Experimental Characterization and Theoretical Modeling of Circular Dorsal Ruffles

• Applicant: Professor Dr. Hans-Günther Döbereiner, Ph.D.
• Subject Area: Biophysics
• Term: from 2013 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 237405144

Circular Dorsal Ruffles (CDRs) are dynamic actin structures propagating on the dorsal cell side. Our goal is to understand the mechanism of CDR propagation. They play an important role in the uptake of growth factors via endocytosis and the reorganization of the cytoskeleton. We are concerned with experimental characterization and theoretical modeling of actin spatiotemporal dynamics with respect to free, branched, and polymerized actin, as well as CDR ultrastructure. We are interested to characterize and model inhibition of actin polymerization. In order to probe the system, we interfere with i) upstream signaling, ii) direct actin regulators, and iii) actin itself by biochemical and optogenetic means. Phenotypic statics and dynamics are altered as a function of growth factor and biochemcial inhibitor concentrations as well as expression levels of selected proteins. CDRs are prepared and observed as lateral waves in the closed circular space between the nucleus and the cellular periphery. They are forced to propagate on ring-like trajectories via disk-shaped micro-contact printed adhesion patches for cells of appropriate size. CDR propagation is characterized by mean velocity, lifetime and repeat frequency. We aim to verify the existing model describing CDRs as bistable states and to investigate the general role of fluctuations. There are two conceptually different effects to be analyzed. First, protein activity and density exhibit temporal fluctuations at overall constant number of molecules. Second, variations in gene expression alter the number of molecular copies. The first effect is mainly relevant on short time scales of a few minutes. On longer timescales the second effect becomes important. We expect random gene expression and drift to affect trajectories in state space, i.e., total (regulating) protein concentrations cannot be considered constant. This means, one is not just observing trajectories in a given fixed phase space, but the phase space itself is changing over time. Experimental tools used include, but are not limited to, optical microscopy (fluorescent, PH, DIC, RICM), microfluidics and microcontact printing, as well as optogenetics. Preparation of defined cell morphologies via adhesion onto disk-like domains ensuring defined boundary conditions turned out to be absolutely critical for reproducible data. Optogenetics allows to manipulate protein expression in vitro and observe in vivo variations in phenotype with genotype. Theoretical tools are image correlation analysis, fitting of numerical solutions to experimental data via parameter matching, and AI-based cluster analysis. Especially important is the identification of type and location of bifurcations of solutions in comparison to experiment. Detailed analysis allows to construct phase diagrams.

• DFG Programme: Research Grants