Die mechanischen Eigenschaften von Zellen charakterisieren und beeinflussen deren Zustand. Die vorliegende Arbeit zielt auf ein besseres Verständnis der biomechanischen Eigenschaften von Zellen ab. Der Fokus lag dabei auf der Biegesteifigkeit von Zellmembranen und der Deformierbarkeit der Zellen. Es werden drei Studien vorgestellt in der diese Materialparameter untersucht wurden. Die erste Studie befasst sich mit der Temperaturabhängigkeit der mechanischen Eigenschaften. Hierbei wurden acht verschieden Zelllienien bei jeweils fünf Temperaturen rheologisch vermessen. Zur Messung wurde der sog. \"optical stretcher\" verwendet der gleichzeitig die Zellen deformieren und aufheizen kann. Die Versuche zeigen, dass eine Zeit-Temperatur superposition dabei nicht für alle Zelltypen funktioniert. In der zweiten Studie wurden die Membransteifigkeit von Gewebeproben von Brust- und Gebärmutterhalskrebspatienten untersucht. Als Kontrollsystem wurde gutartiges Gewebe aus dem Umfeld des Tumors verwendet. Es konnte gezeigt werden, dass die Zellmembranen von Tumorzellen weicher waren als von gesundem Vergleichsgewebe. Die Änderung der Membrankomposition wurde dabei als mögliche Ursache massenspektroskopisch Untersucht und verschieden Ursachen der weichen Membrane diskutiert. Für die dritte Studie wurde der chemische Wirkstoff Soraphen A eingesetzt um die Membransteifigkeit von zwei Zelllienien zu erhöhen. Dies zeigte eine Verringerung von Zellbeweglichkeit und Invasivität.

Diese Arbeit beinhaltet Ergebnisse der ersten klinischen Studie zur Charakterisierung der mechanischen Eigenschaften von Zellen in einem Tumor mit der dafür notwendigen Probengröße. Dies ermöglichte die Erstellung eines umfassenden Bildes von Subpopulationen innerhalb eines Tumors mit großem diagnostischem Potential. Die Änderung der Einzelzellmechanik von Tumorzellen wird durch Veränderung des Zytoskeletts, einem komplexes Polymernetzwerk in Zellen, hervorgerufen. Mit Hilfe von Zellgiften wurde das Zytoskelett gezielt manipuliert, um den Einfluss einzelner Faktoren auf die Biomechanik zu bestimmen. Aus Gewebeproben von Brustkrebspatienten wurden Zellen mit Hilfe enzymatischer Aufspaltung des extrazellulären Kollagennetzwerkes isoliert. Als Kontrollsystem wurden Primärzellen aus Brustreduktionsgewebe und aus Fibroadenomen, gutartigen Gewebeneubildungen der Brustdrüse, verwendet. Unter Einsatz des Optischen Stretchers, einer Zweistrahl-Laserfalle, wurden suspendierte Zellen für zwei Sekunden einer konstanten Zugspannung ausgesetzt und das Deformations- wie auch das anschließende Relaxationsverhalten beobachtet. Dabei ergaben sich wesentliche Unterschiede zwischen Tumor- und Kontrollproben. Neben Zellen mit ähnlichen Steifigkeiten, enthielten Tumorproben Subpopulationen sehr weicher Zellen, wie sie in Normalgewebe nicht zu finden sind. Desweiteren war das Relaxationsverhalten der Tumorzellen stärker elastisch dominiert. Einzelne Zellen kontrahierten sogar aktiv gegen die Zugspannung. Versuche, das Zytoskelett mittels Zellgiften künstlich in einem Zustand zu bringen, der in Krebszellen beobachtet wurde, ergaben zwar ebenfalls die Zunahme weicherer Zellen, jedoch war das Relaxationsverhalten eher viskos dominiert. Fluoreszenzaufnahmen des Aktin-Zytoskeletts sowie der fokalen Adhäsionen, die das Aktin-Netzwerk der Zelle mit dem Substrat verankern, zeigten Veränderungen bei Krebszellen im Vergleich zu Kontrollen. Darüber hinaus wurden Einflussfaktoren auf die Zellmechanik untersucht. Neben Kulturbedingungen, beeinflussen auch Alter und Medikation das biomechanische Verhalten. Die Steifigkeit der Krebszellen scheint vom Ursprungsgewebe beeinflusst zu werden, sodass Zellen verschiedener Krebsarten Steifigkeiten in unterschiedlichen Regimes zeigen. Die Ergebnisse dieser Arbeit liefern wichtige Informationen für unser Verständnis der Karzinogenese und bilden die Grundlage für eine neue diagnostische Methode zur Bestimmung der Tumoraggressivität. Eine gezielte Untersuchung der gefundenen Subpopulationen in einem Tumor könnte dabei helfen, neue Therapieansätze zu entwickeln und damit die hohen Rezidivraten aggressiver Tumore zu vermindern.

I use an integrated approach of experiments, theory, and numerical evaluations to show that stiffening and softening/fluidization are natural consequences of the assumption that the cytoskeleton is mechanically essentially equivalent to a transiently crosslinked biopolymer network. I perform experiments on in vitro reconstituted actin/HMM networks and show that already these simple, inanimate systems display fludization and shake-down, but at the same time stress stiffening. Based on the well-established Wlc theory, I then develop a semi-phenomenological mean-field model of a transiently crosslinked biopolymer network, which I call the inelastic glassy wormlike chain (inelastic Gwlc). At the heart of the model is the nonlinear interplay between viscoelastic single-polymer stiffening and inelastic softening by bond breaking. The model predictions are in good agreement with the actin/HMM experiments. Despite of its simplicity, the inelastic Gwlc model displays a rich phenomenology. It reproduces the hallmarks of the mechanics of adherent cells such as power-law rheology, stress and strain stiffening, kinematic hardening, shake-down, fludization, and recovery. The model also may also be able to provide considerable theoretical insights into the underlying physics. For example, using the inelastic Gwlc model, I am able to resolve the apparent paradox between cell softening and stiffening in terms of a parameter-dependent competition of antagonistic nonlinear microscopic mechanisms. I further shed light on the mechanism responsible for fluidization. I identify pertinent parameters characterizing the microstructure and give criteria for the relevance of various effects, including the effect of catch-bonds on the network response. Finally, a way to incorporate irreversible plastic flow is proposed.

Die mechanischen Eigenschaften von Zellen charakterisieren und beeinflussen deren Zustand. Die vorliegende Arbeit zielt auf ein besseres Verständnis der biomechanischen Eigenschaften von Zellen ab. Der Fokus lag dabei auf der Biegesteifigkeit von Zellmembranen und der Deformierbarkeit der Zellen. Es werden drei Studien vorgestellt in der diese Materialparameter untersucht wurden. Die erste Studie befasst sich mit der Temperaturabhängigkeit der mechanischen Eigenschaften. Hierbei wurden acht verschieden Zelllienien bei jeweils fünf Temperaturen rheologisch vermessen. Zur Messung wurde der sog. \"optical stretcher\" verwendet der gleichzeitig die Zellen deformieren und aufheizen kann. Die Versuche zeigen, dass eine Zeit-Temperatur superposition dabei nicht für alle Zelltypen funktioniert. In der zweiten Studie wurden die Membransteifigkeit von Gewebeproben von Brust- und Gebärmutterhalskrebspatienten untersucht. Als Kontrollsystem wurde gutartiges Gewebe aus dem Umfeld des Tumors verwendet. Es konnte gezeigt werden, dass die Zellmembranen von Tumorzellen weicher waren als von gesundem Vergleichsgewebe. Die Änderung der Membrankomposition wurde dabei als mögliche Ursache massenspektroskopisch Untersucht und verschieden Ursachen der weichen Membrane diskutiert. Für die dritte Studie wurde der chemische Wirkstoff Soraphen A eingesetzt um die Membransteifigkeit von zwei Zelllienien zu erhöhen. Dies zeigte eine Verringerung von Zellbeweglichkeit und Invasivität.

I use an integrated approach of experiments, theory, and numerical evaluations to show that stiffening and softening/fluidization are natural consequences of the assumption that the cytoskeleton is mechanically essentially equivalent to a transiently crosslinked biopolymer network. I perform experiments on in vitro reconstituted actin/HMM networks and show that already these simple, inanimate systems display fludization and shake-down, but at the same time stress stiffening. Based on the well-established Wlc theory, I then develop a semi-phenomenological mean-field model of a transiently crosslinked biopolymer network, which I call the inelastic glassy wormlike chain (inelastic Gwlc). At the heart of the model is the nonlinear interplay between viscoelastic single-polymer stiffening and inelastic softening by bond breaking. The model predictions are in good agreement with the actin/HMM experiments. Despite of its simplicity, the inelastic Gwlc model displays a rich phenomenology. It reproduces the hallmarks of the mechanics of adherent cells such as power-law rheology, stress and strain stiffening, kinematic hardening, shake-down, fludization, and recovery. The model also may also be able to provide considerable theoretical insights into the underlying physics. For example, using the inelastic Gwlc model, I am able to resolve the apparent paradox between cell softening and stiffening in terms of a parameter-dependent competition of antagonistic nonlinear microscopic mechanisms. I further shed light on the mechanism responsible for fluidization. I identify pertinent parameters characterizing the microstructure and give criteria for the relevance of various effects, including the effect of catch-bonds on the network response. Finally, a way to incorporate irreversible plastic flow is proposed.

Diese Arbeit beinhaltet Ergebnisse der ersten klinischen Studie zur Charakterisierung der mechanischen Eigenschaften von Zellen in einem Tumor mit der dafür notwendigen Probengröße. Dies ermöglichte die Erstellung eines umfassenden Bildes von Subpopulationen innerhalb eines Tumors mit großem diagnostischem Potential. Die Änderung der Einzelzellmechanik von Tumorzellen wird durch Veränderung des Zytoskeletts, einem komplexes Polymernetzwerk in Zellen, hervorgerufen. Mit Hilfe von Zellgiften wurde das Zytoskelett gezielt manipuliert, um den Einfluss einzelner Faktoren auf die Biomechanik zu bestimmen. Aus Gewebeproben von Brustkrebspatienten wurden Zellen mit Hilfe enzymatischer Aufspaltung des extrazellulären Kollagennetzwerkes isoliert. Als Kontrollsystem wurden Primärzellen aus Brustreduktionsgewebe und aus Fibroadenomen, gutartigen Gewebeneubildungen der Brustdrüse, verwendet. Unter Einsatz des Optischen Stretchers, einer Zweistrahl-Laserfalle, wurden suspendierte Zellen für zwei Sekunden einer konstanten Zugspannung ausgesetzt und das Deformations- wie auch das anschließende Relaxationsverhalten beobachtet. Dabei ergaben sich wesentliche Unterschiede zwischen Tumor- und Kontrollproben. Neben Zellen mit ähnlichen Steifigkeiten, enthielten Tumorproben Subpopulationen sehr weicher Zellen, wie sie in Normalgewebe nicht zu finden sind. Desweiteren war das Relaxationsverhalten der Tumorzellen stärker elastisch dominiert. Einzelne Zellen kontrahierten sogar aktiv gegen die Zugspannung. Versuche, das Zytoskelett mittels Zellgiften künstlich in einem Zustand zu bringen, der in Krebszellen beobachtet wurde, ergaben zwar ebenfalls die Zunahme weicherer Zellen, jedoch war das Relaxationsverhalten eher viskos dominiert. Fluoreszenzaufnahmen des Aktin-Zytoskeletts sowie der fokalen Adhäsionen, die das Aktin-Netzwerk der Zelle mit dem Substrat verankern, zeigten Veränderungen bei Krebszellen im Vergleich zu Kontrollen. Darüber hinaus wurden Einflussfaktoren auf die Zellmechanik untersucht. Neben Kulturbedingungen, beeinflussen auch Alter und Medikation das biomechanische Verhalten. Die Steifigkeit der Krebszellen scheint vom Ursprungsgewebe beeinflusst zu werden, sodass Zellen verschiedener Krebsarten Steifigkeiten in unterschiedlichen Regimes zeigen. Die Ergebnisse dieser Arbeit liefern wichtige Informationen für unser Verständnis der Karzinogenese und bilden die Grundlage für eine neue diagnostische Methode zur Bestimmung der Tumoraggressivität. Eine gezielte Untersuchung der gefundenen Subpopulationen in einem Tumor könnte dabei helfen, neue Therapieansätze zu entwickeln und damit die hohen Rezidivraten aggressiver Tumore zu vermindern.:Bibliographische Beschreibung 3 1 Introduction 10 2 Background 13 2.1 Cancer development and diagnosis 13 2.2 Biomechanics of cells 16 2.2.1 Cytoskeletal changes in cancer cells 17 2.2.2 Micro-mechanical measurement techniques 21 2.2.3 Interaction of laser light with cells: physical principles of the Optical Stretcher 25 2.2.4 Physical models of cell rheology 29 2.3 Cancer cell motility 35 2.4 Tumor boundaries 38 3 Materials and Methods 40 3.1 Sample preparation 40 3.1.1 Cell lines 40 3.1.2 Primary cells from breast reduction 41 3.1.3 Solid tissue samples 41 3.1.4 Drug treatment 48 3.1.5 Fluorescent staining 49 3.2 The Optical Stretcher 50 3.2.1 Experimental Setup 50 3.2.2 Data analysis 52 3.2.3 Reproducibility of Optical Stretcher measurements 52 3.2.4 Laser induced heating in optical traps 54 3.3 Rheometer measurements of collagen gels 57 4 Experimental Results 58 4.1 Parameter space of Optical Stretcher measurements 58 4.2 How well defined are primary tissues? 60 4.2.1 Cells adapt mechanical properties to culture conditions 60 4.2.2 Individual and cell type differences 64 4.3 Characterization of human primary breast cancer cells 68 4.3.1 Malignant tissues comprise an increased number of softer cells 69 4.3.2 Tumor cells show a strong relaxation behavior 73 4.3.3 Single cells contract against applied stress 75 4.4 Changing the biomechanical phenotype - induced alterations of the F-actin network 76 5 Discussion 80 5.1 Measurement bias and impact factors 80 5.2 Softening as an effect of cytoskeletal reorganization in cancer cells 84 5.3 Mechanism of cytoskeletal reorganization 86 5.4 Cells on their way of breaking the boundary 89 6 Conclusion and Outlook 92 Appendix 94 A Cell culture protocols 94 A.1 Used materials and devices for cell culture 94 A.2 Thawing of cells 95 A.3 Medium exchange 96 A.4 Passaging and preparation for measurement 96 A.5 Collagen gel preparation 97 A.6 Culturing specifics for each cell line used 98 A.7 Tissue dissociation protocol for human mamma carcinoma 98 A.8 Fluorescent staining 99 B Additional graphs 101 Bibliography 103 Acknowledgements 114 Curriculum Vitae 115 Selbständigkeitserklärung 116

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.

No description available.

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.

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