig domain of lamin A. We have investigated by single-molecule force spectroscopy how a stretching mechanical perturbation senses the destabilizing effect of the mutation in the lamin A Ig domain and compared the mechanoelastic properties of the mutant R453W with that of the wild-type in conjunction with steered molecular dynamics. Furthermore, we have shown the interaction of Ig domain with emerin, another key player and interacting partner in the pathogenesis of EDMD, is disrupted in the R453W mutant. This altered mechanoresistance of Ig domain itself and consequent uncoupling of lamin A-emerin interaction might underlie the altered mechanotransduction properties of EDMD affected nuclei.

Lamins at the crossroads of mechanosignaling

“…Exposure of nuclei to shear stress induced stretching of lamin A filaments due to partial ''unfolding'' of the lamin Ig-fold. Interestingly, the Ig-fold in lamin A was recently shown to define the viscoelastic properties of lamin A and the mechanical resilience of lamin A networks (Bera et al 2014).…”

Impaired mechanical response of an EDMD mutation leads to motility phenotypes that are repaired by loss of prenylation

“…Cells depleted of lamin A are less resistant to strain and become more deformable (Broers et al, 2004;Lammerding et al, 2006Lammerding et al, , 2004, whereas a higher expression of lamin A and knockdown of lamin B1 stiffens nuclei and impairs cellular migratory capabilities Rowat et al, 2013;Shin et al, 2013). One of the defining domains in lamin A that determines its visco-elastic properties is the Ig fold (Bera et al, 2014). When lamin A is exposed to shear stress in isolated nuclei, the Ig fold partially unfolds leading to stretching of the lamin molecule .…”

Implications and Assessment of the Elastic Behavior of Lamins in Laminopathies

“…However, it is of interest to elucidate how the mutations that induce secondary and tertiary structural changes at the molecular level [89] play a role in altering the stretching of the domains in response to external forces. Although earlier reports elucidating the mechanical properties of a single vimentin filament had been addressed by atomic force microscopy [90], single molecule pulling experiments on nuclear intermediate filaments using atomic force microscopy were lacking, which has been adequately addressed by Bera et al [91,92]. Single molecule pulling experiments by atomic force microscope cantilevers of defined spring constants have been elegantly used to show a stretching of molecular domains on the application of picoNewton forces.…”

“…Although it has been speculated that the uncoupling of the nucleoskeletal framework corresponds to poor force transduction from the exterior to the nuclear interior, no report has ever shown how the mutations in lamin A that cause laminopathies alter the “springiness” of the otherwise spring-like elastic protein. Recently, how lamin A protein domains unfold in response to externally applied force (Figure 1) and that response is abrogated as a consequence of mutations has been demonstrated via single molecule force spectroscopy [91,92]. In separate experiments, rods 1B and 2B, and Ig-fold domains, were pulled at an average of 1000 nm/s rate.…”

“…R453W in the Ig-fold unfolded at lower force, and this data was corroborated with a steered molecular dynamic simulation where the sequential unfolding of 9 beta strands was observed. It was conclusively shown that R453W, the mutant causing AD-EDMD was intrinsically unfolded, thereby rendering the network weaker and less resilient to force [91]. Interestingly, statistics reveal that the majority of the mutations in the central alpha helical rod domain are principally concentrated in the 1B domain, and this domain possesses a mechanical resilience of up to 200% strain.…”