| dc.description.abstracteng | Cellular shape and the internal structure of living cells are defined by a dense network of three independent filament systems (actins, microtubules and intermediate filaments) termed "the cytoskeleton". Of these three systems, intermediate filaments (IFs) are the most genetically diverse class. A unifying feature of IFs is their formation from extended coiled-coil shaped proteins. Vimentin is one member of the IF protein family that is found in cells of mesenchymal origin such as fibroblasts. In vitro, vimentin filaments assemble hierarchically from tetrameric precursors of about 5 nm diameter and 60 nm length, which form stable complexes at low ionic strength, to several mircometers long, 10 nm wide filaments. This assembly process can be initiated by increasing the ionic strength, e.g. by the addition of monovalent cations, i.e. sodium or potassium. The hierarchical assembly process includes several stages, such as the formation of unit length filaments (ULFs, about 17 nm diameter and 60 nm length). Here, we investigate how the cross-section of both filaments and intermediate stages of the assembly process changes for different cation concentrations.
Small angle X-ray scattering (SAXS) allows us to study the structures of macromolecules in solution. In order to access intermediate stages of the assembly process in situ, we design and characterize microfluidic mixers which are compatible with SAXS. These mixers allow for precise control of the ionic environment of intermediate states.
For vimentin filaments, we observe an increase of diameter with increasing monovalent ion concentration. Divalent ions, such as magnesium, result in significantly thicker filaments and a qualitatively different cross-sectional structure. In addtion, we are able to identify the ULF stage of assembly in situ and to show that its diameter also increases with increasing potassium concentration.
The results of this thesis help to deepen our understanding of the structural changes of vimentin during the later stages of the filament assembly process, thereby providing a building block in the picture of how changes in the vimentin network affect the mechanical properties of cells. | de |