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

Strength of interactions in the Notch gene regulatory network determines patterning and fate in the notochord

Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning is promoted when ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate

The Correlation-Based Method for the Movement Compensation in the Analysis of the Results of FRAP Experiments

This paper presents a computational algorithm for the detection and compensation for intracellular movement in the FRAP experiments with focal adhesions in living cells. The developed approach is based on the calculation of correlation coefficient. It was validated on the series of the experimental datasets and shows the successful results in the comparison with other widelyestablished methods

Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

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

A solid‐phase transfection platform for arrayed CRISPR screens [Corrigendum]

Since the publication of this study, it has come to our attention that a citation to the study by Bulkescher et al (2017) was omitted from the Introduction. The following sentence should have been included in the introduction: “A previously reported solid‐phase reverse transfection method for proteins (Bulkescher et al , 2017) was used for the delivery of RNPs for three endogenous genes suggesting the potential of solid‐phase reverse transfection for CRISPR/Cas9‐based gene editing, despite its low efficiency”. We apologise for any inconvenience this omission may have caused

A solid-phase transfection platform for arrayed CRISPR screens

Arrayed CRISPR‐based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid‐phase transfection platform that enables CRISPR‐based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high‐throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies

A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition

BACKGROUND: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. RESULTS: By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. CONCLUSION: By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression

Algorithms to Recognize Musicial Excerpts, Notes and Chords

Рассмотрены существующие методы распознавания музыки, их применимость к распознаванию акапельного пения, а также способы их улучшения, одним из которых является транскрибирование звука в музыкальную нотацию. Предложены методы выделения нот и аккордов из звукового ряда.= The paper discusses existing methods of music recognition, its adaptability to recognition of acapella singing and methods of its improvement. One of the improvement methods is sound transcription to musical notation. Methods of note and accord extraction from sound series were proposed