Influence of Ions on the Assembly of Vimentin Intermediate Filaments
von Manuela Denz
Datum der mündl. Prüfung:2020-02-10
Betreuer:Prof. Dr. Sarah Köster
Gutachter:Dr. Thomas Burg
Gutachter:Prof. Dr. Ralf Ficner
EnglischThe human body consists of many different cell types, which all exhibit a cytoskeleton. This cytoskeleton is mainly composed of three different types of protein filaments, as well as cross-linkers and motor proteins. The correct interplay of the protein system is necessary for the proper function of the cytoskeleton. To fully understand the physical and chemical properties it is not only important to understand the function of each protein individually, but also the assembly process needs to be understood, as proteins can miss-fold which sometimes leads to aggregates which are not fully functional anymore. Intermediate filaments (IFs) are one of the three cytoskeletal filaments, which in contrast to the other two, have a hierarchical assembly pathway. In this thesis, the influence of ions on the assembly of vimentin, an IF often found in cells of mesenchymal origin, is investigated. By combining small angle x-ray scattering (SAXS) with microscopy (atomic force microscopy and fluorescence microscopy), information about the cross-section of the filament as well as the overall structure, e.g. single filaments or networks, can be obtained. Before the influence of ions with different valencies could be investigated, a new buffer system was established, which does not complex with the ions used in this study. By adding ions of different valencies to vimentin, it could be shown that the presence of monovalent ions leads to single filaments with a radius of roughly 6 nm, whereas multivalent ions (di-, tri-, and tetravalent) induce network formation and the filaments reach radii of roughly 10 nm before collapsing into very dense and insoluble networks. In addition to static experiments, the assembly process in flow is investigated by combining SAXS with microfluidics. In a first step, a reliable x-ray compatible microfluidic device solely made out of one material is established and characterized using gold colloids. In a second step the very early time points of vimentin assembly in the presence of potassium is observed. The results of this thesis helped to get a better understanding of the influence of ions on the assembly of vimentin IFs. It could be shown that there is a large difference in the assembly of vimentin in the presence of monovalent and multivalent ions. We could furthermore introduce a new x-ray compatible microfluidic device and monitor the first few ms of the assembly process.
Keywords: small angle x-ray scattering; intermediate filaments; vimentin; biophysics; protein self-assembly; microfluidic devices