Vimentin Intermediate Filament Softening - Recovery Behavior and Post-Translational Modifications

The capability of eukaryotic cells to resist large deformations, to transport intracellular cargo and to adapt the cell shape, for example during migration, depends on the cytoskeleton, a composite network of three types of filamentous proteins: actin filaments, microtubules and intermediate filaments (IFs). The physical properties of cells are controlled by the architecture of these cytsokeletal filaments and networks and different expression levels of various proteins or protein modifications lead to variation of the mechanical properties of cells. The individual components of the cytoskeleton differ in their diameter, their assembly process, their mechanical properties and interaction partners as well as their function. Whereas actin and tubulin are expressed throughout all cell types, different types of IF proteins are expressed in a cell-type specific manner. In general, IFs are believed to function as supporters for the mechanical resistance of cells. Over the past few years, particularly the role of the IF protein vimentin gained importance for its role in cell mechanics. Furthermore, different IF proteins seem to be highly involved in diseases such as cancer: the IF protein vimentin, for instance, is linked to tumorigenesis, epithelial-to-mesenchymal transition and the metastatic spread of cancer. In this thesis the mechanics of individual vimentin filaments are probed by using an optical trapping setup which is combined with microfluidics and confocal imaging. Optical traps allow for contact-free measurements of individual proteins in pure buffer and by combining them with microfluidics, the protein environment can be varied easily while simultaneously observing changes in the mechanics. To be able to compare the results of single filament mechanics to network mechanics, we measure vimentin networks at different conditions with passive microrheology. This method allows us to obtain mechanical properties of networks by tracking micrometer-sized particles embedded in the network. From these particle tracks the mean-squared displacement can be calculated and thereby the viscoelastic response of the network can be derived.2021-09-2