Simulation and computer-assisted analysis of actin cytoskeleton dynamics in living cells using fluorescence microscopy methods

Controlled in time and space by a variety of actin-binding proteins, assembly and disassembly of the actin cytoskeleton is involved in many biological and biophysical phenomena. In order to understand the dynamics of such a complex intracellular system quantitative time-lapse imaging approaches are required.
This thesis presents the results of the interdisciplinary project aimed at the quantitative evaluation of the effects of actin-binding proteins on actin turnover under physiological conditions. It combines fluorescence microscopy experiments, development of mathematical models and data processing tools to understand how regulatory proteins control actin dynamics. Confocal-microscopy-based Fluorescence Recovery After Photobleaching (FRAP) technique is a major experimental tool to measure the exchange of actin and actin-binding proteins between polymerised and monomeric pools. The developed models and computational algorithms allow to predict actin dynamics depending on regulatory proteins and to connect the experimental data to parameters characterising polymerisation dynamics, length and structures of actin filaments and activities of tested regulatory proteins.
To test the models and to evaluate the activities of actin binding proteins without cellular constrains I combined quantitative FRAP analysis with a biomimetic assay which allows to reproduce major features of actin motility. This combination of techniques was utilised to measure the influence of previously characterised actin-binding proteins working together on actin dynamics in a concentration-dependent manner. In particular we investigated how capping and severing of actin filaments influences actin exchange in a bulk meshwork generated from spatially restricted nucleation.
The experimental and analysis methods I developed were also used to investigate the dynamics of the actin cytoskeleton at focal adhesions of living cells. Taking as an example the focal adhesion protein zyxin and its binding partner Tes we addressed how the interactions of these proteins with actin regulate cytoskeleton dynamics.
Taken together, the developed approaches and collected data help to better understand how regulatory proteins control actin dynamics in living cells.