Etude des facteurs régulant la production de force de traction par les cellules

Mechanical forces are involved in many physiological processes including morphogenesis, migration, division and differentiation. All these events involve a tight regulation of both the magnitude and spatial distribution of the contractile forces at the cell and tissue-level. Although we know that at the molecular level, the regulation of force production and transmission relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton, we still do not have a comprehensive understanding of the mechanisms supporting force generation at the entire cell level. In addition, the magnitude of the traction forces exerted by cells on their underlying extracellular matrix in culture and as such the cell to cell variation of theses forces remain difficult to predict, largely because of the difficulty to characterize precisely how molecular components forming the actomyosin and adhesion networks, individually or via their specific interplay, are related to the force magnitude exerted by these cells. In this context, the aim of my PhD project was to investigate how key biological parameters are precisely related to force generation and regulation process.The first part of my study thereby focused on looking into the effect of the progression of the cell cycle on cell to cell heterogeneity in traction forces. I demonstrated that although the cell cycle status of the cells had a major impact on the magnitude of forces exerted by cells, it was not impacting the overall cell to cell variability in the traction force exerted. I also examined next the possibility that some internal/subcellular contractile efforts could be dissipated instead of being transmitted to cell anchorages by looking at the interplay between actin dynamics and traction forces. The analysis of actin turnover in stress fibers showed that although variations in strain energies were associated to variations in actin dynamics, they were not significant enough to explain the large cell to cell heterogeneities measured in traction force. Finally, I conducted a study dedicated to the characterization of the biochemical composition of the actomyosin network and adhesion pattern of cells in relationship with the force generated and transmitted by cells. To that end, I implemented the standard TFM assay in order to introduce an intermediate labeling step allowing for simultaneous measurement of traction forces and intracellular protein contents. This assay was then used to characterize the content of molecules of the actomyosin cytoskeleton and of the adhesions, either alone or in combination, and force. This work first demonstrated that the vinculin content measured at the level of the entire cell and the area of the focal adhesions, represented good predictors of force. I then showed that actin and myosin displayed broader deviations in their linear relationship to the strain energies, and thus appeared as less reliable predictors of force. Instead, my data suggested that their relative cellular amount plays a key role in setting the magnitude of force exerted by cells. I finally demonstrated that although the alpha-actinin content was not correlated at all with force magnitude, the relative amount of alpha-actinin as compared to actin content was of key importance to regulate force production.In conclusion, these results identified the biochemical contant of focal adhesion and the relative amounts of molecular motors and crosslinkers per actin as key parameters involved in setting the magnitude of force exerted by cells and thereby shed new light on the mechanisms supporting force generation at the entire cell level.La production de forces mécaniques est impliquée dans de nombreux processus physiologiques incluant la morphogénèse, la migration, la division ou encore la différentiation. Tous ces évènements requièrent une régulation précise de la magnitude des forces et de leur distribution spatiale à l’échelle de la cellule et du tissu. Bien que la production de ces forces au niveau moléculaire repose sur l’interaction complexe d’un petit nombre de protéines bien identifiées, la régulation de ces forces aussi bien que leur mécanisme de transmission à l’échelle de la cellule entière demeure encore mal compris. D’autre part, prédire la magnitude des forces exercées par les cellules sur leur matrice extracellulaire ou leur variation s’avère impossible, en particulier à cause de la difficulté à caractériser précisément comment la composition des réseaux intracellulaires d’acto-myosine et celle des adhésions est reliée de manière individuelle ou combinée, à ces dernières. Dans ce contexte, l’objectif de ma thèse a été d’investiguer comment des paramètres biologiques clés sont impliqués précisément dans les processus de génération des forces et leur régulation.La première partie de mon étude s’est portée sur l’effet de la progression dans le cycle cellulaire sur l’hétérogénéité des forces développées par les cellules. J’y ai ainsi démontré que le statut des cellules dans le cycle cellulaire, bien qu’ayant un impact majeur sur la magnitude des forces exercées par les cellules, n’impactait pas la variation des forces intercellulaires. J’ai par la suite examiné la possibilité qu’une partie des efforts contractiles internes/sub-cellulaires soit dissipée au lieu d’être transmise aux ancrages cellulaires en étudiant l’interconnexion entre la dynamique de l’actine et les forces de traction. L’analyse du renouvellement dynamique de l’actine dans les fibres de stress a ainsi montré que bien que le turnover de l’actine variait en relation avec les modulations de forces, ces variations n’étaient pas suffisamment significatives pour expliquer l’hétérogénéité des forces mesurées entre les cellules. Pour finir, j’ai conduit une étude dédiée à la caractérisation de la composition biochimique du réseau d’actine et des adhésions en relation avec la force exercée et transmise par les cellules. Pour se faire, j’ai tout d’abord implémenté la technique classique de TFM en y incorporant une étape intermédiaire d’immunomarquage afin de pouvoir suivre simultanément les forces exercées par les cellules ainsi que leur composition biochimique. Cet essai a ensuite été appliqué à la caractérisation du lien entre le contenu intracellulaire de plusieurs protéines du cytosquelette d’actomyosine et des adhésions, seules ou combinées entre elles, avec la force. Ces travaux ont tout d’abord démontré que le contenu en vinculine, mesuré à l’échelle de la cellule entière, et l’aire des adhésions, constituaient de bons prédicteurs des forces cellulaires. J’ai par ailleurs pu montrer que l’actine et la myosine exhibaient quant à elles des déviations plus larges dans leur relation linéaire avec l’énergie mécanique des cellules, et représentaient ainsi des prédicteurs moins directs des forces. Au contraire, les données obtenues suggèrent que c’est leur composition relative dans la cellule qui détermine la magnitude des forces exercées par les cellules. J’ai enfin pu mettre en évidence que la quantité d’alpha-actinine cellulaire seule ne corrélait pas du tout avec les forces, mais que sa quantité relative par rapport à l’actine jouait un rôle clé pour réguler la production des forces.En conclusion, ces résultats ont permis d’identifier la composition biochimique des adhésions, ainsi que le contenu relatif des moteurs moléculaires et crosslinkers par rapport à l’actine comme des paramètres essentiels de la régulation de la production des forces

Climate of a school class

The theoretical part of the thesis deals predominantly with the climate of a school class, with the class being viewed as a social group. The focus is on the pupil as a member of both formal as well as informal groups and on his/her social behaviour

Regional variations in the nonlinearity and anisotropy of bovine aortic elastin

Arterial walls have a regular and lamellar organization of elastin present as concentric fenestrated networks in the media. In contrast, elastin networks are longitudinally oriented in layers adjacent to the media. In a previous model exploring the biomechanics of arterial elastin, we had proposed a microstructurally motivated strain energy function modeled using orthotropic material symmetry. Using mechanical experiments, we showed that the neo-Hookean term had a dominant contribution to the overall form of the strain energy function. In contrast, invariants corresponding to the two fiber families had smaller contributions. To extend these investigations, we use biaxial force-controlled experiments to quantify regional variations in the anisotropy and nonlinearity of elastin isolated from bovine aortic tissues proximal and distal to the heart. Results from this study show that tissue nonlinearity significantly increases distal to the heart as compared to proximally located regions (). Distally located samples also have a trend for increased anisotropy (), with the circumferential direction stiffer than the longitudinal, as compared to an isotropic and relatively linear response for proximally located elastin samples. These results are consistent with the underlying tissue histology from proximally located samples that had higher optical density (), fiber thickness (), and trend for lower tortuosity () in elastin fibers as compared to the thinner and highly undulating elastin fibers isolated from distally located samples. Our studies suggest that it is important to consider elastin fiber orientations in investigations that use microstructure-based models to describe the contributions of elastin and collagen to arterial mechanics

The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces

International audienceThe regulation of cellular force production relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton: actin, myosin, and α-actinin. Despite our deep knowledge of the role of these proteins in force production at the molecular scale, our understanding of the biochemical regulation of the magnitude of traction forces generated at the entire-cell level has been limited, notably by the technical challenge of measuring traction forces and the endogenous biochemical composition in the same cell. In this study, we developed an alternative Traction-Force Microscopy (TFM) assay, which used a combination of hydrogel micropatterning to define cell adhesion and shape and an intermediate fixation/immunolabeling step to characterize strain energies and the endogenous protein contents in single epithelial cells. Our results demonstrated that both the signal intensity and the area of the Focal Adhesion (FA)–associated protein vinculin showed a strong positive correlation with strain energy in mature FAs. Individual contents from actin filament and phospho-myosin displayed broader deviation in their linear relationship to strain energies. Instead, our quantitative analyzes demonstrated that their relative amount exhibited an optimum ratio of phospho-myosin to actin, allowing maximum force production by cells. By contrast, although no correlation was identified between individual α-actinin content and strain energy, the ratio of α-actinin to actin filaments was inversely related to strain energy. Hence, our results suggest that, in the cellular model studied, traction-force magnitude is dictated by the relative numbers of molecular motors and cross-linkers per actin filament, rather than the amounts of an individual component in the cytoskeletal network. This assay offers new perspectives to study in more detail the complex interplay between the endogenous biochemical composition of individual cells and the force they produce

MCF7 cells at the time of release from circle (A), square (B) and cross (C) shaped constraints are shown.

<p>A representation of the regions chosen as the vertices (red), sides (blue) and intersections (green) in the three shapes are indicated in the boxes. Scale bar = 500 μm.</p

Cluster speeds show temporal differences in the different regions of the circle (A), square (B) and cross (C) shapes respectively.

<p>Speeds from the sides increase (I), plateau (II) and finally decrease (III) over time. These results show clear differences in cluster edge speeds which depend on spatial location in the geometry. Significant differences (p<0.05) in speeds are indicated in each figure.</p

Velocity fields and vorticity maps for MDCK cells are shown at 2 hours (A, D), 8 hours (B, E) and 16 hours (C, F) after removal of constraint.

<p>The cluster border is also shown at 0 hours for reference. Cells in the vertices show minimal movement as compared to those in the sides and intersections (D, E, F). We see the development of vortices, in both clockwise (blue) and counter clockwise (yellow-red) directions at intersections by 16 hours. Scale bar = 250 μm.</p

Migrations of MCF7 cells located on the advancing edges of the circle (A), square (B) and cross shapes (C) were obtained using individual cell tracking and are illustrated on a windrose plot to show the cell migration trajectories from the cluster edge.

<p>We see differences in motility that depend on the cell spatial positions which are labelled corresponding to sides, vertices and intersections.</p

Velocity and vorticity fields in MCF7 cell monolayers in the circle (A), square (B), cross vertex (C) and intersections (D) at 30 hours summed every hour.

<p>The solid black line is the cluster border at the start of migration. Scale bar = 250 μm.</p