Monitoring intermediate filament assembly by small-angle x-ray scattering reveals the molecular architecture of assembly intermediates

Abstract

Intermediate filaments (IFs), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF ''building block'' have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris⅐HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coeficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A 11 mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section.3D structure ͉ vimentin

Hierarchical Structure Controls Nanomechanical Properties of Vimentin Intermediate Filaments

“…The total length of the dimer without the head and tail domain is <48.7 nm. This result is in agreement with experimental results where a range of 46-49 nm has been reported earlier [15,27]. From dynamical analyses of the molecular dynamics trajectory of the dimer at 300 K, we observe that the linker domains represent soft, hinge-like structures that connect much stiffer coiled-coil segments (see Movie S1).…”

“…The average diameter of the cross section of the dimer is d dimer <2 nm, and the average diameter of the tetramer is measured to be d tetramer <3.6 nm, in close agreement with experimental measurements of 3.4 nm [15] (the small difference may be caused by the interaction with other dimers in larger filaments that contain many dimers, which may lead to increased compaction of the structure). Based on these geometric measurements we estimate that the diameter of a non-compacted unit length filament is d ULF <18.3 nm, which is closely within the range measured in experiment, where values of 16-20 nm have been reported [33].…”

A novel coiled-coil repeat variant in a class of bacterial cytoskeletal proteins

“…This supports the hypothesis that the minimal assembly unit of FilP is a dimer, but that a dimer of dimers is a stable association, mediated by some part of the N-terminal region of each dimer; these would interact with each other or with a more C-terminal part of the partner dimer. This is reminiscent of the assembly of dimers-of-dimers in some IF proteins (Sokolova et al, 2006), but key interactions of the latter involve large, non-coiled-coil head domains which Scy and FilP appear to lack. The segment of AbpS implicated in mediating assembly beyond dimers straddles both CC7 and CC51 (resi- Table 7 Potential charge interactions in the hendecad units of the Scy CC51 domain.…”

“…Homologues of both actins and tubulins have been found in prokaryotes and they are essential for cell division and bacterial shape via the control of cell wall synthesis (Löwe and Amos, 2009;Jacobs-Wagner, 2005, 2007). A third class of cytoskeletal (and nucleoskeletal) protein, the intermediate filament (IF), has a variety of globular head and tail domains, but its filamentous nature results from its long a-helical domains winding around each other to form coiled-coil ''rods" which then bundle together (Herrmann and Aebi, 2004;Sokolova et al, 2006). Although a-helical coiled coils are ubiquitous in all kingdoms of life, true IF proteins have been found throughout metazoans but not conclusively in bacteria or plants.…”

Lateral association and elongation of vimentin intermediate filament proteins: A time-resolved light-scattering study

“…A ULF is 60 nm long and 17 nm in diameter, and thus SAXS is a highly suitable technique to observe the lateral assembly from tetramers to ULFs (5,11). Recently fluorescence labeling techniques have been developed for vimentin IFs (12,13), enabling us to observe the elongational growth of filaments 1 μm long or longer.…”