Schmidt, Christoph CFSLi, Mingru2025-07-022025-07-022025https://hdl.handle.net/10161/32788<p> Many processes of eukaryotic cells involve motion and deformation in overdamped environments, which require constant force generation. To calculate forces from observed deformations, we need to know the mechanical response properties of the structures making up cells. Using optical trapping technology and microscopy, I studied the mechanical properties in three different systems: individual suspended cells, isolated nuclei, and other cellular components. The main contributor to the mechanical response of suspended A431 cells is the actin cortex. I attached polystyrene beads directly to the actin cortex and used optical tweezers to measure force-displacement relationships and force fluctuations from individual cells under different conditions. The qualitative changes in mechanical responses to external forces were first examined using the Kelvin-Voigt model. This simplified model was applied to the force-displacement curves to extract an effective stiffness and an effective damping coefficient. In growth medium, I found that A431 cells have an apparent stiffness of 50~310pN/µm, and an apparent damping coefficient of 5 „ 35pN sec/µm. Drug interference tests were conducted to examine the roles of different components of the actin cortex. Actin polymerization inhibition led to a significant decrease in effective stiffness, whereas myosin activity inhibition only led to lower-amplitude force fluctuations. The effect of non-physiological osmolarity was also tested. Hypoosmolarity showed negligible effects on the mechanical responses. In contrast, in a hyperosmotic environment, cells showed a stiffer response on average and start to show signs of cytoplasmic jamming transition. Because the Kelvin-Voigt model only delivers global values for mechanical response, and does not permit any conclusions on the material properties of specific cellular structures, I next used a more detailed model for finite-element analysis consisting of an elastic spherical shell surrounding a viscous interior, with a volume constraint, to derive elastic moduli of the cell cortex and viscosity of the cytoplasm from the force-displacement curves. The Young’s modulus of the elastic shell for cells in growth medium was found to be 6~20kPa, which is close to that of actomyosin networks measured in in vitro experiments. The viscosity of the interior ranges from 10 to 40pN sec/µm, which is only slightly higher than what has been measured in the cytoplasm. The second system I studied were isolated eukaryotic cell nuclei. To accurately capture the mechanical properties of the cell nucleus, we optimized an isolation method that can extract nuclei that are still encapsulated with a cell membrane, which keeps them alive for a couple of hours. The isolated nuclei were placed under an atomic force microscope to measure the force-indentation relation. As expected, the elastic response was highly nonlinear and shows hysteresis effects due to the viscosity of the cytoplasm. I further studied two model systems in vitro. First, the mechanical properties of an artificial peptide-RNA polymer, which forms condensates under correct conditions, were measured using one-point microrheology. The derived viscosity of the condensate in the low frequency regime is roughly three orders of magnitude higher than that of water, which is comparable to that of the cytoplasm. Second, we constructed a model system for collective cytoskeletal motility from the cytoplasmic extracts of Xenopus laevis eggs, enclosed in water-in-oil emulsion droplets. The dynamics of the network showed different collective non-equilibrium motion patterns and phase-separation behavior that depended on the droplet size. </p>https://creativecommons.org/licenses/by-nc-nd/4.0/BiophysicsActin CortexCell MechanicsMicrorheologyNucleusOptical TweezersDissertation