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Temperature has a reliable and nearly instantaneous influence on mechanical responses of cells. As recently published, MCF-10A normal epithelial breast cells follow the time-temperature superposition (TTS) principle. Here, we measured thermorheological behaviour of eight common cell types within physiologically relevant temperatures and applied TTS to creep compliance curves. Our results showed that superposition is not universal and was seen in four of the eight investigated cell types. For the other cell types, transitions of thermorheological responses were observed at 36 °C. Activation energies (EA) were calculated for all cell types and ranged between 50 and 150 kJ mol-1. The scaling factors of the superposition of creep curves were used to group the cell lines into three categories. They were dependent on relaxation processes as well as structural composition of the cells in response to mechanical load and temperature increase. This study supports the view that temperature is a vital parameter for comparing cell rheological data and should be precisely controlled when designing experiments.

abstract: This dissertation features a compilation of studies concerning the biophysics of multicellular systems. I explore eukaryotic systems across length scales of the cell cytoskeleton to macroscopic scales of tissues. I begin with a general overview of the natural phenomena of life and a philosophy of investigating developmental systems in biology. The topics covered throughout this dissertation require a background in eukaryotic cell physiology, viscoelasticity, and processes of embryonic tissue morphogenesis. Following a brief background on these topics, I present an overview of the Subcellular Element Model (ScEM). This is a modeling framework which allows one to compute the dynamics of large numbers of three-dimensional deformable cells in multi-cellular systems. A primary focus of the work presented here is implementing cellular function within the framework of this model to produce biologically meaningful phenotypes. In this way, it is hoped that this modeling may inform biological understanding of the underlying mechanisms which manifest into a given cell or tissue scale phenomenon. Thus, all theoretical investigations presented here are motivated by and compared to experimental observations. With the ScEM modeling framework I first explore the passive properties of viscoelastic networks. Then as a direct extension of this work, I consider the active properties of cells, which result in biological behavior and the emergence of non-trivial biological phenotypes in cells and tissues. I then explore the possible role of chemotaxis as a mechanism of orchestrating large scale tissue morphogenesis in the early embryonic stages of amniotes. Finally I discuss the cross-sectional topology of proliferating epithelial tissues. I show how the Subcellular Element Model (ScEM) is a phenomenological model of finite elements whose interactions can be calibrated to describe the viscoelastic properties of biological materials. I further show that implementing mechanisms of cytoskeletal remodeling yields cellular and tissue phenotypes that are more and more biologically realistic. Particularly I show that structural remodeling of the cell cytoskeleton is crucial for large scale cell deformations. I provide supporting evidence that a chemotactic dipole mechanism is able to orchestrate the type of large scale collective cell movement observed in the chick epiblast during gastrulation and primitive streak formation. Finally, I show that cell neighbor histograms provide a potentially unique signature measurement of tissue topology; such measurements may find use in identifying cellular level phenotypes from a single snapshot micrograph. / Dissertation/Thesis / Ph.D. Physics 2011

Temperature has a reliable and nearly instantaneous influence on mechanical responses of cells. As recently published, MCF-10A normal epithelial breast cells follow the time-temperature superposition (TTS) principle. Here, we measured thermorheological behaviour of eight common cell types within physiologically relevant temperatures and applied TTS to creep compliance curves. Our results showed that superposition is not universal and was seen in four of the eight investigated cell types. For the other cell types, transitions of thermorheological responses were observed at 36 °C. Activation energies (EA) were calculated for all cell types and ranged between 50 and 150 kJ mol-1. The scaling factors of the superposition of creep curves were used to group the cell lines into three categories. They were dependent on relaxation processes as well as structural composition of the cells in response to mechanical load and temperature increase. This study supports the view that temperature is a vital parameter for comparing cell rheological data and should be precisely controlled when designing experiments.

Temperature has a reliable and nearly instantaneous influence onmechanical responses of cells.As recently published, MCF-10Anormal epithelial breast cells follow the time–temperature superposition (TTS) principle. Here,wemeasured thermorheological behaviour of eightcommoncell types within physiologically relevant temperatures and appliedTTS to creep compliance curves.Our results showed that superposition is not universal and was seen in four of the eight investigated cell types. For the other cell types, transitions of thermorheological responses were observed at 36 °C.Activation energies (EA)were calculated for all cell types and ranged between 50 and 150 kJmol−1.The scaling factors of the superposition of creep curves were used to group the cell lines into three categories. They were dependent on relaxation processes aswell as structural composition of the cells in response tomechanical load and temperature increase.This study supports the view that temperature is a vital parameter for comparing cell rheological data and should be precisely controlledwhen designing experiments.

La mécanique cellulaire a gagné un intérêt croissant en raison de son implication fondamentale dans des nombreux processus cellulaires, notamment la migration, la division, la différentiation et l’apoptose. Entre autres techniques, la microscopie à force atomique (AFM) s’est avérée particulièrement utile pour la caractérisation mécanique des cellules vivantes. Dans cette thèse, deux aspects différents ont été étudiés par AFM. Dans un premier temps, l’élasticité des cellules épithéliales étalées sur des micropatterns adhésifs a été cartographiée. Cette étude montre que l’élasticité d’une cellule varie en fonction de sa géométrie d’adhésion à la fois au niveau global et subcellulaire. La deuxième partie de cette thèse est dédiée à la caractérisation de la réponse viscoélastique d’une cellule à un stimulus mécanique oscillatoire à haute fréquence. Des études précédentes montrent que la réponse des cellules est dominée par un stress élastique et suive une loi de puissance faible à basse fréquence. Une réponse cellulaire essentiellement visqueuse est attendue à haute fréquence, mais jusqu’à présent les limitations techniques ont empêché l’évaluation de cette propriété. Dans ma thèse, ces limitations ont été dépassées grâce à la modification d’un AFM à grande vitesse (HS-AFM). Des mesures de rhéologie active sur fibroblastes ont été réalisées entre 1Hz et 120 kHz, permettant d’étendre de deux ordres de grandeur l’échelle de fréquences explorée. Ce travail montre une réponse cellulaire aux stimulations à haute fréquence plus visqueuse qu’à basse fréquence, mais suggèrent aussi une réponse bien plus complexe qu’attendue. / The field of cell mechanics gained a growing interest because of its fundamental implication in several cellular processes, such as migration, division, differentiation and apoptosis. Among other techniques, atomic force microscopy (AFM) demonstrated particularly useful for the mechanical characterization of living cells. In this thesis, two different aspects were investigated by AFM. In the first part, the elastic properties of epithelial cells grown on adhesive micropatterns were mapped. This study shows that the elasticity of a cell varies as a function of the geometry of its adhesive environment on both global and subcellular scales. The second part of this thesis focuses on the characterization of the viscoelastic response of a cell subjected to an oscillatory mechanical stimulus at high frequency. Previous studies show that the response of cells to such stimuli is mainly dominated by elastic stress and follows a weak power law at low frequency. Instead, a predominantly viscous behavior is expected at high frequency. Up to now, technical limitations prevented the experimental validation of this property. In this thesis, these limitations were overcome thanks to the modification of a high-speed AFM (HS-AFM). With this setup, active rheological measurements of living fibroblasts could be performed from 1 Hz to 120 kHz, extending of two orders of magnitude the frequency scale explored until now. This work highlights a response of cells to high-frequency stimuli which is more viscous than at low frequency, but also suggests a more complex response than expected.

From a physical perspective biological cells consist of active soft matter that exist in a thermodynamic state far from equilibrium. Not only in muscles but also during cell proliferation, wound healing, embryonic development, and many other physiological tasks, generation of forces on the scale of whole cells is required. To date, cellular contractions have been ascribed to adhesion dependent processes such as myosin driven stress fiber formation and the development of focal adhesion complexes. In this thesis it is shown for the first time that contractions can occur independently of focal adhesions in single suspended cells. To measure mechanical properties of suspended cells the Optical Stretcher – a dualbeam laser trap – was used with phase contrast video microscopy which allowed to extract the deformation of the cell for every single frame. For fluorescence imaging confocal laser scanning microscopy was employed. The ratio of the fluorescence of a temperature sensitive and a temperature insensitive rhodamine dye was utilized to determine the temperatures inside the optical trap during and after Optical Stretching. The rise in temperature at a measuring power of 0.7W turned out to be enough to open a temperature sensitive ion channel transfected into an epithelial cell line. In this way a massive Ca2+ influx was triggered during the Optical Stretcher experiment. A new setup combining Optical Stretching and confocal laser scanning microscopy allowed fluorescence imaging of these Ca2+ signals while the cells were deformed by optically induced surface forces, showing that the Ca2+ influx could be manipulated with adequate drugs. This model system was then employed to investigate the influence of Ca2+ on the observed contractions, revealing that they are partially triggered by Ca2+. A phenomenological mathematical model based on the fundamental constitutive equation for linear viscoelastic materials extended by a term accounting for active contractions allowed to quantify the activity of the measured cells. The skewness and the median of the strain distributions were shown to depend on the activity of the cells. The introduced model reveals that even in measurements, that seemingly are describable by passive viscoelasticity, active contractililty might be superimposed. Ignoring this effect will lead to erroneous material properties and misinterpretation of the data. Taken together, the findings presented in this thesis demonstrate that active processes are an essential part of cellular mechanics and cells can contract even independently of adhesions. The results provide a method that allows to quantify active contractions of suspended cells. As the proposed model is not based on specific assumptions on force generating processes, it paves the way for a thorough investigation of different influences, such as cytoskeletal structures and intra-cellular signaling processes, to cellular contractions. The results present an important contribution for better mechanical classification of cells in future research with possible implications for medical diagnosis and therapy.

Cette thèse interdisciplinaire a été dédiée à la caractérisation des propriétés mécaniques de myoblastes (murins et humains) et de myotubes (murins) à l'aide de la microscopie à force atomique (AFM). En modifiant ou en inhibant la dynamique du cytosquelette (CSK) d’actine de ces cellules, nous avons pu montrer que ces propriétés mécaniques variaient. L’enregistrement de courbes de force indentation nous a permis de montrer que la présence de cellules adhérentes introduisait sur les leviers d’AFM un amortissement visqueux supplémentaire à celui d’une paroi solide, et que cet amortissement visqueux dépendait de sa vitesse d’approche et que celui-ci restait non négligeable pour les plus faibles vitesses (1μm/s). Nous avons observé que les propriétés mécaniques des précurseurs de muscles devenaient non linéaires (comportement plastiques) pour des grandes déformations (>1μm) et qu’elles dépendaient de l’état, du type de cellule et de leur environnement. En combinant des expériences d’AFM, des modèles visco-élastiques et des méthodes d'analyse multi-échelle basées sur la transformation en ondelettes, nous avons illustré la variabilité des réponses mécaniques de ces cellules (de visco-élastiques à visco-plastiques). À l'aide de courbes de force-indentation, de l’imagerie morpho-structurale (DIC, microscopie à fluorescence) et de traitements pharmacologiques, nous avons éclairé le rôle essentiel des processus actifs (dépendants de l’ATP) dans la mécanique de myoblastes, en discutant tout particulièrement ceux des moteurs moléculaires (myosine II) couplés aux filaments d’actine. En particulier, nous avons montré que les fibres de stress du cytosquelette d’actine situées autour du noyau pouvaient présenter des évènements de remodelage soudains (ruptures) et que ces ruptures étaient une mesure indirecte de l’aptitude de ces cellules à tendre leur CSK. Nous avons enfin montré qu’il était possible de généraliser cette approche à des cas cliniques humains, en l’occurrence des myoblastes primaires de porteurs sains et de patients atteints de dystrophie musculaire de Duchenne, ouvrant la voie à des études plus larges sur d’autres types cellulaires et pathologies. / This interdisciplinary thesis was dedicated to the atomic force microscopy (AFM) characterization of the mechanical properties of myoblasts (murine and human) and myotubes (murine). We reported that the mechanical properties of these cells were modified when their actin cytoskeleton (CSK) dynamics was inhibited or altered. Recording single AFM force indentation curves, we showed that adherent layers of myoblasts and myotubes introduced on the AFM cantilever an extra hydrodynamic drag as compared to a solid wall. This phenomenon was dependent on the cantilever scan speed and not negligible even at low scan velocities (1μm/s). We observed that the mechanical properties of the muscle precursor cells became non-linear (plastic behaviour) for large local deformations (>1μm) and that they varied depending on the state, type and environment of the cells. Combining AFM experiments, viscoelastic modeling and multi-scale analyzing methods based on the wavelet transform, we illustrated the variability of the mechanical responses of these cells (from viscoelastic to viscoplastic). Through AFM force indentation curves analysis, morpho-structural imaging (DIC, fluorescence microscopy) and pharmacological treatments, we enlightened the important role of active (ATP-dependent) processes in myoblast mechanics, focusing especially on those related to the molecular motors (myosin II) coupled to the actin filaments. In particular, we showed that the perinuclear actin stress fibers could exhibit some abrupt remodelling events (ruptures), which are characteristic of the ability of these cells to tense their CSK. Finally, we showed that this approach can be generalized to some human clinical cases, namely primary human myoblasts from healthy donors and patients affected by Duchenne muscular dystrophy, paving the way for broader studies on different cell types and diseases.

From a physical perspective biological cells consist of active soft matter that exist in a thermodynamic state far from equilibrium. Not only in muscles but also during cell proliferation, wound healing, embryonic development, and many other physiological tasks, generation of forces on the scale of whole cells is required. To date, cellular contractions have been ascribed to adhesion dependent processes such as myosin driven stress fiber formation and the development of focal adhesion complexes. In this thesis it is shown for the first time that contractions can occur independently of focal adhesions in single suspended cells. To measure mechanical properties of suspended cells the Optical Stretcher – a dualbeam laser trap – was used with phase contrast video microscopy which allowed to extract the deformation of the cell for every single frame. For fluorescence imaging confocal laser scanning microscopy was employed. The ratio of the fluorescence of a temperature sensitive and a temperature insensitive rhodamine dye was utilized to determine the temperatures inside the optical trap during and after Optical Stretching. The rise in temperature at a measuring power of 0.7W turned out to be enough to open a temperature sensitive ion channel transfected into an epithelial cell line. In this way a massive Ca2+ influx was triggered during the Optical Stretcher experiment. A new setup combining Optical Stretching and confocal laser scanning microscopy allowed fluorescence imaging of these Ca2+ signals while the cells were deformed by optically induced surface forces, showing that the Ca2+ influx could be manipulated with adequate drugs. This model system was then employed to investigate the influence of Ca2+ on the observed contractions, revealing that they are partially triggered by Ca2+. A phenomenological mathematical model based on the fundamental constitutive equation for linear viscoelastic materials extended by a term accounting for active contractions allowed to quantify the activity of the measured cells. The skewness and the median of the strain distributions were shown to depend on the activity of the cells. The introduced model reveals that even in measurements, that seemingly are describable by passive viscoelasticity, active contractililty might be superimposed. Ignoring this effect will lead to erroneous material properties and misinterpretation of the data. Taken together, the findings presented in this thesis demonstrate that active processes are an essential part of cellular mechanics and cells can contract even independently of adhesions. The results provide a method that allows to quantify active contractions of suspended cells. As the proposed model is not based on specific assumptions on force generating processes, it paves the way for a thorough investigation of different influences, such as cytoskeletal structures and intra-cellular signaling processes, to cellular contractions. The results present an important contribution for better mechanical classification of cells in future research with possible implications for medical diagnosis and therapy.