"Cell traction force is generated in the cytoskeleton by actomyosin activity and plays an important role in many cellular processes. In previous cell traction force experiments performed by our lab, unexpectedly large variations were measured. Because these experiments were utilizing a cell population of randomized phase, and there had been no documented investigation into whether cell phase affected cell traction force generation or propagation, it was hypothesized that there would be a significant difference in traction force between S phase and the other phases of interphase, as the physical and chemical changes happening within the nucleus at this time might elicit changes within the cytoskeleton. To test this hypothesis, we characterized the time-evolution of traction forces from a population of synchronized 3T3 fibroblasts. 3T3 fibroblasts were synchronized in G1-phase via serum starvation. The transition times between cellular phases during the first cell cycle after synchronization were identified by BrdU and Hoechst staining at different time points. After phase transition times were approximated, the traction forces of 9 cells were measured in 4-hour intervals for 24 hours. The differences between traction forces measured in G1, S, and G2 phases are not significant, demonstrating that cellular phase does not significantly affect traction force magnitude."

The ability of cells to probe their mechanical environment and react to external stimuli is critical for maintenance of their normal structure and function. Through connections to the extracellular matrix, cells can sense mechanical cues such as substrate rigidity and stretch, and through force transmission across their contractile cytoskeleton can react accordingly to those signals by applying contractile traction forces to their surrounding environment. Healthy cells can react to these mechanical cues to maintain their cytoskeletal prestress (tension) at a set or homeostatic level over time, a phenomenon known as tensional homeostasis. Progression of certain diseases such as asthma, atherosclerosis, and cancer have been linked to a loss of tensional homeostasis. As such, tools for quantifying the traction forces that adhered cells apply to their substrate are crucial for gaining a better understanding of not just how healthy cells interact mechanically with their environment, but also how changes to the extracellular matrix or mutations within the cell can impact their ability to maintain tensional homeostasis and therefore remain both functional and viable. Our group has previously developed a method of quantifying cellular traction forces using indirectly pattered, soft hydrogel substrates known as micropattern traction microscopy. This method was initially developed to create discrete grid micropatterns, which while useful for measuring cellular traction forces does not offer any ability for the user to control cell growth area shape or size. This technique was further improved on through the creation of a protocol for changing discrete grid patterns into isolated island micropatterns, but this two-step process was challenging and generated islands of inconsistent shape and size. Here, we propose a new method for generating isolated island micropatterns of essentially any desired shape and size in a single step, as well as a corresponding image analysis algorithm for calculating cellular traction forces from these island micropatterns. Additionally, this dissertation also includes an investigation into the impact of distinct Epithelial-cadherin mutations on the ability of gastric adenocarcinoma (AGS) cells to achieve tensional homeostasis. Disruption of tensional homeostasis in the epithelium is a hallmark of certain cancers, and mutations in E-cadherin proteins have been identified in malignant epithelial cells. Here, through analysis of AGS cell traction force data collected previously by Dr. Han Xu during her dissertation work, we have found that two distinct mutations in the intracellular domain of E-cadherins have an impact on the ability of AGS cells to achieve tensional homeostasis.

"In this thesis, a cell traction force microscopy method is developed for measuring traction forces of connective tissue cells. This method includes an improved methodology in traction force microscopy of live cells cultured on an elastic substrate. Tissue cells, such as skin and muscle cells respond to the mechanical stimuli of their microenvironment by adhering to their substrate and exerting forces on the proteins of the extracellular matrix (ECM). These forces are called cell traction forces. Fibroblasts are grown on polyacrylamide (PA) gels embedded with fluorescent beads and coated with different types of ECM ligands. Traction forces of NIH 3T3 fibroblasts are calculated from the measured deformations of PA gels by using a 3-D finite element method. The advantages of this method compared to the traditional methods of cell traction force microscopy (CTFM) are that this method takes into account the finite thickness of the substrate by applying a 3-D FEM analysis to reduce the errors of using an infinite half space approximation for a substrate with a finite thickness and that it uses a novel method for embedding the substrate with fluorescent markers that decreases the measurement uncertainties. In our approach fluorescent beads were embedded on the top of substrate instead of getting mixed with the gel. This decreases the effect of out-of-focus fluorescent beads on the measured deformation fields which enhances the accuracy of cell traction force measurements."

Les dysfonctionnements vasculaires ou les blessures induites par l'âge, le tabac, les traumatismes ou une hyperlipidémie font partie de la myriade de facteurs de risques qui contribuent à la pathogénèse de nombreuses maladies cardiovasculaires. Un objectif important de la biologie vasculaire est de comprendre les processus cellulaires qui favorisent ou protègent contre ces maladies vasculaires. Cette pathogénèse est étroitement associée avec le dysfonctionnement de la paroi interne des vaisseaux sanguins. Cette paroi est constituée par une monocouche de cellules endothéliales qui forment l'endothélium vasculaire. La réparation de l'endothélium implique le remodelage des adhésions focales (AF) et des jonctions adhérentes (JA). Des modifications dans la composition protéique de ces structures adhésives génèrent des forces qui sont à la base du remodelage et de la réparation de l'endothélium. Dans la littérature, les forces cellulaires sont étudiées sur des cellules isolées, des doublets de cellules ou des ilots de cellules en croissance mais les forces mécaniques qui s'exercent au sein d'un tissu doivent encore être caractérisées. Dans cette thèse, nous utilisons la Microscopie à Traction de Force (TFM) sur des substrats en polyacrylamide pour étudier l'équilibre mécanique entre les jonctions intercellulaires et les adhésions cellule/substrat. Nous analysons dans quel mesure la TFM peut être utilisée pour étudier des monocouches cellulaires et présentons une nouvelle approche pour extraire les forces contractiles exercées par un tissu endothéliale. Finalement, nous utilisons cette méthode pour caractériser les forces transmises par les cellules à leur substrat et les forces contractiles pour une monocouche endothéliale. Cette méthode fournit un outil intéressant pour étudier la contribution de certaines protéines des jonctions adhérentes sur les forces transmises au sein de l'endothélium. / Vascular dysfunction or injury induced by aging, smoking, inflammation, trauma, hyperlipidaemia are among a myriad of risk factors that contribute to the pathogenesis of many cardiovascular diseases. An important objective in vascular biology is to understand cellular processes that promote or protect against cardiovascular diseases. This pathogenesis is very closely associated with dysfunction of the inner face of the vessel wall. The inner face of the vessel wall is lined by a monolayer of endothelial cells forming the vascular endothelium. Reparation of the endothelium involves remodelling of focal adhesionns (FA) and adherent junctions (AJ). Modifications in the protein composition of these adhesive structures generate forces at the basis of endothelium remodelling and reparation. In the literature, cellular forces are studied on single cells, epithelial cell doublets or cell aggregates in growth but mechanical forces inside tissues remains to be characterized. In this thesis, we use traction force microscopy (TFM) on polyacrylamide substrates to study the mechanical equilibrium between intercellular junctions and cell/substrate adhesion. We analyse to which extent TFM can be used for studying monolayers and present a novel approach to extract contractile forces exerted by an endothelial tissue. Finally, we use this methodology to characterize forces transmitted to the substrate and the contractile forces of endothelial monolayers. This method provides an interesting tool to study the contribution of some proteins of the adherent junctions to force transmission within the endothelium.

No / The ability of a cell to adhere and transmit traction forces to a surface reveals the cytoskeleton integrity of a cell. Shear sensitive liquid crystals were discovered with new function in sensing cell traction force recently. This liquid crystal has been previously shown to be non-toxic, linear viscoelastic and sensitive to localized exerted forces. This paper reports the possibility of extending the application of the proposed liquid crystal based cell force sensor in sensing traction forces of osteoblast-like (MG-63) and human keratinocyte (HaCaT) cell lines exerted to the liquid crystal sensor. Incorporated with cell force measurement software, force distributions of both cell types were represented in force maps. For these lowly contractile cells, chondrocytes expressed regular forces (10 – 90 nN, N = 200) around the circular cell body whereas HaCaT projected forces (0 – 200 nN, N = 200) around the perimeter of poly-hedral shaped body. These forces are associated with the organisation of the focal adhesion expressions and stiffness of the LC substrate. From the results, liquid crystal based cell force sensor system is shown to be feasible in detecting forces of both MG63 and HaCaT.

Ce manuscrit de thèse est composé de trois parties dédiées aux développements technologiques nécessaires à l'étude de la polarité et des contraintes mécaniques dans les cellules épithéliales. La première partie décrit les développements technologiques et méthodologiques qui ont été réalisés en micro-fabrication et traitement de surface, acquisition et analyse d'image, et mesure des forces de traction. La deuxième partie décrit l'étude de l'organisation spatiale du système d'adhérence des cellules épithéliales. De la régulation de leur polarité à celle de leur fonction, l'architecture des cellules épithéliales est profondément liée à leur système d'adhérence. Nous avons utilisé les micropatrons adhésifs pour contrôler la géométrie de la matrice extra-cellulaire pour examiner l'effet de l'adhérence des cellules avec la matrice sur la position des zones d'adhérence intercellulaire. Nos résultats montrent que l'organisation spatiale de l'adhérence cellule-matrice joue un rôle déterminant sur celle de l'adhérence intercellulaire. Ils montrent également que cette organisation dirige ensuite la position du centrosome et l'orientation de l'ensemble de la polarité interne. Lors d'une réorganisation spatiale de l'épithélium, comme c'est le cas au cours de la transition épithélium-mésenchyme, les systèmes d'adhérence et la polarité interne subissent tous les deux de profondes modifications. Néanmoins, les cellules semblent capables de les réguler de façon indépendante selon le type de stimulus qui induit la réorganisation. La dernière partie est une analyse des paramètres physiques impliqués dans l'architecture épithéliale. En parallèle des régulations biochimiques, les contraintes mécaniques jouent également un rôle fondamental dans la régulation des processus morphogenétiques. L'association de l'ensemble de nos développements technologiques (patterning de substrat déformable, logiciel de détection et de mesure de force, contrôle du positionnement des cellules) nous a permis d'analyser précisément les propriétés mécaniques des architectures multicellulaires. Nous avons découvert que l'organisation spatiale du système adhérence était un régulateur majeur de l'intensité et de la répartition des forces intra-cellulaires. Cette observation nous a permis de proposer une modification du modèle actuel de distribution des contraintes dans un épithélium qui prend en compte l'anisotropie des forces inter-cellulaires en réponse à l'hétérogénéité de la matrice extra-cellulaire. Ce nouveau modèle physique permet de rendre compte des positions adoptées par les cellules en réponse aux différentes géométries de la matrice extra-cellulaire. / This thesis dissertation is comprised of three major parts. The first part devotes to all the technological developments that have been realized in my thesis study. These developments in microfabrication, in image acquisition and analysis, and in the traction force analysis had solved various problems we have encountered during our study of epithelial architecture. The second part describes the study of the spatial organization of the adhesion systems in epithelia. From their polarity, their functioning, to their remodeling, the epithelial architecture is deeply linked with the adhesion systems. With the capability to well define the location of cell-matrix interaction, we examined how the intercellular adhesion was organized according to the cell-matrix adhesion. Our results highlighted the instructive role of cell-matrix adhesion in organizing the intercellular adhesion. This organization subsequently governed the internal polarity which was indicated by the centrosome positioning. During epithelial remodeling, both the adhesion system and internal polarity were subjected to modification. Nevertheless they could be regulated differently depending on the context of remodeling. The last part is focused on the physical aspect of the epithelial architecture. Apart from the biochemical signaling network, mechanical force is also a substantial ingredient in morphogenesis. Together with our techniques in micropatterning the soft gel, the development of software for traction force microscope, and our knowledge of cell-cell positioning, we were able to analyze precisely the mechanical property of the multicellular architecture. We found that the cellular contractility was modulated by the spatial organization of the adhesion system. It permitted us to complete the current physical model of epithelial geometry with an anisotropic term for contractility. This new physical model could effectively account for the cell positioning on various matrix geometries.

Ce travail nous a permis de proposer un cadre pour sonder la morphogenèse d'une micro-colonie bidimensionnelle. Plus particulièrement, nous avons exploré la manière dont les effets individuels de croissance et d'adhésion se combinaient au cours de la croissance de la micro-colonie. Nous avons montré (i) que l'adhésion de cellules isolées est asymétrique du fait d'un vieux pôle plus ancré et (ii) que l'allongement des bactéries peut induire des forces de poussée à l'intérieur des colonies. Dans la mesure où ces deux effets, combinés à l'échelle d'une micro-colonie, sont susceptibles de générer des contraintes mécaniques, nous avons développé une technique pour mesurer les forces d'adhésion résultantes à l'aide de substrats déformables. Nous avons ainsi démontré que des adhésions focales sont créées et rompues dynamiquement, avec un biais au vieux pôle des cellules. Nous avons aussi examiné le rôle de l'adhésion sur la forme des colonies. Nous avons montré que l'adhésion polaire était responsable de la transition d'un régime de croissance linéaire à un régime bidimensionnel qui est observée après la première division. Pour des colonies de taille plus importante, le niveau d'adhésion était aussi corrélé avec la forme globale des colonies. Enfin, l'adhésion est aussi impliquée dans la transition d'une colonie bidimensionnelle à une colonie tridimensionnelle. L'ensemble de ces résultats suggère que l'expression des adhésines ainsi que leur localisation à la surface des cellules pourraient permettre aux bactéries de moduler activement la forme du groupe dans lequel elles vivent. / In this work, we propose a framework to understand the morphogenesis of two-dimensional microcolonies. In particular, we have explored how growth and adhesion of individual cells compete during microcolony extension. We have shown (i) that isolated cells display an asymmetry in their adhesion, which is higher at the old pole, (ii) that bacterial elongation can result in pushing forces inside the colony. Since the combination of these two effects is expected to produce mechanical stress at the scale of the microcolony, we have developed a method to measure the resulting adhesion forces using deformable substrates. We have demonstrated that focal adhesions are dynamically established and ruptured, with a bias towards the old poles. We have also probed the role of adhesion in the shape of the colony. We have shown that polar adhesion drives the transition from a linear to a two-dimensional growth after the first division. At larger colony sizes, the level of adhesion continues to correlate with the global shape of the colony. Finally, adhesion is involved in the transition from a two-dimensional to a three-dimensional colony. Taken together, our results suggest that the expression of adhesins and their location at the surface of the cells could be levers by which bacteria actively modulate the shape of the group in which they reside.

Calcific aortic valvular disease (CAVD) is the most common valvular pathology in the developed world. CAVD results in calcifications forming on the aortic valve leaflets, inhibiting proper closure and causing complications of stenosis and regurgitation. Although, the mechanisms behind the disease initiation are unknown, it is believed to be a cell-mediated phenomenon, and not the result of passive degradation of the valve as once believed due to the increased prevalence with age. Currently, there are no pharmaceutical options for the prevention or reversal of calcifications, the only treatment option is complete valve replacement, an imperfect solution. Hindering the development of potential therapeutics is that currently there are no adequate animal models which replicate the calcification and cell death seen in disease explanted valves. An in vitro model has been develop where valvular interstitial cells (VICs), the main cell type of the valve, are seeded at high density into tissue culture polystyrene dishes and cultured with TGF-β1. This results in VICs activating to the myofibroblast phenotype and forming cell aggregates. Due to currently unknown mechanisms, apoptosis occurs within the center of the aggregates and calcification ensues. Although simplistic, this model has been used to show that rate and frequency of aggregation is affected by cellular tension; conditions of high tension increase aggregation response, while conditions of low tension prevent aggregation and calcification from occurring. It is important to note; however, that despite its wide usage, the current model is limited as the aggregation and subsequent calcification are random occurrences and are not consistent across literature where same conditions for control samples are used. The motivation of the presented work is two-fold. First, high intracellular tension has been suggested as one of the mechanisms leading to disease in the valve. Despite the clear and important role of cell tension, VIC tension has never before been measured in a dynamic environment. The ways in which dynamic stimulation affects individual VIC tension is not known. In aim one, a method is developed to allow for long-term cyclic stretch of VICs with measurement of cell traction force. It was found that cyclic stretch decreased cell tension in cells with high prestress and increased cell tension for conditions of low prestress. Combined, these findings indicate a homeostatic cellular tension which is dependent upon the mechanical environment. In the second aim, a novel method for creating VIC aggregates is validated. Micro-contact printing, essentially “stampingâ€� of a protein in a defined pattern, is used to create circular aggregates on polyacrylamide gels. This method allows for the separation of the aggregation from the subsequent calcification, an improvement over the current in vitro model. The method is then used to explore the role of the distribution of tension in the initiation of disease

In this dissertation, I utilize digital holographic microscopy (DHM) to study the motility of biological cells. As an important feature of DHM, quantitative phase microscopy by digital holography (DH-QPM) is applied to study the cell-substrate interactions and migratory behavior of adhesive cells. The traction force exerted by biological cells is visualized as distortions in flexible substrata. Motile fibroblasts produce wrinkles when attached to a silicone rubber film. For the non-wrinkling elastic substrate polyacrylamide (PAA), surface deformation due to fibroblast adhesion and motility is visualized as tangential and vertical displacement. This surface deformation and the associated cellular traction forces are measured from phase profiles based on the degree of distortion. Intracellular fluctuations in amoeba cells are also analyzed statistically by DH-QPM. With the capacity of yielding quantitative measures directly, DH-QPM provides efficient and versatile means for quantitative analysis of cellular or intracellular motility. Three-dimensional profiling and tracking by DHM enable label-free and quantitative analysis of the characteristics and dynamic processes of objects, since DHM can record real-time data for micro-scale objects and produce a single hologram containing all the information about their three-dimensional structure. Here, I utilize DHM to visualize suspended microspheres and microfibers in three dimensions, and record the four-dimensional trajectories of free-swimming cells in the absence of mechanical focus adjustment. The displacement of microfibers due to interactions with cells in three spatial dimensions is measured as a function of time at sub-second and micrometer levels in a direct and straightforward manner. It has thus been shown that DHM is a highly efficient and versatile means for quantitative tracking and analysis of cell motility.

Ce manuscrit de thèse est composé de trois parties dédiées aux développements technologiques nécessaires à l'étude de la polarité et des contraintes mécaniques dans les cellules épithéliales. La première partie décrit les développements technologiques et méthodologiques qui ont été réalisés en micro-fabrication et traitement de surface, acquisition et analyse d'image, et mesure des forces de traction. La deuxième partie décrit l'étude de l'organisation spatiale du système d'adhérence des cellules épithéliales. De la régulation de leur polarité à celle de leur fonction, l'architecture des cellules épithéliales est profondément liée à leur système d'adhérence. Nous avons utilisé les micropatrons adhésifs pour contrôler la géométrie de la matrice extra-cellulaire pour examiner l'effet de l'adhérence des cellules avec la matrice sur la position des zones d'adhérence intercellulaire. Nos résultats montrent que l'organisation spatiale de l'adhérence cellule-matrice joue un rôle déterminant sur celle de l'adhérence intercellulaire. Ils montrent également que cette organisation dirige ensuite la position du centrosome et l'orientation de l'ensemble de la polarité interne. Lors d'une réorganisation spatiale de l'épithélium, comme c'est le cas au cours de la transition épithélium-mésenchyme, les systèmes d'adhérence et la polarité interne subissent tous les deux de profondes modifications. Néanmoins, les cellules semblent capables de les réguler de façon indépendante selon le type de stimulus qui induit la réorganisation. La dernière partie est une analyse des paramètres physiques impliqués dans l'architecture épithéliale. En parallèle des régulations biochimiques, les contraintes mécaniques jouent également un rôle fondamental dans la régulation des processus morphogenétiques. L'association de l'ensemble de nos développements technologiques (patterning de substrat déformable, logiciel de détection et de mesure de force, contrôle du positionnement des cellules) nous a permis d'analyser précisément les propriétés mécaniques des architectures multicellulaires. Nous avons découvert que l'organisation spatiale du système adhérence était un régulateur majeur de l'intensité et de la répartition des forces intra-cellulaires. Cette observation nous a permis de proposer une modification du modèle actuel de distribution des contraintes dans un épithélium qui prend en compte l'anisotropie des forces inter-cellulaires en réponse à l'hétérogénéité de la matrice extra-cellulaire. Ce nouveau modèle physique permet de rendre compte des positions adoptées par les cellules en réponse aux différentes géométries de la matrice extra-cellulaire.