This thesis presents a whole-embryo finite element model of neurulation -- the first of its kind. An advanced, multiscale finite element approach is used to capture the mechanical interactions that occur across cellular, tissue and whole-embryo scales. Cell-based simulations are used to construct a system of constitutive equations for embryonic tissue fabric evolution under different scenarios including bulk deformation, cell annealing, mitosis, and Lamellipodia effect. Experimental data are used to determine the parameters in these equations. Techniques for obtaining images of live embryos, serial sections of fixed embryo fabric parameters, and material properties of embryonic tissues are used. Also a spatial-temporal correlation system is introduced to organize and correlate the data and to construct the finite element model. Biological experiments have been conducted to verify the validity of this constitutive model. A full functional finite element analysis package has been written and is used to conduct computational simulations. A simplified contact algorithm is introduced to address the element permeability issue. Computational simulations of different cases have been conducted to investigate possible causes of neural tube defects. Defect cases including neural plate defect, non-neural epidermis defect, apical constriction defect, and convergent extension defect are compared with the case of normal embryonic development. Corresponding biological experiments are included to support these defect cases. A case with biomechanical feedbacks on non-neural epidermis is also discussed in detail with biological experiments and computational simulations. Its comparison with the normal case indicates that the introduction of biomechanical feedbacks can yield more realistic simulation results.

Cell adhesion to the extracellular matrix (ECM) provides tissue structure and integrity as well as triggers signals that regulate complex biological processes such as cell cycle progression and tissue-specific cell differentiation. Hence, cell adhesion is critical to numerous physiological and pathological processes, including embryonic development, cancer metastasis, and wound healing, as well as biotechnological applications, such as host responses to implanted devices and integration of tissue-engineered constructs. During the adhesion process, integrin surface receptors bind ECM proteins, cluster, and associate with the actin cytoskeleton. Subsequent strengthening of the integrin/actin cytoskeleton interaction occurs via complexes of proteins known as focal adhesions. Due to the close association between biochemical and biophysical processes within adhesion complexes, mechanical analyses can provide important new insights into structure/function relationships involved in regulating the adhesion process. The objective of this project was to investigate the role of the protein tyrosine kinase FAK in cell adhesion strengthening. Our central hypothesis was that FAK regulates adhesion strengthening by modulating interactions between integrins and FA structural components. Using a novel combination of genetically engineered cells to control the interactions of FAK, a spinning disk adhesion assay with micropatterned substrates to obtain reproducible and sensitive measurements of adhesion strength, and quantitative biochemical assays for analyzing changes in adhesive complexes, we demonstrate that FAK modulates adhesion strengthening via two distinct mechanisms: (1) FAK expression results in elevated integrin activation leading to regulation of strengthening rate and (2) FAK regulates steady-state adhesion strength via vinculin recruitment to focal adhesions. We also show that the autophosphorylation and catalytic sites of FAK are critical to this regulation of adhesion strengthening. This work is significant because it both identifies functional mechanisms of FAK and provides the first evidence that focal adhesion signaling regulates the adhesion strengthening process. Furthermore, this research demonstrates that the dependency of migration on adhesion strength is highly complex and establishes a need for adhesion strengthening metrics in analyzing the functional mechanisms of molecules within adhesion complexes.

L’ingénierie tissulaire est une stratégie médicale qui repose sur la régénération de tissu par les cellules avec ou sans matériaux. Pour maîtriser cette synthèse, il faut comprendre la cellule comme une part intégrante du tissu. Hormis ses interactions biochimiques avec son support, la cellule interagit également mécaniquement avec son environnement. Elle s’accroche à ce dernier et évalue sa dureté pour adapter sa réponse biologique. Dans cette étude, j’ai développé des modèles numériques pour analyser l’influence de la rigidité du substrat sur le comportement mécanique de la cellule, sur sa structure contractile interne et les efforts qu’elle génère. En d’autres termes, j’ai essayé de comprendre comment la cellule ressent la rigidité de son environnement. De plus, au lieu de me focaliser sur les propriétés mécaniques quantitatives, j’ai cherché à développer un modèle conceptuel simplifié plus proche de la structure cellulaire. / Tissue engineering is a medical strategy based on utilizing cells and materials to regenerate a new tissue. Yet, it involves intertwined interactions that allow cells to act as integrated parts of an organ. In addition to chemical reactions, the cell interacts mechanically with its environment by sensing its rigidity. Here, we used several computational models to understand how substrate rigidity affects a cell’s structure as it adheres and spreads on it. In other words we tried to understand the way a cell feels how soft or hard it surrounding is, how it affects its internal structure and the forces that transit within it. In addition, instead of focusing on mechanical properties, we developed a simplified, yet coherent conceptual understanding of the cellular structure.

Au cours de la ventilation liquide totale (VLT), les cellules pulmonaires sont exposées à des perfluorocarbones (PFC) dont les propriétés physiques diffèrent fortement du milieu standard de culture cellulaire (DMEM) et encore plus des propriétés de l'air. Dans cette thèse nous étudions les effets d’une exposition au PFC sur la réponse des cellules épithéliales pulmonaires, en effectuant une étude approfondie des propriétés structurales, mécaniques et fonctionnelles. La réponse des cellules A549 (alvéolaire), HBE (bronchique) et AM (Macrophage alvéolaire) exposées au PFC est étudiée par comparaison au DMEM. Les variations de la structure de F-actine, de la densité d'adhésion focale et de la distribution du glycocalyx sont évaluées par fluorescence. Les changements de propriétés mécaniques et de paramètres d’adhésion sont mesurés par la Magnétocymétrie (MTC) étendue à l’analyse multiéchelle. La mécanique cellulaire est caractérisée par deux modèles microrhéologiques reflétant deux types de comportement possibles du cytosquelette (CSK). L'adhésion à la matrice cellulaire est analysée par un modèle stochastique de dé-adhésion, décrivant la composante non-réversible de la réponse cellulaire. Les rôles fondamentaux de la structure de F-actine et de la couche de glycocalyx sont respectivement évalués par dépolymérisation de F-actine et en dégradant le glycocalyx. Les résultats montrent que l'exposition au PFC induit un remodelage de la structure de F-actine, un affaiblissement du CSK et une diminution de l'adhésion. Ces résultats démontrent que le PFC déclenche une réponse particulière des cellules épithéliales caractérisée par une diminution de la tension intracellulaire, l'affaiblissement de l'adhésion et la redistribution du glycocalyx. L’origine de cette adaptation cellulaire est physique et très probablement reliée à l’augmentation de l'énergie interfaciale associée à la basse tension de surface d’un PFC chimiquement apolaire. La faible tension de surface du PFC est également responsable d'une augmentation de la compliance pulmonaire pendant VLT et a des impacts profonds dans les paramètres respiratoires, parallèlement à la modification de la réponse cellulaire. / During Total Liquid Ventilation (TLV), lung cells are exposed to perfluorocarbon (PFC) whose physical properties highly differ from aqueous medium (DMEM) standardly used for cell culture and farther air properties. In this thesis, we study the effects of PFC exposure on the response of pulmonary epithelial cell by performing a thorough assessment of their structural, mechanical and functional properties. The response of A549 cells (alveolar), HBE (bronchial), and AM (alveolar macrophages) exposed to PFC is studied by comparison to DMEM. Changes in F-actin structure, focal adhesion size and density and glycocalyx expression are evaluated by fluorescence. Changes in cell mechanics and adhesion parameters are measured by a multiscale Magnetic Twisting Cytometry (MTC) method. Cell mechanics is analyzed by two microrheological models reflecting two possible cytoskeleton features. Cell-matrix adhesion is analyzed by a stochastic multibond de-adhesion model describing the non-reversible component of the cell response by MTC. The key roles of F-actin structure and glycocalyx layer are established by respectively depolymerising F-actin and degrading glycocalyx. Results show that PFC exposure induces F-actin remodelling, cytoskeleton softening and adhesion weakening. They demonstrate that PFC triggers an epithelial cell response which is characterized by decay in intracellular tension, adhesion weakening and glycocalyx redistribution. The origin of this cellular adaptations is physical and most likely related to the increase in interfacial energy, associated to the low surface tension of the non polar perflurorocarbon, The low surface tension of PFC is also responsible for an increase in lung compliance during TLV and has deep impacts during ventilation parallel to the modification of cell response.

Because cells are machines, their structure determines their function (health). But their structure also determines cells’ mechanical properties. So if we can understand how cells’ mechanical properties are influenced by specific structures, then we can observe what’s happening inside of cells via mechanical measurements. The Atomic Force Microscope (AFM) has become a standard tool for investigating the mechanical properties of cells. In many experiments, an AFM is used to ‘poke’ adherent cells with nanonewton forces, and the resulting deformation observed via, e.g. Laser Scanning Confocal Microscopy. Results of such experiments are often interpreted in terms of continuum mechanical models which characterize the cell as a linear viscoelastic solid. This “top-down” approach of poking an intact cell —complete with cytoskeleton, organelles etc.— can be problematic when trying to measure the mechanical properties and response of a single cell component. Moreover, how are we to know the sensitivity of the cell’s mechanical properties to partial modification of a single component (e.g. reducing the degree of cross- linking in the actin cortex)? In contrast, the approach taken here —studying the deformation and relaxation of lipid bilayer vesicles— might be called a “bottom-up” approach to cell mechanics. Using Coarse- Grained Molecular Dynamics simulations, we study the deformation and relaxation of bilayer vesicles, when poked with constant force. The relaxation time, equilibrium area expansion, and surface tension of the vesicle membrane are studied over a range of applied forces. Interestingly, the relaxation time exhibits a strong force-dependence. Force-compression curves for our simulated vesicle show a strong similarity to recent experiments where giant unilamellar vesicles were compressed in a manner nearly identical to that of our simulations.

Les cellules peuvent migrer sous différentes conditions qui dépendent de l’environnement biochimique ou mécanique. Connaître les mécanismes de la migration, les protéines impliquées et leur régulation est essentiel pour comprendre les processus de morphogénèse ou certaines situations pathologiques. Dans ce contexte, la migration collective des cellules est un processus clé qui intervient pendant le développement ainsi que dans la vie adulte. Elle joue un rôle très important pour la formation et l’entretien des couches épithéliales, notamment au cours du développement embryonnaire et pendant la cicatrisation des trous épithéliaux résultant, par exemple, d’une blessure. Lorsque l’épithélium présente une discontinuité, des mécanismes actifs qui impliquent une migration coordonnée des cellules sont nécessaires pour préserver l’intégrité des tissus. Dans ce travail, nous avons étudié les mécanismes impliqués dans la fermeture des trous dans un épithélium. Pour des blessures de faible taille, le mode de fermeture dit de purse string est souvent évoqué, impliquant la contraction d’un anneau contractile d’acto-myosine qui ferme la blessure. Pour des blessures de tailles plus importantes, il est courant d’observer un mécanisme différent conduisant { la migration active des cellules du bord qui couvrent la surface “libre”.Pour étudier ces aspects de manière quantitative et reproductible, nous avons développé une nouvelle méthode basée sur des techniques de microfabrication et de lithographie dite « molle » qui permet de faire une étude quantitative de la fermeture des trous épithéliaux. Nous avons fabriqué des substrats de micropiliers de diamètre et de forme variés dans les quels les cellules sont libres de pousser entre les microstructures. Lorsqu’elles sont parvenues à confluence, on retire le substrat qui laisse apparaître des trous contrôlés.De cette manière, nous avons observé que les cellules épithéliales forment des lamellipodes pour la fermeture de ces trous. Le mécanisme de fermeture dépend de la taille des trous et nous avons pu observer différents régimes en fonction de diamètre des piliers. Les trous petits (de la taille d’une seule cellule) sont fermés par un mécanisme passif alors que la fermeture de trous plus larges nécessite un mécanisme actif de migration conduisant à la formation de lamellipodes et à des modes de migration collective. Par la suite, nous nous sommes intéressés à l’aspect mécanique de la fermeture des trous épithéliaux. Pour cela, nous avons utilisé un système d’ablation laser pour rompre quelques cellules dans une monocouche épithéliale. Nous avons alors mesuré les forces de traction que les cellules exercent au substrat et leur évolution temporelle et spatiale. Nous avons pu mettre en évidence différents modes de traction: au début, les cellules exercent des forces de traction importantes sur leur substrat pour laisser place à des contraintes mécaniques qui sont davantage issues d’un processus collectif au travers de la formation d’un câble multicellulaire qui les relie les cellules de bord entre elles. En conclusion, ce travail nous a permis d’obtenir des informations sur les mécanismes dynamiques de fermeture des tissus épithéliaux qui sont évidemment impliqués dans la cicatrisation des blessures mais aussi dans certains problèmes de malformations congénitales lors l’embryogenèse. / Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. 11Abstract (English)Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. Next, we also addressed the mechanical component of epithelial gap closure. In this study, we took advantage of a laser-ablation system to disrupt some cells within an epithelial monolayer, and study how the remaining cells sealed that gap. By measuring the traction forces that cells exert on the substrate along the closure, we observed that cells first pulled on the substrate to propel themselves. By the last steps of closure, there is a transition in the direction of the force, so that cells are pulled to the center of the gap due to the assembly of a supracellular actin cable. Altogether, this work provides valuable knowledge on the current understanding of the mechanisms accounting for epithelial gap closure. We believe that a better comprehension of these mechanisms can help to shed light in clinically relevant situations where epithelial gap closure is impaired.

Comment les cellules établissent leurs formes et organisations internes est un problème biologique fondamental. Au cours de cette thèse, j’ai étudié le développement de la forme cellulaire et de la polarité chez la cellule de levure fissipare. Ces études sont fondées sur l’exploration de la façon dont les petites spores symétriques de levures se développent et s’organisent pour briser la symétrie pour la définition de leur tout premier axe de polarité. Dans une première partie, j’ai étudié les couplages entre la mécanique de surface de la paroi cellulaire des spores et la stabilité de domaines de polarité de Cdc42 qui contrôlent les aspects spatio-temporelles de la brisure de symétrie de ces spores. Dans une seconde partie, j’ai étudié les mécanismes par lesquels ces domaines de polarité contrôlent leur taille et l'adapte à la géométrie de la cellule, un processus vraisemblablement pertinents pour comprendre comment des domaines fonctionnels corticaux s’adaptent à la taille des cellules. Globalement, ces nouvelles recherches focalisant sur la façon dont les cellules développent dynamiquement leur forme et polarité de novo, permettent de mettre en évidence des couplages complexes dans la morphogenèse qui ne peuvent pas être testés en regardant les cellules à « l’état stationnaire» ou avec des outils génétiques. / How cells establish their proper shapes and organization is a fundamental biological problem. In this thesis, I investigated the dynamic development of cellular form and polarity in the rod-shape fission yeast cell. These studies are based on monitoring how small symmetric fission yeast spores grow and self-organize to break symmetry for the definition of their very first polarity axis. In a first part, I studied interplays between surface mechanics of the spore cell wall and the stability of Cdc42-based polarity domains which control spatio-temporal aspects of spore symmetry breaking. In a second part, I studied mechanisms by which these polarity domains control their width and adapt it to cell surface geometry, a process likely relevant to understand how functional cortical domains scale to cell size. Overall these novel investigations focusing on how cells dynamically develop their form and polarity de novo highlight complex feedbacks in morphogenesis that cannot be evidenced by looking at cells at “steady state” or with genetics.

Almost all animal cells maintain a thin layer of actin filaments and associated proteins underneath the cell membrane. The actomyosin cortex is subject to internal stress patterns which result from the spatiotemporally regulated activity of non-muscle myosin II motors in the actin network. We study how these active stresses drive changes in cell shape and flows within the cortical layer, and how these cytoskeletal deformations and flows govern processes such as cell migration, cell division and organelle transport. Following a continuum mechanics approach, we develop theoretical descriptions for three different cellular processes, to obtain - in collaboration with experimental groups - a detailed and quantitative understanding of the underlying cytoskeletal mechanics. We investigate the forces and cortex flows involved in adhesion-independent cell migration in confinement. Many types of cell migration rely on the extension of protrusions at the leading edge, where the cells attach to the substrate with specific focal adhesions, and pull themselves forward, exerting stresses in the kPa range. In confined environments however, cells exhibit migration modes which are independent of specific adhesions. Combining hydrodynamic theory, microfluidics and quantitative imaging of motile, non-adherent carcinosarcoma cells, we analyze the mechanical behavior of cells during adhesion-independent migration. We find that the accumulation of active myosin motors in the rear part of these cells results in a retrograde cortical flow as well as the contraction of the cell body in the rear and expansion in the front, and we describe how both processes contribute to the translocation of the cells, depending on the geometric and mechanical parameters of the system. Importantly, we find that the involved propulsive forces are several orders of magnitude lower than during adhesive motility while the achieved migration velocities are similar. Moreover, the distribution of forces on the substrate during non-adhesive migration is fundamentally different, giving rise to a positive force dipole. In contrast to adhesive migration modes, non-adhesive cells move by exerting pushing forces at the rear, acting to expand rather than contract their substrate as they move. These differences may strongly affect hydrodynamic and/or deformational interactions between collectively migrating cells. In addition to the work outlined above, we study contractile ring formation in the actin cytoskeleton before and during cell division. While in disordered actin networks, myosin motor activity gives rise to isotropic stresses, the alignment of actin filaments in the cortex during cell division introduces a preferred direction for motor-filament interactions, resulting in anisotropies in the cortical stress. Actin filaments align in myosin-dependent shear flows, resulting in possible feedback between motor activity, cortical flows and actin organization. We investigate how the mechanical interplay of these different cortical properties gives rise to the formation of a cleavage furrow during cell division, describing the level of actin filament alignment at different points on the cortex with a nematic order parameter, in analogy to liquid crystal physics. We show that cortical anisotropies arising from shear-flow induced alignment patterns are sufficient to drive the ingression of cellular furrows, even in the absence of localized biochemical myosin up-regulation. This mechanism explains the characteristic appearance of pseudocleavage furrows in polarizing cells. Finally, we study the characteristic nuclear movements in pseudostratified epithelia during development. These tissues consist of highly proliferative, tightly packed and elongated cells, with nuclei actively travelling to the apical side of the epithelium before each cell division. We explore how cytoskeletal properties act together with the mechanics of the surrounding tissue to control the shape of single cells embedded in the epithelium, and investigate potential mechanisms underlying the observed nuclear movements. These findings form a theoretical basis for a more detailed characterization of processes in pseudostratified epithelia. Taken together, we present a continuum mechanics description of the actomyosin cell cortex, and successfully apply it to several different cell biological processes. Combining our theory with experimental work from collaborating groups, we provide new insights into different aspects of cell mechanics.

Ce manuscrit détaille le développement d'un montage de pince optique permettant d'étudier les propriétés mécaniques des cellules endothéliales, impliquées dans le développement de l'athérosclérose. Le but est de déterminer les propriétés viscoélastiques des cellules, et de suivre la propagation d’une contrainte mécanique au sein de la cellule. Cette contrainte mécanique est appliquée via une bille liée à la membrane de la cellule et soumise à un piège optique.Le dispositif réalisé combine le piégeage optique et la microscopie à contraste de phase, permettant d'exercer une force tout en imageant les cellules via le même objectif de microscope. L'originalité du montage de pince optique repose sur la détection du signal rétrodiffusé par la bille piégée, dans un plan conjugué du plan focal arrière de l'objectif, afin de mesurer la position relative de la bille par rapport au piège.Une part importante de ce travail a consisté à comprendre l'allure du signal détecté présentant un système d'interférences en anneaux, et à l’expliquer par un modèle simple. Ce modèle a permis de comprendre la présence d’artefacts de mesure de position dus à la superposition de l'anneau de phase sur la figure d’interférence. Pour y remédier, l'anneau de phase est déporté dans un plan conjugué intervenant uniquement dans l'imagerie de l'échantillon.La figure d'interférence présente un atout majeur : elle donne accès à la hauteur précise de la bille piégée, généralement difficile à mesurer. Cette information est nécessaire pour calibrer la constante de raideur du piège optique à la hauteur des cellules, que ce soit par l'analyse de la densité spectrale de puissance du mouvement brownien de la bille piégée ou par sa réponse à un échelon de position du piège. Ces deux méthodes de calibration, ainsi que l'application du théorème d’équipartition et l'analyse par inférence bayésienne, ont été mises en œuvre. Tous les résultats s'avèrent en bon accord. La calibration complète du dispositif en fait un outil prêt à l'emploi pour exercer des forces locales contrôlées en direction et en amplitude sur les cellules. / This manuscript details the development of an optical tweezer setup to study the mechanical properties of endothelial cells, involved in the development of atherosclerosis. The goal is to determine the viscoelastic properties of the cells, and to follow the propagation of the mechanical constraint inside the cell. This mechanical constraint is applied via a bead attached to the cell membrane and subjected to an optical trap.The setup built combines optical trapping with phase contrast microscopy, to apply a force while imaging the cells with the same microscope objective. The originality of the optical tweezer setup relies on the detection of the signal backscattered by the trapped bead, in a plane conjugate to the back focal plane of the objective, in order to measure the relative position of the bead with respect to the center of the trap.An important part of this work was dedicated to the understanding of the detected signal presenting an interference pattern with rings, explained by a simple model. This model provides an explanation for the position measurement artifacts arising from the superposition of the phase ring and the interference pattern. To solve the problem, the phase ring was moved in a conjugate plane involved only in the imaging path of the sample.The interference pattern has the main advantage of giving access to the precise height of the trapped bead, usually difficult to measure. This information is necessary to calibrate the optical trap stiffness at the height of the cells, either by the power spectrum analysis of the Brownian motion of the trapped bead, or by its response to a step motion of the trap. These two calibration methods, along with the application of the equipartition theorem and Bayesian inference analysis, were implemented and their results compared, showing a good agreement. The complete calibration of the setup makes it a ready-to-use tool to exert local forces controlled in direction and amplitude on cells.

Mechanical properties of cells and their environment have an undeniable impact on physiological and pathological processes such as tissue development or cancer metastasis. Hence, there is a pressing need for establishing and validating methodologies for measuring the mechanical properties of cells, as well as for deciphering the molecular underpinnings that govern the mechanical phenotype. During my doctoral research, I addressed these needs by pushing the boundaries of the field of single-cell mechanics in four projects, two of which were method-oriented and two explored important biological questions. First, I consolidated real-time deformability cytometry as a method for high-throughput single-cell mechanical phenotyping and contributed to its transformation into a versatile image-based cell characterization and sorting platform. Importantly, this platform can be used not only to sort cells based on image-derived parameters, but also to train neural networks to recognize and sort cells of interest based on raw images. Second, I performed a cross-laboratory study comparing three microfluidics-based deformability cytometry approaches operating at different timescales in two standardized assays of osmotic shock and actin disassembly. This study revealed that while all three methods are sensitive to osmotic shock-induced changes in cell deformability, the method operating at the shortest timescale is not suited for detection of actin cytoskeleton changes. Third, I demonstrated changes in cell mechanical phenotype associated with cell fate specification on the example of differentiation and de-differentiation along the neural lineage. In the process of reprogramming to pluripotency, neural precursor cells acquired progressively stiffer phenotype, that was reversed in the process of neural differentiation. The stiff phenotype of induced pluripotent stem cells was equivalent to that of embryonic stem cells, suggesting that mechanical properties of cells are inherent to their developmental stage. Finally, I identified and validated novel target genes involved in the regulation of mechanical properties of cells. The targets were identified using machine learning-based network analysis of transcriptomic profiles associated with mechanical phenotype change, and validated computationally as well as in genetic perturbation experiments. In particular, I showed that the gene with the best in silico performance, CAV1, changes the mechanical properties of cells when silenced or overexpressed. Identification of novel targets for mechanical phenotype modification is crucial for future explorations of physiological and pathological roles of cell mechanics. Together, this thesis encompasses a collection of contributions at the frontier of single-cell mechanical characterization across timescales and cell state transitions, and lays ground for turning cell mechanics from a correlative phenomenological parameter to a controllable property.:Abstract Kurzfassung List of Publications Contents Introduction Chapter 1 — Background 1.1. Mechanical properties as a marker of cell state in health and disease 1.2. Functional relevance of single-cell mechanical properties 1.3. Internal structures determining mechanical properties of cells 1.4. Cell as a viscoelastic material 1.5. Methods to measure single-cell mechanical properties Aims and scope of this thesis Chapter 2 — RT-DC as a versatile method for image-based cell characterization and sorting 2.1. RT-DC for mechanical characterization of cells 2.1.1. Operation of the RT-DC setup 2.1.2. Extracting Young’s modulus from RT-DC data 2.2. Additional functionalities implemented to the RT-DC setup 2.2.1. 1D fluorescence readout in three spectral channels 2.2.2. SSAW-based active cell sorting 2.3. Beyond assessment of cell mechanics — emerging applications 2.3.1. Deformation-assisted population separation and sorting 2.3.2. Brightness-based identification and sorting of blood cells 2.3.3. Transferring molecular specificity into label-free cell sorting 2.4. Discussion 2.5. Key conclusions 2.6. Materials and experimental procedures 2.7. Data analysis Chapter 3 — A comparison of three deformability cytometry classes operating at different timescales 3.1. Results 3.1.1. Representatives of the three deformability cytometry classes 3.1.2. Osmotic shock-induced deformability changes are detectable in all three methods 3.1.3. Ability to detect actin disassembly is method-dependent 3.1.4. Strain rate increase decreases the range of deformability response to actin disassembly in sDC 3.2. Discussion 3.3. Key conclusions 3.4. Materials and methods Chapter 4 — Mechanical journey of neural progenitor cells to pluripotency and back 4.1. Results 4.1.1. fNPCs become progressively stiffer during reprogramming to pluripotency 4.1.2. Transgene-dependent F-class cells are more compliant than ESC-like iPSCs 4.1.3. Surface markers unravel mechanical subpopulations at intermediate reprogramming stages 4.1.4. Neural differentiation of iPSCs mechanically mirrors reprogramming of fNPCs 4.1.5. The closer to the pluripotency, the higher the cell stiffness 4.2. Discussion 4.3. Key conclusions 4.4. Materials and methods Chapter 5 — Data-driven approach for de novo identification of cell mechanics regulators 5.1. Results 5.1.1. An overview of the mechanomics approach 5.1.2. Model systems characterized by mechanical phenotype changes 5.1.3. Discriminative network analysis on discovery datasets 5.1.4. Conserved functional network module comprises five genes 5.1.5. CAV1 performs best at classifying soft and stiff cell states in validation datasets 5.1.6. Perturbing expression levels of CAV1 changes cells stiffness 5.2. Discussion 5.3. Key conclusions 5.4. Materials and methods Conclusions and Outlook Appendix A Appendix B Supplementary Tables B.1 – B.2 Supplementary Figures B.1 – B.9 Appendix C Supplementary Tables C.1 – C.2. Supplementary Figures C.1 – C.5 Appendix D Supplementary Tables D.1 – D.6 Supplementary Figures D.1 – D.7 List of Figures List of Tables List of Abbreviations. List of Symbols References Acknowledgements