Post-Translational Modifications Soften Vimentin Intermediate Filaments

Abstract

The mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of the cytoskeletal proteins, modulation of the network architecture and the interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording precise force-strain curves using optical traps. Whereas full phosphorylation leads to disassembly of IFs, partial phosphorylation results in softening of the filaments. We show that binding of the protein 14-3-3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14-3-3 may serve to preserve the softening and thereby the altered cell mechanics. By employing phosphomimetic mutants and complementary Monte Carlo simulations, we explain our observation through the additional charges introduced during.The cytoskeleton of eukaryotes consists of three filament systems -microtubules, actin filaments and intermediate filaments (IFs) -which form a complex biopolymer network. The exact composition of the cytoskeleton and the interplay between the three filament types are of great importance as they define the mechanical properties of different cell types. 1Microtubules and actin filaments are highly conserved throughout cell types and between organisms, whereas more than 70 human genes encode for IFs. 2 Although different IFs are expressed in a cell type specific manner, they all share the same hierarchical assembly pathway from monomers to filaments. The secondary structure of the monomers consists of an α-helical rod domain, flanked by intrinsically disordered head and tail domains. 3,4 During assembly, two monomers align and form parallel coiled-coil dimers, two dimers form antiparallel, half-staggered tetramers, and multiple tetramers constitute a unit-length filament (ULF) (see Fig. 1a). Subsequent longitudinal annealing yields elongated filaments with a diameter of about 10 nm. 5 This hierarchical structure of IFs, in contrast to polar microtubules or actin filaments that elongate by rapid growth at the plus-end, grants IFs their unique mechanical properties. [6][7][8] Here, we study the most abundant IF, vimentin, which is found in cells of mesenchymal origin. 5,9 Single vimentin filaments are highly extensible and can be elongated up to at least 4.5 fold. 7,10,11 During elongation, three regimes are observed in the force-strain curves: an initial linear (elastic) increase, a plateau region and a subsequent stiffening. 6,7,12,13 These

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging

“…Furthermore, because IF assembly and disassembly are regulated by phosphorylation and other posttranslational modifications (18), it will be interesting to investigate whether these modifications alter the axial repeat of IFs, reflecting changes in their structural organization. In vitro work already showed that phosphorylation of vimentin by adenosine 3′,5′monophosphate-dependent protein kinase A soften the filaments (44). Moreover, it would be also relevant to determine whether other IF types, such as keratins or neurofilaments, also have an axial repeat and also unfold their  helices cooperatively during filament stretching.…”

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging

“…Furthermore, because IF assembly and disassembly are regulated by phosphorylation and other post-translational modifications (18), it will be interesting to investigate whether these modifications alter the axial repeat of IFs, reflecting changes in their structural organization. In vitro work already showed that phosphorylation of vimentin by cAMP-dependent protein kinase A soften the filaments (44).…”

The vimentin cytoskeleton: when polymer physics meets cell biology

“…The Nterminus of vimentin is targeted by several protein kinases that phosphorylate neutral serine residues to bestow a negative charge, and protein arginine deiminases that convert positively-charged arginine to neutral citrulline [10]. These alterations of charge can lead to filament disassembly, and at lower extents of alteration can decrease the stiffness of the filament [33].…”