Purification of Mammalian Vimentin via the Reassembly of BJ5TAa IF Method

Abstract

The protein vimentin is an intermediate filament and is part of the cytoskeleton in cells and tissues. The cytoskeleton is comprised of three total protein-based components that undergo liquid-liquid phase separation (LLPS). Liquid-liquid phase separation occurs when proteins or nucleic acids (macromolecules) condense into what resembles liquid droplets. A major factor concerning whether a solution will undergo LLPS is the concentration and identities of the macromolecules and the solution. Vimentin is associated with multiple human diseases including cancer, Crohn’s disease, HIV, and cataracts. One area that has not been studied fully is the purification of mammalian vimentin, which is an intermediate filament. Intermediate filaments, along with microfilaments and microtubules, are the basis of the cytoskeleton in eukaryotic cells. These entities are interconnected, helping to give the cell its shape while also regulating mechanical, spatial, and biochemical properties of the cell. The name intermediate filament was first established when it was discovered that IF were localized between actin and myosin in muscle cells. The Weitz Lab has reported the formation of a reconstituted network containing physiologically relevant concentrations of all three cytoskeletal proteins: F-actin, microtubules, and vimentin intermediate filaments (VIFs). They have characterized the structure of the network using scanning and transmission electron microscopy as well as confocal fluorescence microscopy. In this work, multiple purification methods for vimentin were considered; ultimately, the method that was selected for the vimentin purification was reassembly of BJ5TAa IF in MEF cells.