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Table of contents
Chapter 1: General Introduction to Cell Motility and the Actin Cytoskeleton
1.1 Cell shape changes and motility
1.2 Structures of cell motility
1.2.1 Lamellipodia
1.2.2 Filopodia
1.2.3 The cell cortex
1.2.4 Stress fibers and focal adhesions
1.3 Actin polymerization and dynamics
1.3.1 Actin in general
1.3.2 From monomers to filaments
1.3.3 Assembly dynamics
1.4 Actin polymerization regulatory proteins
1.4.1 G-actin binding proteins
1.4.2 F-actin regulating proteins
1.4.3 Cross-linkers of actin networks
1.4.4 Molecular motors
1.4.5 Actin nucleating proteins
1.4.6 Activators of actin polymerization
1.4.7 Putting all the ingredients together
1.5 Biomimetic approaches to study actin dynamics and actin-based motility
1.5.1 Listeria monocytogenes motility
1.5.2 Reconstitution of actin polymerization
1.5.3 Symmetry breaking and movement generation
1.5.4 Diversity of biomimetic systems
Chapter 2: Ena/VASP Proteins
2.1 Ena/VASP proteins in general
2.3 Role of Ena/VASP proteins in cells and in vivo
2.3.1 In lamellipodia and cell motility
2.3.2 In filopodia
2.3.3 In cell-substrate adhesions and stress fibers
2.3.4 In cancer
2.2 Ena/VASP domains and their functions
2.2.1 EVH1 domain
2.2.2 Proline rich domain
2.2.3 EVH2 domain
2.4 Modes of action of Ena/VASP and controversy
2.4.1 Nucleation activity
2.4.2 Anti-capping activity
2.4.3 Effect on barbed end elongation
2.4.4 Effect on Arp2/3 complex branching
Chapter 3: Experimental Methods in vitro
3.1. Actin network reconstitution on beads
3.1.1. DNA and proteins
3.1.2. Bead preparation
3.1.3. Actin polymerization on beads
3.1.4 Two-color experiments
3.1.5 Bead observation and data processing
3.2 Actin polymerization assessment by pyrene assay
3.3 Single filament assay by TIRF microscopy
3.4 Photoswitchable Arp2/3 complex inhibitors
Chapter 4: Ena/VASP Affects Polarized Actin Network Growth and Architecture
4.1 Introduction and open questions concerning the mode of action of Ena/VASP proteins
4.2 Results
4.2.1 Mouse VASP restores polarized actin network growth in the absence of capping protein
4.2.2 Mouse VASP is a barbed end elongation enhancement protein
4.2.3 Which VASP domains are necessary for restoring polarized growth in the absence of capping protein?
4.2.4 Aggressive nucleation at the surface can compensate for the absence of capping protein
4.2.5 VASP can compensate for reduced Arp2/3 complex in the network polarity establishment.
4.2.6 Actin network density and Arp2/3 complex levels increase at the bead surface in the presence of VASP
4.3 Conclusion and perspectives
Chapter 5: Small Molecule Photoswitchable Inhibitors of the Arp2/3 Complex
5.1 Introduction to inhibition of the Arp2/3 complex
5.1.1 CK-666
5.1.2 Photoswitchable Arp2/3 complex inhibitors based on CK-666
5.2.3 Attempts to improve solubility of LU06-type compounds
5.3 Conclusions and perspectives.
Chapter 6: Exploring Actin Architecture in vivo in Nematode Embryos
6.1 Introduction
6.1.1 Goal of the study
6.1.2 Asymmetric cell division
6.1.3 Symmetry breaking
6.1.4 Polarity establishment
6.1.5 Spindle positioning
6.2 Preliminary results actin visualization
6.2.1 Actin labeling of live embryos
6.2.2 Phalloidin labeling of fixed samples
6.2.3 Conclusions actin visualization
6.3 First tests rheology of nematode embryos
6.3.1 Optical trapping of endogenous granules
6.3.2 Tests with bead injection
6.3.3 Conclusions rheology
6.4 Overall conclusion and perspectives
6.5 Procedures and solution recipes
6.5.1 Worm manipulation and embryo isolation
6.5.2 Solutions
6.5.3 Polylysine slides
General Conclusion
References
