Mechanobiological Control of Circular Dorsal Ruffle Dynamics

Abstract

Dynamic structures of polymerized actin play a crucial role in different cellular processes. These include different kinds of actin waves in a multitude of cell types, like Dictyostelium, neutrophiles, macrophages and fibroblasts. These actin waves are connected to a remodeling of the cytoskeleton, cell protrusion and migration as well as the uptake of extracellular fluids, but their specific functions are still debated. One type of them are circular dorsal ruffles (CDRs), actin-based ring-like membrane undulations on the dorsal cell side of fibroblasts, which emerge after growth factor stimulation. A large number of macromolecules were shown to be localized in CDRs and to be crucial for CDR formation. However, to date, the detailed signaling pathway and the underlying mechanism of CDR formation including their molecular main players remain unknown. Different studies on CDRs described them as actin waves in an excitable system or as wavefronts in a bistable regime between two stable states of actin. However, other studies focused on the interaction between actin polymerization and the cell membrane via the interplay of curved membrane protein complexes. This thesis further investigates the mechanism underlying CDR formation. For this study, the morphology of cells is an essential effector for the dynamics of actin waves. Their complexity and dynamical remodeling pose a challenge to the comparability of data. Therefore, in this work, fibroblasts are shaped into well-defined morphologies by seeding them on disk-like adhesion patterns made of fibronectin. This enables to identify long-range interactions between different CDRs combined with the influence of stochastic perturbations and thus uncovers the important role of the membrane tension in CDR dynamics. In combination with microfluidics, the response of the actin wave machinery to biochemical interference with drugs that target different parts of the actin machinery is investigated. The system allows systematical measurements of CDR velocities, periodicities and lifetimes that are performed to carry out a before/after comparison of the treated cells for examining the influence of actin, PIP3 and N-WASP. It is observed a dependence of CDR velocities, periodicities and lifetimes on the total amount of actin leading to the conclusion of a direct regulating role of actin in CDR formation and propagation. Furthermore, it is found that the actin nucleator N-WASP plays a fundamental role in CDR formation but not in CDR propagation. Numerical solutions of wavefronts in a bistable regime of a model system on an annulus domain resemble experimentally gained data and further uncover a dependence of the stimulation threshold for propagating wavefronts on the total actin concentration. The results underline the hypothesis that CDRs can be considered as wavefronts in a bistable regime between two stable states of actin.