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Significant efforts have addressed the role of vimentin intermediate filaments (VIF) in cell motility, shape, adhesion and their connections to microfilaments (MF) and microtubules (MT). The present work uses micropatterned substrates to control the shapes of mouse fibroblasts and demonstrates that the cytoskeletal elements are dependent on each other and that unlike MF, VIF are globally controlled. For example, both square and circle shaped cells have a similar VIF distribution while MF distributions in these two shapes are quite different and depend on the curvature of the shape. Furthermore, in asymmetric and polarized shaped cells VIF avoid the sharp edges where MF are highly localized. Experiments with vimentin null mouse embryonic fibroblasts (MEFs) adherent to polarized (teardrop) and un-polarized (dumbbell) patterns show that the absence of VIF alters microtubule organization and perturbs cell polarity. The results of this study also demonstrate the utility of patterned substrates for quantitative studies of cytoskeleton organization in adherent cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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 protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

Abstract: Although intermediate filaments are one of three major cytoskeletal systems of vertebrate cells, they remain the least understood with respect to their structure and function. This is due in part to the fact that they are encoded by a large gene family which is developmentally regulated in a cell and tissue type specific fashion. This article is in honor of Ueli Aebi. It highlights the studies on IF that have been carried out by our laboratory for more than 40years. Many of our advances in understanding IF are based on conversations with Ueli which have taken place during adventurous and sometimes dangerous hiking and biking trips throughout the world. [Copyright &y& Elsevier]