Vimentin Filament Assembly is Altered by Substrate Elastic Modulus

Abstract

Background: The cytoskeletal protein vimentin is involved in the determination of cell mechanical properties, shape and motile behavior. We are investigating how vimentin participates in the response to external mechanical cues.Methods: Normal and mouse embryo fibroblasts (mEF) harvested from the vimentin-knockout mouse are grown on fibronectin-coated polyacrylamide gels (Fn-PAA) of 0.5-40 kPa. Protein expression levels are assayed by Western blotting, and vimentin network distribution by immunofluorescence. Rates of vimentin subunit turnover are assayed by fluorescence recovery after photobleaching (FRAP) experiments; and atomic force microscopy (AFM) is used to measure cells’ elastic and viscoelastic-plastic properties.Results: Vimentin protein expression levels do not change in response to substrate stiffness. Whereas FRAP results in other cell types - which are less phenotypically responsive to substrate stiffness - show no difference in the rate of vimentin subunit turnover across various substrates, early results suggest that subunit turnover increases when fibroblasts are grown on physiological-range stiffnesses (∼6 kPa). Also, many short vimentin filaments and squiggles (<15 µm in length) are evident that appear not to be connected to the extended vimentin network, and vimentin is more detergent-soluble under these conditions. The responses (e.g. shape, motility, etc.) of vimentin-null fibroblasts confirm vimentin's participation in these processes across the range of substrate stiffnesses. Finally, vimentin-null mEF are less stiff than mEF on ∼6 kPa Fn-PAA, and initial experiments show that whether vimentin makes cells softer or stiffer depends upon the substrate elastic modulus.Conclusion: Vimentin is expressed at consistent levels across conditions that cause changes in cell stiffness, shape and motility, yet the response of vimentin-knockout cells to the same conditions demonstrates vimentin's role in response to mechanical stimuli. We hypothesize that modulation of vimentin's assembly state underlies its contribution to cell mechanics.NIH-NHLBI NRSA, NIH PO1GM096971