Active and Passive Microrheology of F-Actin Membrane Composites

Abstract

The complex mechanical properties of a living cell are not only a function of its structural components but also of the organizational super-structure and the dynamic interconnection of filaments, proteins and the plasma membrane. Especially, the F-actin cortex plays a central role in cell adhesion, migration, division, growth and differentiation. Abnormalities in these essential cellular processes are tightly connected to diseases such as cancer and malaria. It is thus of pivotal interest to study the determinants of cellular mechanics.The main part of this thesis is dedicated to the investigation of a minimal cortex model of the F-actin cortex. A pre-polymerized network of semi-flexible F-actin polymers is attached to a lipid bilayer by the physiological cross-linker ezrin. A pseudophosphorylated mutant of ezrin, T567D, is used to study the impact of transient membrane linkage on the frequency dependent viscoelastic properties. Here, passive as well as active microrheological measurements are established on these thin composite materials and frequency spectra ranging from 10 -3 -10 2 Hz are measured. In an intermediate frequency regime (10 -2 -10 1 Hz) the elastic properties dominate the force response of the model system. The stiffness of the system is dominated by the mesh size of the self-organized model cortex, which is in turn a function of the availability of pinning-points in the membrane. These findings suggest the formation of an affine network. The low frequency regime (10 -3 -10 -2 Hz) of the shear modulus is dominated by the transient binding kinetic of the membrane cross-link ezrin. An apparent unbinding rate constant is determined from the microrheological data and the transient nature of the membrane attachment is supported by a low energy barrier for the unbinding. For the high frequency regime (10 1 -10 2 Hz) deviations from the typically found power law scaling of ¾ are observed and discussed in the context of increased inertia upon F-actin attachment to a solid supported model membrane. The minimal actin cortex model is compared to entangled networks of the semi-dilute F-actin filament as well as F-actin membrane composites isolated from the apical cortex of living cells by the sandwich cleavage method.In the second part of this thesis, the focus is set on mechanotransduction and rigidity sensing in epithelial monolayers. Cell mechanics in response to surface elasticity are studied by active atomic force microscopy based microrheology and interpreted in terms of (active) soft glassy rheology. Epithelial cells from the kidney (MDCK II) and mammary gland (MCF-10A) are studied as a function of substrate elasticity (E=1-100 kPa). Cells cultured on soft substrates (1 kPa) exhibit similar frequency dependent viscoelastic properties as cells, which are F-actin depleted by latrunculin A. Both cell lines behave stiffer when cultured on surfaces of higher elasticity. Above a certain threshold of substrate stiffness no further changes can be observed upon increase in surface stiffness. This final cortic...