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Table of contents
1 The cytoskeleton
1.1 A network of crosslinked filamentous biopolymers
1.2 Functions of the cytoskeleton in eukaryotic cells
1.2.1 Actin filaments regulate cell shape and movement
1.2.2 Intermediate filaments maintain cellular integrity
1.2.3 Microtubules provide a structural framework and orchestrate intracellular trafficking
1.3 Intermediate filaments
1.3.1 Diversity of intermediate filaments in eukaryotic cells
1.3.2 Structure and assembly of intermediate filaments
1.3.3 Vimentin, an intermediate filament protein present in many cells
1.4 Microtubules
1.4.1 Dynamics of microtubules
1.4.2 Microtubules and post-translational modifications
2 Mechanical properties of the cytoskeleton
2.1 Measuring mechanical properties in cell biology
2.1.1 Cytoskeletal mechanics measurements in vitro
2.1.2 Whole-cell-scale, cortical and intracellular force measurements
2.1.3 Our approach: combining optical tweezers-based intracellular rheology with live cell imaging
2.2 Mechanics of microtubules in vitro
2.2.1 Anisotropic stiffness of microtubules
2.2.2 Variability in the measurements of microtubule flexural rigidity
2.2.3 Microtubule response to repeated mechanical stress
2.3 Mechanics of intermediate filaments in vitro
2.3.1 Networks of intermediate filaments: highly deformable and almost unbreakable
2.3.2 Individual intermediate filaments exhibit nonlinear strain-stiffening
2.4 Microtubules and intermediate filaments in cellulo
2.4.1 Measuring mechanics of cytoskeletal filaments in cellulo
2.4.2 Mechanical contribution of microtubules in cells
2.4.3 Mechanical contribution of intermediate filaments in cells
3 Mechanical coupling within the cytoskeleton
3.1 Cytoskeletal crosstalk involving vimentin intermediate filaments and microtubules
3.1.1 Crosstalk through molecular motors
3.1.2 Crosstalk through crosslinking proteins
3.2 Impact of cytoskeletal crosstalk on network organization and cell mechanics
3.2.1 Synergistic organization of cytoskeletal networks
3.2.2 Mechanical reinforcement mediated by cytoskeletal interactions
4 Aims of the PhD project
5 Materials and Methods
5.1 Cell culture
5.2 Immunofluorescence staining and fixed cell imaging
5.3 Live cell imaging of vimentin and tubulin in cellulo
5.4 Drugs targeting microtubules
5.5 ATP depletion
5.6 Optical tweezer-based microrheology
5.7 Data analysis: from the movies to the effective stiffness
5.8 Statistical tests
6 Results and Discussion
6.1 Mechanics of vimentin bundles and microtubules
6.1.1 In cellulo, vimentin bundles are stiffer than microtubules
6.1.2 Sequential deflections make vimentin bundles more rigid
6.2 Mechanical coupling between microtubules and vimentin intermediate filaments
6.2.1 The vimentin network does not play a key role in the mechanical properties of microtubules
6.2.2 Modifying microtubule stability affects vimentin mechanical behaviour
6.3 Study of a post-translational modification: acetylation
6.3.1 Acetylation leads to microtubule softening
6.3.2 Acetylated microtubules impact vimentin bundle mechanics
6.4 Preliminary results: role of ATP in cytoskeletal mechanics
7 Conclusion and Perspectives
