Dynamics and mechanics of adherent cells in the context of environmental cues
Impact of substrate topology, chemical stimuli and Janus nanoparticles on cellular properties
by Jan Henrik Rother
Date of Examination:2014-06-11
Date of issue:2015-03-25
Advisor:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Mikael Simons
Referee:Prof. Dr. Dirk Görlich
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Sarah Köster
Referee:Dr. Michael Meinecke
Persistent Address:
http://dx.doi.org/10.53846/goediss-4990
Files in this item
Name:150211_Dissertation_Rother_2014_SUB150.pdf
Size:9.94Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
Cellular behavior is influenced by many biochemical but also physical factors in the direct cellular environment. Thereby, cells not only react to external cues, the interaction between cells and their environment is also dependent on the properties of the cell itself. The endocytosis of nanoparticles for example depends on the intermolecular forces between plasma membrane and particle as well as on the mechanical properties of the membrane. In the first part of this thesis I focus on the interaction between inorganic Janus nanoparticles, a new type of nanomaterials, which possess amphiphilic properties, and model membranes. In coarse grain simulations it has been demostrated that incubation of membranes with these particles lead either to pore formation in the lipid bilayer or to tubulation and vesiculation by long-range attractive interaction between particles bound to the membrane. Conducting surface plasmon resonance spectroscopy experiments I show that the binding energy of the used inorganic Janus particles to a solid supported monolayer could be sufficient to induce tubulation of tension-free membranes but is to small to provide the energy necessary to form a vesicle. This result is confirmed by fluorescence microscopic examination of giant unilamellar vesicles serving as a model system for the plasmamembrane, which were treated with Janus particles. Vesicles incubated with Janus particles show inwards directed membrane tubes, while incubation of vesicles with isotropic control particles had no effect on the membrane or could be attributed to an osmotic gradient. However, uptake experiments into living cells and cytotoxicity assays show no obvious difference between spherical particles and Janus particles, which hints for a negligible contribution of nanoparticle-induced tubulation or vesiculation to cellular uptake of nanoparticles and cytotoxicity. On the one hand mechanical properties of the cell influence the interaction between the cell and its environment. On the other hand, mechanical properties of cells change in response to environmental cues. Therefore, in the next part, atomic force microscopy-based microrheology is used to measure frequency-dependent mechanical properties of cells in different conditions. Fixation of cells with different chemical fixatives and transformation of epithelial cells to mesenchymal cells lead to more solid-like mechanical properties, while interaction with the actin cytoskeleton lead to more fluid-like properties. A comparison between malignant cells and non-malignant cells shows that malignant cells are more fluid-like compared to their non-malignant counterparts. Furthermore, the influence of substrate topology on cellular mechanics and cytoskeletal arrangement is examined. Changing physical properties of the substrate such as stiffness or topography has been shown to affect plenty of cellular processes like migration, proliferation, morphology or differentiation. Here, I investigate the impact of porous substrates on cellular morphology, cytoskeletal organization and elasticity in the context of confluent epithelial monolayers. I found that cells eventually self-organize to match the geometry of the pore pattern and remodel their actin cytoskeleton to reinforce their adhesion zone. Cells fluidize with increasing pore size up to 2 µm but eventually become stiffer if grown on very large pores up to 5 µm. The adhesion of cells to substrates is further researched by application of metal-induced energy transfer fluorescence lifetime imaging, which is used for the first time for this purpose. The fluorescence lifetime of a fluorophore in proximity to a metal layer is a function of the distance between fluorophore and metal layer. Applying a quantitative model of this interaction facilitates locating the fluorophore with nanometer precision in the axial direction up to 200 nm above the metal layer. By staining of the plasmamembrane I was able to image to basal membrane of three different cell lines and follow spreading of cells with high axial resolution. The introduced method is not restricted to measurement of cell/substrate distance and can be used for applications, which necessitate axial nanometer resolution in a range up to 200 nm.
Keywords: Janus particles; porous substrate; atomic force microscopy; cell/substrate distance; microrheology