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.

Actin stress fibers (SFs) are mechanosensitive structural elements that respond to applied strain to regulate cell morphology, signal transduction, and cell function. The purpose of this dissertation is to elucidate the effects of mechanical stretch on cell mechanobiology via the following three aims. First, a sarcomeric model of SFs was developed to describe the role of actomyosin crossbridge cycling in SF tension regulation and reorientation in response to various modes of stretch. Using model parameters extracted from literature, this model described the dependence of cyclic stretch-induced SF alignment on a two-dimensional (2-D) surface on positive perturbations in SF tension caused by the rate of lengthening, which was consistent with experimental findings. Second, the sarcomeric model was used to predict how stretch-induced pro-inflammatory mechanotransduction depends on the mode of strain application. Together with experimental data, the results indicated that stretch-induced stress fiber alignment, MAPK activations and downstream pro-inflammatory gene expressions are dependent on SF strain rate (and related changes in SF tension) rather than SF turnover. Third, to produce biocompatible materials that are both mechanically resilient under (physiological) load and also mechanosensitive, a novel hybrid engineered tissue was developed that transmits strain stimuli to cells residing in three-dimensional (3-D) collagen microspheres. However, the macroscopic stress is largely borne by a more resilient acellular polyethylene glycol diacrylate (PEGDA) hydrogel supporting the microspheres. Careful analysis indicated that cell alignment occurs prior to significant collagen fibril alignment.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23929号 / 工博第5016号 / 新制||工||1783(附属図書館) / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 山本 量一, 教授 宮原 稔, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.

abstract: The atomic force microscope (AFM) is capable of directly probing the mechanics of samples with length scales from single molecules to tissues and force scales from pico to micronewtons. In particular, AFM is widely used as a tool to measure the elastic modulus of soft biological samples by collecting force-indentation relationships and fitting these to classic elastic contact models. However, the analysis of raw force-indentation data may be complicated by mechanical heterogeneity present in biological systems. An analytical model of an elastic indentation on a bonded two-layer sample was solved. This may be used to account for substrate effects and more generally address experimental design for samples with varying elasticity. This model was applied to two mechanobiology systems of interest. First, AFM was combined with confocal laser scanning fluorescence microscopy and finite element analysis to examine stiffness changes during the initial stages of invasion of MDA-MB-231 metastatic breast cells into bovine collagen I matrices. It was determined that the cells stiffen significantly as they invade, the amount of stiffening is correlated with the elastic modulus of the collagen gel, and inhibition of Rho-associated protein kinase reduces the elastic modulus of the invading cells. Second, the elastic modulus of cancer cell nuclei was investigated ex situ and in situ. It was observed that inhibition of histone deacetylation to facilitate chromatin decondenstation result in significantly more morphological and stiffness changes in cancerous cells compared to normal cells. The methods and results presented here offer novel strategies for approaching biological systems with AFM and demonstrate its applicability and necessity in studying cellular function in physiologically relevant environments. / Dissertation/Thesis / Doctoral Dissertation Physics 2015

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17556号 / 工博第3715号 / 新制||工||1566(附属図書館) / 30322 / 京都大学大学院工学研究科機械理工学専攻 / (主査)教授 富田 直秀, 教授 井手 亜里, 教授 安達 泰治 / 学位規則第4条第1項該当

Mechanical cues have been recognized to be critically important in the regulation of cells. A myriad of cellular processes including differentiation, proliferation, and gene expression are all affected by physical forces from the extra- and intra-cellular microenvironments. Despite recent advances in nano-technologies, many questions still surround how cells sense and respond to forces. Through a series of studies, we demonstrate how both the structure and inherent mechanical properties of the cell affect their response to mechanical cues. We first develop a methodology to mechanically manipulate cells while simultaneously characterizing their deformations. Using combined atomic force and confocal microscopy techniques and through systematic examination we demonstrate the role of the cytoskeleton and nucleus in the deformability and shape change of epithelial cells. Mechanical properties have been used in recent years to identify diseased states, including cancer. With this in mind, we used HeLa cells as a model and characterized significant deformability of their plasma membrane and underlying cortex. Importantly, we demonstrate and characterize their ability to recover from large shape changes, which we also observed in other epithelial cells. Shape recovery is shown to be rapid and reliant upon the actin cytoskeleton and intracellular fluid flow. Although the nucleus does not contribute significantly to the deformation and recovery of HeLa cells, the importance of nuclear mechanics cannot be forgone. In vitro studies have shown that mechanical forces transmitted through the cell’s cytoskeleton critically affect nuclear mechanics and gene transcription processes. Many others have used simple models and isolated nuclei in an attempt to characterize nuclear properties. Thus, in a subsequent study, we examine the nucleus within intact cells. Nuclear shape change, in response to force, is shown to be complex and cannot be well-characterized by isotropic mechanical properties. Characterization of the mechanics of the cell, as demonstrated through our findings, is crucial in the field of biological physics. The aforementioned studies, written as scientific articles, are presented in the body of this thesis (Chapters 2-5). A review article that focuses on mechanotransduction and relevant examples using AFM as a tool for its examination acts as an introductory chapter.

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