Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly.

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with ß-catenin at cell-cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network.

Vimentin Is at the Heart of Epithelial Mesenchymal Transition (EMT) Mediated Metastasis.

Epithelial-mesenchymal transition (EMT) is a reversible plethora of molecular events where epithelial cells gain the phenotype of mesenchymal cells to invade the surrounding tissues. EMT is a physiological event during embryogenesis (type I) but also happens during fibrosis (type II) and cancer metastasis (type III). It is a multifaceted phenomenon governed by the activation of genes associated with cell migration, extracellular matrix degradation, DNA repair, and angiogenesis. The cancer cells employ EMT to acquire the ability to migrate, resist therapeutic agents and escape immunity. One of the key biomarkers of EMT is vimentin, a type III intermediate filament that is normally expressed in mesenchymal cells but is upregulated during cancer metastasis. This review highlights the pivotal role of vimentin in the key events during EMT and explains its role as a downstream as well as an upstream regulator in this highly complex process. This review also highlights the areas that require further research in exploring the role of vimentin in EMT. As a cytoskeletal protein, vimentin filaments support mechanical integrity of the migratory machinery, generation of directional force, focal adhesion modulation and extracellular attachment. As a viscoelastic scaffold, it gives stress-bearing ability and flexible support to the cell and its organelles. However, during EMT it modulates genes for EMT inducers such as Snail, Slug, Twist and ZEB1/2, as well as the key epigenetic factors. In addition, it suppresses cellular differentiation and upregulates their pluripotent potential by inducing genes associated with self-renewability, thus increasing the stemness of cancer stem cells, facilitating the tumour spread and making them more resistant to treatments. Several missense and frameshift mutations reported in vimentin in human cancers may also contribute towards the metastatic spread. Therefore, we propose that vimentin should be a therapeutic target using molecular technologies that will curb cancer growth and spread with reduced mortality and morbidity.