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

Doctoral thesis (2012)

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 ... [more ▼]

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. [less ▲]

Quantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.

in PloS one (2010), 5(2), 9210

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers ... [more ▼]

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion. [less ▲]