Thesis presented December 18, 2020

Abstract:
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.

Keywords:
Traction force, biochemical composition, actin

On-line thesis.