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

Abstract

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 β-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.

Transcriptome analysis reveals vimentin-induced downregulation of cell-cell associations augments cancer cell migration

“…Although it is not clear how Liu and co-workers observed the effect of vimentin at individual cell level [4], it could be due to the tag used in their study. In this regard, we have recently shown that the N-terminally tagged vimentin only forms aggregates that align along intercellular junctions in an epithelial colony, and although the tagged vimentin integrates into the pre-existing network, the laments were found to be weaker [33].…”

“…The full-length human vimentin cDNA was sub-cloned in pLPChygro as described previously [33]. The constructs were named pLPChygro-FV (full-length vimentin) and pLPChygro-CV (control vector).…”

“…When 70-80% con uent, cells were washed twice with PBS and lysed using 250 µl/well Laemmli lysis buffer (2% sodium dodecyl sulphate (SDS), 20% glycerol, 125 mM Tris-HCl, pH 6.8). SDS gel electrophoresis and western blotting were performed as described previously [37]. ECL prime detection reagent (cat # PRN2232, GE Healthcare, UK) and ChemiDoc (BioRad, UK) were used to visualise protein bands.…”

Transcriptome Analysis Reveals Vimentin-Induced Disruption of Cell–Cell Associations Augments Breast Cancer Cell Migration

“…When 70–80% confluent, cells were washed twice with PBS and lysed using 250 μL/well Laemmli lysis buffer (2% sodium dodecyl sulphate (SDS), 20% glycerol, 125 mM Tris-HCl, pH 6.8). SDS gel electrophoresis and Western blotting were performed as described previously [ 55 ]. ECL prime detection reagent (Cat # PRN2232, GE Healthcare, Lisburn, UK) and ChemiDoc (BioRad, Watford, UK) were used to visualise protein bands.…”

“…The full-length human vimentin cDNA was sub-cloned into pLPChygro as described previously [ 55 ]. The constructs were named pLPChygro-FV (full-length vimentin) and pLPChygro-CV (control vector) .…”