Active Matter in Confined Geometries - Biophysics of Artificial Minimal Cortices
by Hanna Hubrich
Date of Examination:2020-12-07
Date of issue:2020-12-18
Advisor:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Sarah Köster
Referee:Dr. Sarah Adio
Referee:Prof. Dr. Michael Meinecke
Referee:Prof. Dr. Jörg Großhans
Referee:Prof. Dr. Silvio O. Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-8373
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Abstract
English
Essential physiological processes, such as cell motility, adhesion, growth and differentiation are determined by the ability of cells to modify their mechanical properties. Consequently, both the vitality as well as malignancy and thus the fate of the cells are often reflected in alterations of their viscoelastic properties. The viscoelasticity of cells is believed to be predominately regulated by the actin cortex, a thin transiently cross-linked actomyosin network containing several hundred actin binding proteins (ABPs), which is dynamically coupled to the plasma membrane by proteins of the ERM (ezrin-radixin-moesin) family. The multitude of different ABPs ensures on the one hand the resilience of the cell body and on the other hand, due to their transient nature, also provides sufficient fluidity to enable cell shape changes on long time scales. The highly dynamic biochemical processes in the active adaptive actin cortex and how they influence cell mechanics are still not fully understood. In this thesis, the contribution of the actin cortex to the viscoelasticity of cells based on the concept of continuum mechanics was studied – with the focus on architecture, motor activity and membrane-attachment – using different model systems as a mimic of the cellular cortex. First experiments on purely entangled 3D actin gels showed that network architecture and mechanics can be altered by the experimental setup, such as the construction of the measuring chamber and the choice of the illumination technique of the measuring method. In bead tracking microrheology, a light-induced softening of the network was found by fluorescent imaging, while non-fluorescent imaging showed no effect. Network softening was also observed in bulk rheology measurements of 3D actin gels in presence of the motor fragment protein heavy meromyosin (HMM) under ATP consumption. In minimal cell compartments (MCCs) created by polymerization of F-actin within water-in-oil droplets, full-length myosin II motors generated non-thermal fluctuations under excess ATP. These fluidization and fluctuations are proposed to be caused by filament sliding movements induced by active motors during ATP hydrolysis, mimicking the contractility of the cell cortex. In comparison to 3D actin networks, a significant increase of the network stiffness by a factor of about 6 was found in minimal actin cortices (MACs), where a quasi-two-dimensional F-actin network was crowded by methylcellulose (MC) on a solid supported membrane (SSM) and coupled to the membrane via an active mutant of ezrin and the receptor lipid phosphatidylinositol-4,5-bisphosphate (PIP2). In addition, actin filaments where observed to be about a quarter of the size in MACs compared to 3D networks. These results indicate that the dynamic attachment of F-actin to the membrane plays a key role in self-organization and viscoelasticity of the actin cortex. In order to measure rheological properties of the entire actin cortex components, in absence of the crowded cytoplasm and the stiff nucleus, apical cell cortices were isolated from living MDCK II cells, deposited on porous silica matrices and locally deformed with a sharp atomic force microscope (AFM) tip. The obtained force cycle data was described with a viscoelastic cortex model using a time-dependent area compressibility modulus that obeys the same universal scaling behavior as applied for living cells. In comparison to living cells, the apical cell cortices showed a reduced fluidity, which was partially restored by addition of exogenous ATP to reactivate myosin motors. A comparison with MACs showed higher cortex fluidity due to the absence of cross-links. From this it can be concluded that viscoelasticity of cells to external deformation is mainly determined by the components of the membrane cortex, in particular the actin cortex dynamically connected to the plasma membrane and the myosin motor activity controlled in an ATP-dependent manner.
Keywords: Top-down approach; Bottom-up approach; Myosin motor protein; F-actin; Minimal actin cortex; Apical cell cortex; Microrheology; Bulk rheology; Indentation
