Structure and Mechanics of Membrane-bound Vimentin Filaments and Networks under Strain
von Sarmini Nageswaran
Datum der mündl. Prüfung:2022-08-29
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Sarah Köster
Diese Datei ist bis 28.08.2023 gesperrt.
EnglischThe shape and mechanical properties of eukaryotic cells are determined by the cytoskeleton, composed of actin filaments, microtubules and intermediate filaments (IFs). Among these, IFs are considered to be the main determinants of cell stiffness and strength. The reason for this is that they can withstand much larger deformations than the other two filament classes. Previous studies have revealed a rim-and-spoke arrangement of IFs, suggesting that they are involved not only in themechanical stability of the cytoplasm but also of the plasma membrane. Therefore, the organization of IFs at the plasmamembrane and their influence on the mechanical properties of cells under a variety of strains are of great interest. To mimic the rim arrangement at the plasma membrane under strain, the aim of this work was the development of an in vitro model system, with focus on the membranebound vimentin intermediate filaments (VIFs) that allows for uniaxial stretching. To investigate the rim arrangement, an elastic solid support, namely polydimethylsiloxane (PDMS), was used to enable lateral stretching of the composite system due to its molecular flexibility. The hydrophobic PDMS with embedded fluorescent beads was oxidized to render the surface hydrophilic, and by spreading small unilamellar vesicles (SUVs), a lipid bilayer was formed. The assembled VIFs coupled to ATTO647N and biotin were linked to the biotin-decorated lipid bilayer via a neutravidin linker. A lipid reservoir in the formof SUVs was added to provide the system with excess lipid material. Stretching of this composite system was achieved by a uniaxial motor-driven stretching device. Performing stretching of individual membrane-bound VIFs revealed mechanical and entropic stretching. The former means that elongation of the filament’s contour length upon stretching occurs, while for the latter pulling out the thermal fluctuations is observed. If the strain rate and pinning point density were increased, mechanical stretching rather than entropic stretching was increasingly observed. Additionally, the underlying lipid bilayer contributed to the reorientation of VIFs by diffusive reorganization or affected the strain transmission due to sliding and rupturing. The found properties of single membrane-bound VIFs might contribute to the mechanical response of membrane-bound VIF networks. Even though filaments in the networks mainly responded with entropic stretching, so far, there has been no access if mechanical stretching occurs or not. However, it can be speculated that the applied strains might be too low to observe mechanical stretching. In summary, an artificial model system was established that opens up a pathway to study the structural and mechanical properties of cytoskeletal components under strain in order to imitate the cell cortex in nature.
Keywords: supported lipid bilayer; fluorescence microscopy; atomic force microscopy; biotin-neutravidin linkages; vimentin intermediate filaments