title: Divalent cations crosslink vimentin intermediate filament tail domains to regulate network mechanics.
abstract: Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca(2+) and Mg(2+), showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l(P) approximately 0.5 mum, and Young's modulus, Y approximately 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.
authors.affiliation: Department of Physics, Harvard University, Pierce 231, 29 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: Yi-Chia
authors.initials: YC
authors.lastname: Lin
authors.forename: Chase P
authors.initials: CP
authors.lastname: Broedersz
authors.forename: Amy C
authors.initials: AC
authors.lastname: Rowat
authors.forename: Tatjana
authors.initials: T
authors.lastname: Wedig
authors.forename: Harald
authors.initials: H
authors.lastname: Herrmann
authors.forename: Frederick C
authors.initials: FC
authors.lastname: Mackintosh
authors.forename: David A
authors.initials: DA
authors.lastname: Weitz
mesh_terms: D000595:Amino Acid Sequence; D001696:Biomechanical Phenomena; D002413:Cations, Divalent / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D055119:Elastic Modulus; D006801:Humans; D066298:In Vitro Techniques; D007382:Intermediate Filaments / Q000737:chemistry / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D012212:Rheology; D014746:Vimentin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure
chemical_list: D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014746:Vimentin