The unexpected structure and dynamics of vimentin networks

The cytoskeleton controls the physical properties of cells by regulating the shape and the internal organization changes of the cells when they grow, divide, and move (1). The main components of the cytoskeleton are actin, microtubules, and intermediate filaments. Their interactions build up the cytoskeleton as a composite material with complex mechanical properties, implement biological functions, and modulate the cell’s properties and behavior in response to biochemical signals as well as mechanical forces.

While the structure and dynamics of actin and microtubules in cells have been characterized in much detail, this is true to a lesser extent for intermediate filaments. Intermediate filaments are cell type specific, reflecting different mechanical properties required for different cell types. The best-studied member of the intermediate filament family, vimentin, is expressed in mesenchymal cells such as fibroblasts. It forms dense networks near the nucleus, which become sparser toward the cell periphery (Fig. 1). Imaging the structure of the vimentin network in cells is a challenge due to the high density of filaments. Even in the peripheral regions, where the network is sparse, distinguishing individual filaments from bundles of filaments or assemblies of parallel filaments remains challenging. The small persistence length of about 1 μm of vimentin filaments, which reflects their high flexibility, also contributes to the difficulty of observing individual filaments. Therefore, the organization of vimentin (and other intermediate filaments) in cells remains poorly understood.

Related to the question of how intermediate filaments are organized in cells is the question of how they interact with microtubules. Again, some things are known for vimentin. Vimentin filaments form parallel arrays closely associated with microtubules, and vimentin filaments and microtubules template each other to maintain persistent cell polarity during migration (3). Depolymerization of microtubules results in a collapse of vimentin filaments, and microtubules associated with vimentin filaments are more stable against drug-induced depolymerization (4). However, microtubule curvature is unaffected by vimentin density (5). Moreover, vimentin has been shown to move along microtubules (6).

Even though microtubules and vimentin filaments are known to interact, the mechanism of their interaction, especially within the native environment of a cell, remains a mystery. It could be based on bundling by perpendicular interactions, which would result in strong co-alignment, on sparse cross-linking, which would allow for a looser organization, or even only on the transport of vimentin along the microtubules, with transient and mobile contacts between the two filaments. Correspondingly, multiple proteins have been proposed to mediate interactions between microtubule and vimentin filaments, including kinesin (7), dynein (8), plectin (9), and microtubule-actin cross-linking factor (10), and direct interactions have been shown by in vitro optical trapping experiments (11).

A third question concerns the dynamics of vimentin filaments. The vimentin network is a dynamic structure, as vimentin is transported along microtubules. However, the unsolved challenge of discerning individual filaments in a vimentin network also means that it is unclear whether vimentin is moved along microtubules as individual filaments, bundles, or other larger assemblies.

To address these questions, Renganathan et al. (2) used a combination of two powerful methods that together give unprecedented details on the organization of the vimentin network in cells. To resolve the spatial organization of the vimentin network, they used a 3D EM method (focused ion beam scanning EM, FIB-SEM), in which a stack of SEM images is produced by ablating the cryofixed sample layer by layer with an ion beam. They imaged subvolumes of the cytoplasm in the vimentin-rich perinuclear region and in the peripheral region with a more dilute vimentin network, and reconstructed the vimentin network and its interaction with microtubules. With this method, they obtained a detailed 3D picture of the spatial organization of these filaments (Fig. 1).

The reconstructions reveal the intricate structure of the vimentin network and show that filaments are organized into diverse interconnected higher-order assemblies. The images show individual vimentin filaments, loosely connected assemblies of multiple filaments, as well as tightly coordinated bundles of vimentin filaments, in particular in the perinuclear region. Individual filaments span through different assemblies, providing a loose coupling between neighboring bundles. Likewise, the thickness of a filament assembly and the coupling between its constituent filaments vary along the length of the assembly, from a single disconnected filament to a loosely connected assembly to a tightly coordinated bundle.

The authors’ analysis shows that vimentin filaments are globally aligned approximately with microtubules but do not follow them closely locally. Rather than being precisely parallel to microtubules, individual vimentin filaments splay out of vimentin bundles to interact with microtubules at localized contact points. Comparing the organization near the nucleus and at the cell periphery, vimentin is more bundled in the vimentin-dense region near the nucleus and more closely aligned with microtubules in the cell periphery.

While the FIB-SEM approach provides detailed 3D structural information, it only provides a static picture. To visualize the dynamics of individual vimentin filaments, Renganathan et al. also developed a single-particle tracking approach based on SunTag labeling of vimentin, a vimentin fusion protein linked to a synthetic scaffold with many binding sites for fluorescent reporter proteins. This construct effectively places multiple copies of green fluorescent protein on a single vimentin subunit, resulting in bright and long-lived fluorescence. By limiting the expression level of the vimentin-SunTag, they achieved a low density of labeling, with less than one labeled vimentin subunit per filament and thus one bright spot per filament. This approach allowed the authors to track the motion of individual filaments over a long time and in an environment densely packed with other vimentin filaments.

They observed a variety of motility patterns, including unidirectional, bidirectional, and nondirectional motion, stationary filaments, and many combinations of these. Only a small fraction (8–10%) of all vimentin spots showed filament motility, similar in the perinuclear and peripheral region, indicating sporadic motility. The motility was dependent on microtubules, and the tracks of motile vimentin filaments followed microtubules via the molecular motors kinesin and dynein.

Most importantly, analyzing the correlations of the displacements between different filaments in the vimentin-rich region did not show the strong correlation expected for filaments in a tightly coordinated bundle that move together. Rather it appears that individual filaments move independently within the dense region, consistent with the loosely bundled structures seen in the FIB-SEM reconstructions.

Taken together, by combining two innovative imaging methods, the work of Renganathan et al. provides an unprecedented view of the intermediate filament network in cells, revealing both the structure of the vimentin network and giving a detailed view of the motility of individual vimentin filaments. Far from being a relatively static structural support, the vimentin network is shown as a versatile and dynamic component of the cytoskeleton. The results open up the exciting prospect for further research: In particular, it should be interesting to compare the intermediate filament networks in different cell types that express different intermediate filaments, to relate the network structure to the mechanical properties of the cell, and to examine how different properties of filaments (12) and different network structures work together to determine the mechanical properties of cells.