withaferin a alters intermediate filament organization, cell shape and behaviorwithaferin改变中间丝状体组织,细胞形状和行为.pdfVIP

Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior 1 1 2 1 1 Boris Grin , Saleemulla Mahammad , Tatjana Wedig , Megan M. Cleland , Lester Tsai , 2 1 Harald Herrmann , Robert D. Goldman * 1 Department of Cell and Molecular Biology, Feinberg School of Medicine at Northwestern University, Chicago, Illinois, United States of America, 2 Division Biophysics of Macromolecules, German Cancer Research Center, Heidelberg, Germany Abstract Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet