Misdirected vimentin messenger RNA alters cell morphology and motility

Abstract

Localized messenger RNAs were first observed as embryonic determinants that altered development when mislocalized. In recent years localized mRNAs have been found for several cytoskeletal proteins, including actin, vimentin and several microtubule associated proteins. We sought to determine whether redirecting mRNA for a cytoskeletal protein to an inappropriate address would alter cellular phenotypes. To do so we generated vimentin mRNAs with a myc epitope tag and the (beta)-actin 3′ untranslated region (3′ UTR) as a localization signal. When misdirected vimentin mRNAs are expressed in either fibroblasts or SW13 cells, cells develop numerous, extremely long processes; these cells also move more slowly to enter a wound of the monolayer. In situ hybridization revealed that the misdirected mRNA was often localized in the processes, in contrast to endogenous vimentin mRNA. The processes usually contained actin distal to the transgenic vimentin and microtubules proximal to it. SW13 cells lacking vimentin produced fewer and shorter processes, suggesting a dominant negative effect that involves recruitment of endogenous vimentin. Control experiments that transfected in constructs expressing tagged, correctly localized vimentin, or (beta)-galactosidase that localized through the (beta)-actin 3′ UTR, indicate that neither the shape nor the motility changes are solely due to the level of vimentin expression in the cell. This is direct evidence that the site of expression for at least one cytoskeletal mRNA alters the phenotype of the cell in which it is expressed. Messenger RNA localization is proving to be as essential for the normal maintenance of somatic cell phenotypes as embryonic determinants are for embryogenesis.