Deciphering vimentin assembly: Bridging theoretical models and experimental approaches

Abstract

The intricate assembly process of vimentin intermediate filaments (IFs), key components of the eukaryotic cytoskeleton, has yet to be elucidated. In this work, we investigated the transition from soluble tetrameric vimentin units to mature 11-nm tubular filaments, addressing a significant gap in the understanding of IF assembly. Through a combination of theoretical modeling and analysis of experimental data, we propose a novel assembly sequence, emphasizing the role of helical turns and gap filling by soluble tetramers. Our findings shed light on the unique structural dynamics of vimentin and suggest broader implications for the general principles of IF formation.

Keywords: Cytoplasmic intermediate filament; Structural assembly; Theoretical modeling; Tube-like filament; Vimentin.

Fig. 1

The configurations for vimentin assembly. (A) Representation of the vimentin monomer as a cylindrical model. The head and tail regions, depicted as slim cylinders, contrast with the thicker cylinders representing the rod domain. Color coding was used for clarity: coil 1a is green, coil 1b is orange, coil 2 is violet. The boundary of the stutter (residues 344-354) is depicted as a dotted line. The midpoint residues listed in Table 1 are represented by gray shapes: a circle represents Glu191, a triangle represents Val258, and a star represents Arg342. The black dotted lines indicated in the figure represent the positions of the residues mentioned in Table 1. (B) The central rod domains of the A11 tetramer were arranged around Glu191 in a staggered fashion. This structure has a total length of approximately 65 nm, with a central overlapping region measuring 27-nm and a 19-nm overhang from coil 2. The midpoint of the A11 arrangement is presented as a gray circle and labeled, although the arrangement is indicated by a red dotted line. (C) A pair of A11 tetramers in the A22 arrangement (indicated by a red dotted line) centered around Arg342 are represented by a gray triangle and labeled. This complex spans more than 110 nm in length. Its cross-section mainly contains 4 alpha helices, but in the ACN overlap region, the interaction between the C-terminal section of coil 2 and the N-terminal part of coil 1a creates a 6 alpha helix cross-section. Adding another A11 tetramer increases the length of the complex by 45 nm. (D) A pair of A11 tetramers in the A12 configuration centered around Val258. The midpoint is illustrated with a label, while the central axis is denoted by a red dotted line. The A12 configuration of 2 A11 tetramers results in an 86-nm long assembly. The cross-section features 8 alpha helices in the interacting region. The addition of another A11 tetramer extends the assembly length by 20 nm.

Fig. 2

Comparison of high-order arrangements with multiple A11 tetramers. (A) Elongation of filaments occurs exclusively through the A22 configuration, which is illustrated schematically, resulting in filaments with a thickness of 3.5 nm resembling the lamin protofilament. The A22 interaction features a 45 nm repeat spacing with the overlapping region that consists of the 6-helix cross-section spaced 23 nm and 22 nm apart. The gray square box at the bottom indicates the expected electron density of the filament cross-section, with the darker gray shading indicating more densely packed helical regions. (B) A filament was grown in the A12 configuration, constructed using A11 tetramer units with a uniform spacing of 20 nm. The entire structure is characterized by a staggered parallelogram configuration with an estimated thickness of 6 to 7 nm. The lower part of the figure illustrates the expected perpendicular projection, as observed via transmission electron microscopy (TEM) (Herrmann et al., 1996).

Fig. 3

A tubular representation of vimentin (Top) and its cross-sectional cut (Bottom) through the vimentin bundle are shown. The cylinder is composed of 10 coiled-coil structures, each featuring 40 helices in its cross-section. The planar representation in the bottom panel corresponds to the two-dimensional surface lattice model for vimentin intermediate filaments proposed by Peter M. Steinert and colleagues (Steinert et al., 1993). The interlock, or ACN arrangement, overlaps with the green lines. The directions of the four threads are indicated by arrows matching the colors of the A11 tetramers. The pitch (P) and lead (L) of the spirals are described.

Fig. 4

A possible pathway for merging four A12-driven parallelogram structures. (A) The growth of a filament via the A12 arrangement is depicted as a long, striped-pattern ribbon with a distinct twist, indicative of its initial configuration. This ribbon is subject to fluctuations, leading to subsequent twisting and rolling motions. (B) The lateral edges of filaments are susceptible to separation due to their exposure and weak interactions. This results in the unraveling of the coiled-coil termini, creating an open configuration at the edges. This parallelogram linearly exhibited the capacity for reversible lateral interactions with other parallelogram vimentin structures followed by the A22 arrangement. (C) The complete assembly model, featuring 4 parallelograms arranged in an A22 layout, each driven by the A12 configuration. The segments are colored violet, orange, cyan, and brown. The structure is notable for its flexibility and is able to bend or twist along the axis of the resulting complex's growth.

Fig. 5

Overall mechanism of the transition from the primer to the mature vimentin tubular structure. (A) The complete assembly model from Figure 4A with an altered aspect ratio was extended vertically to enhance the understanding of the vimentin assembly configuration. This model consists of 4 threads, indicated by the arrows in the color scheme: violet, orange, cyan, and brown. Each thread represents the A12-driven parallelogram structure in the same color scheme as the arrow. The folding lines of the layers are drawn as gray dotted lines. The layers are sequentially labeled from the (n)th to the (n + 10)th layer. Each layer represents a lamin-like protofilament. The orange and red bars mark the locations where the A22 interaction occurs; these are identified as the sites produced by outward folding along the dotted folding lines between the layers. (B) The primer structure was notably altered by internal A22 interactions (red and orange bars). Both the frontal and lateral perspectives demonstrate the folding of a parallelogram plane comprising 10 layers and 4 threads. The lateral view particularly highlights the twist of the parallelogram, with the (n + 10)th layer looping back to the (n)th layer in a helical pattern. The left panel shows a cross-section of the primer, where the red spiral symbolizes the N-terminal head domain extending from the rod domain, which plays a role in spiral interactions, as mentioned previously (Eibauer et al., 2024). (C) The stabilization of the primer structure through the attachment of A11 tetramers to the indentations at both ends results in the formation of a mature nucleus for filament elongation. The magnified view inside a black box highlights how a tetramer integrates into the indentation, facilitated by both A12 and A22 interactions. (D) Elongation model of vimentin filaments originating from the stabilized nucleus. New A11 tetramers are incorporated into the nucleus through indentation at both ends. For clarity in distinguishing the 4 threads, the direction of each thread is indicated by arrows in the same color code. The model details the pitch, indicative of the protrusion of an A11 tetramer into the adjacent thread, measuring 45 nm. Additionally, the length of the spiral arrangement within the same thread was shown to be 180 nm, aligning with the structure observed in cryo-ET (Eibauer et al., 2024).