Establishment Of Stable Lens Cell Lines Expressing Wild-type And E151K Mutant Vimentins

Vimentin is a type III intermediate filament protein that is expressed in endothelial and other mesenchymal cells. In the eye lens epithelial cells,vimentin is abundantly expressed with important functions. Structurally,vimentin has three domains. The central rod domain contains α helix connecting both the N-terminal head domain and the C-terminal tail domain. As a major cytoskeletal protein, vimentin in involved in connecting the nucleus and membrane as well as mediating signal transduction. The normal function of vimentin influences differentiation of lens cells, and assures cell movement since it is part of the pseudopodia moving machinery. The function of vimentin is affected by phosphorylation in which the protein kinase C is known to act on some of the phosphorylation sites. In addition, vimentin is highly expressed in cancer cells that acquire invasiveness. The normal function of the lens is closely related to vimentin function since mutations in vimentin gene cause its assembly disorder. A classic example is G596 A change in vimentin which codes for exon 1 with the production of the mutant protein E151 K. This mutant protein leads to vimentin assembly disorder,causing cataract formation. Since the substrate binding with SUMO generally has the same motif ΨKx E / D, where Ψ is a hydrophobic aminoacid, K is target lysine, x is any amino acid, so E151 K mutation will produce a very conservative SUMO sites. We speculate that the E151 K mutation may lead to sumoylation of the vimentin monomers, causing absence of vimentin assembly and resulting in cataractogenesis. To prove this hypothesis, we cloned the vimentin cDNA from mouse lens epithelial cells and conducted in vitro mutagenesis to generate the wild type and mutant E151 K vimentin expression constructs. Then, we transfected these constructs into mouse lens epithelial cells, α-TN4-1 and cultured the transfected cells in the presence of the screening drug, G418(600 ug/ml).After 4 to 6 weeks of screening with cell density of 40% confluence, the stable clones expressing both wild type and E151 K mutant vimentin were obtained. These cells were further examined by PCR, Western Blot and immunofluorescence to confirm their stable expression of either wild type or mutant vimentin. The establishment of the stable expression cell lines lays the necessary foundation for further determination whether E151 K caused vimentin assembly disorders by sumoylation.A comparison of the parent, wild type and E151 K mutant vimentin-transfected cells revealed contrast morphology of the three types of cells: the vimentin in normal α-TN4-1 cells in the far end is straight and slender; the wild type vimentin-transfected cells displayed an elongated extension of vimentin with obvious bending in extending area.The E151K-transfected cells, however, did not show extension, or thearea of extension is significantly reduced, and the vimentin around the nucleus displayed irregular shape.