Doctor of Philosophy

dissertationCells encounter mechanical cues from the environment to which they sense and respond. The actin cytoskeleton is the main network that can not only sense mechanical changes, but can also reorganize in response. Actin stress fibers are predominant in cultured fibroblast cells and are load-bearing structures of the cell. Here, in collaboration with others, I have investigated the mechanisms of stress fiber strain response and remodeling using fluorescently-labeled cytoskeletal proteins and live cell microscopy, traction force microscopy, and genetic manipulation to assess these mechanisms. High resolution image acquisition and analysis have provided novel insight into the mechanosensitivity of actin stress fibers. Specifically, the actin-associated protein zyxin has been implicated in an actin repair mechanism with mechanical consequences. We discovered a novel zyxin-mediated actin repair mechanism that restored structural and mechanical integrity to stress fibers following a hyperleongation event in a single stress fiber sarcomere. We also discovered that while these spontaneously occurring hyperelongation events impact single sarcomeres along a stress fiber, they coincide with compensatory shortening in the near-by regions of stress fiber sarcomeres, suggesting there is active remodeling that occurs in actin stress fibers in order to maintain the structure and mechanical homeostasis in live cells. Lastly, we designed a computational model to test whether actin and myosin-based mechanical changes drive some of these dynamic changes in stress fibers. We discovered that variable differences in actin stiffness and myosin contractility may be the main factors in spontaneous changes in iv stress fiber sarcomere length. The findings presented in this dissertation have made exciting contributions to the field of actin cytoskeletal dynamics, and will provide groundwork to future studies dissecting the role of actin-associated proteins in structural and mechanical homeostasis in stress fibers

A SINGLE CELL PAIR MECHANICAL INTERROGATION PLATFORM TO STUDY CELL-CELL ADHESION MECHANICS

Cell-cell adhesion complexes are macromolecular adhesive organelles that integrate cells into tissues. Perturbations of the cell-cell adhesion structure or relatedmechanotransduction pathways lead to pathological conditions such as skin and heart diseases, arthritis, and cancer. Mechanical stretching has been used to stimulate the mechanotransduction process originating from the cell-cell adhesion and cell-extracellular matrix (ECM) complexes. The current techniques, however, have limitations on their ability to measure the cell-cell adhesion force directly and quantitatively. These methods use a monolayer of cells, which makes it impossible to quantify the forces within a single cell-cell adhesion complex. Other methods using single cells or cell pairs rely on cell-ECM adhesion to find the cell-cell adhesion forces and consequently, they indirectly measure the junctional forces. In the current study, we designed and developed a single cell-cell adhesion interrogation and stimulation platform based on nanofabricated polymeric structures. The platform employs microstructures fabricated from biocompatible materials using two photon polymerization (TPP), a process that enables direct 3D structure writing with nanometer precision. The microdevice allows a pair of epithelial cells to form a mature cell junction. The single matured cell junction is stretched with controlled strain until cell-cell junction ruptures while the forces within the cell-junction-cell system are recorded. Using this platform, we have conducted mechanical characterization of a single cell junction with strain-stress analysis. The strain dependency of the junction has been investigated through the stretch test with four different strain rates. The results showed that the junction behaves in a strain-rate dependent manner, where high strain-rates lead to decreased viscosity property, a characteristic for a shear-thinning viscoelastic material. This also confirms our hypothesis that strain-rate plays an important role in the cell mechanical behavior, particularly the cytoskeleton dominant cell mechanics. The maturation of this technology can pave the way for the in situ investigation of mechano-chemical signaling pathways mediated by cell-cell junctions and potentially reveal novel disease mechanisms in which defects in cell-cell adhesion play a significant role in the disease pathology. Advisor: Ruiguo Yan

Continuum Models of Collective Migration in Living Tissues

This dissertation investigates the physical mechanics of collective cell migration in monolayers of epithelial cells. Coordinated cell motion underlies a number of biological processes, including wound healing, morphogenesis and cancer metastasis, and is controlled by the interplay of single cell motility, cell-cell adhesions, cell-substrate interaction, and cell contractility modulated by the acto-myosin cytoskeleton. Here we examine the competing roles of these mechanisms via a continuum model of a tissue as an active elastic medium, where mechanical deformations are coupled to and feed back onto chemical signaling. We begin in Chapter 1 with a brief review of cell migration at both the single-cell and many-cell levels, and of the experimental tools used to probe the mechanical properties of cells and tissues. In Chapter 2 we formulate our minimal continuum model of a tissue as an overdamped active elastic medium on a frictional substrate. The model couples mechanical deformations in the tissue to myosin-based contractile activity and to cell polarization. Two new ingredients of our model are: (i) a feedback between the on-off dynamics of myosin motors and the active contractile stresses they induce in the tissue, and (ii) the coupling of cell directed motion or polarization to tissue strain. In the following two chapters we employ this model to describe collective cell dynamics in expanding (Chapter 3) and confined (Chapter 4) tissues and compare with experiments. In expanding monolayers, as realized for instance in wound healing assays where an initially confined tissue is allowed to expand freely on a substrate, our model reproduces the propagating waves of mechanical stress observed in experiments and believed to play a key role in controlling the transmission of information across the tissue and mediating coordinated cell motion. Combining analytical and numerical work we construct a phase diagram that identifies various dynamical regimes in terms of single-cell properties, such as contractility and stiffness. In Chapter 4, we use our model to describe collective dynamics of cells confined to a circular geometry. In this case the propagating waves are replaced by standing sloshing waves guided by both contractility and polarization. The work on confined tissues was carried out in collaboration with the experimental group of Jeff Fredberg at the Harvard School of Public Health. By combining theory and experiment we can provide a quantitative understanding of how contractility and polarization regulate the mechanics of the tissue by renormalizing the tissue elastic moduli and controlling the frequency of oscillatory modes

Multiscale mechanobiology: computational models for integrating molecules to multicellular systems

Mechanical signals exist throughout the biological landscape. Across all scales, these signals, in the form of force, stiffness, and deformations, are generated and processed, resulting in an active mechanobiological circuit that controls many fundamental aspects of life, from protein unfolding and cytoskeletal remodeling to collective cell motions. The multiple scales and complex feedback involved present a challenge for fully understanding the nature of this circuit, particularly in development and disease in which it has been implicated. Computational models that accurately predict and are based on experimental data enable a means to integrate basic principles and explore fine details of mechanosensing and mechanotransduction in and across all levels of biological systems. Here we review recent advances in these models along with supporting and emerging experimental findings.National Cancer Institute (U.S.) (U01-CA177799

Zellen als lebende Materialien: Kraftspektroskopische Untersuchung der Mechanotransduktion

Mechanotransduction describes a cellular mechanism of sensing and converting mechanical cues into biochemical signals to regulate cell processes, such as adhesion, migration, proliferation and/or apoptosis. Thus, becoming an ever-growing field of research with high potential for medical applications. I present a new strategy towards reliable microindentation measurements, which is essential for investigating mechanotransduction using soft substrates. I show a precise, reproducible determination of Young’s moduli through an automatic analysis of indentation data. The algorithm presented detects Young’s moduli in a region without dependence on indentation depth while minimizing the fitting error. This strategy is a step towards a comprehensive study of soft materials on a spatial scale similar to cell interactions. It has broad applicability ranging from fundamental research to developing innovative implants that match the in vivo situation. Also, I present novel approaches for multifaceted cellular manipulation. I show that layer thickness of a soft material fixed to a stiff underlying substrate can be crucial for cell adhesion. These findings are pioneer for new implant designs and advanced application fields. I present two atomic force microscopy-based manipulation systems that allow applying specific mechanical stimuli to single cells and a subsequent correlation to whole cell detachment and single bond strengths. The unique AFM-based shear system presented combines application of shear stimuli and cell detachment measurements, whereas the AFM-based modulation system combines oscillatory pushing and pulling with cell detachment measurements. Both shear and oscillatory forces are essential in our body. Thus, the strategies presented in this thesis are of significant medical interest allowing an overarching study of mechanotransduction and may pave the way towards smart stimulation devices that allow cell adhesion on demand

The effect of cell morphology on the permeability of the nuclear envelope to diffusive factors

A recent advance in understanding stem cell differentiation is that the cell is able to translate its morphology, i.e., roundish or spread, into a fate decision. We hypothesize that strain states in the nuclear envelope (NE) cause changes in the structure of the nuclear pore complexes. This induces significant changes in the NE’s permeability to the traffic of the transcription factors involved in stem cell differentiation which are imported into the nucleus by passive diffusion. To demonstrate this, we set up a numericalmodel of the transport of diffusivemolecules through the nuclear pore complex (NPC), on the basis of the NPC deformation. We then compared the prediction of the model for two different cell configurations with roundish and spread nuclear topologies with those measured on cells cultured in both configurations. To measure the geometrical features of the NPC, using electron tomography we reconstructed three-dimensional portions of the envelope of cells cultured in both configurations. We found non-significant differences in both the shape and size of the transmembrane ring of single pores with envelope deformation. In the numerical model, we thus assumed that the changes in pore complex permeability, caused by the envelope strains, are due to variations in the opening configuration of the nuclear basket, which in turn modifies the porosity of the pore complex mainly on its nuclear side. To validate the model, we cultured cells on a substrate shaped as a spatial micro-grid, called the “nichoid,” which is nanoengineered by two-photon laser polymerization, and induces a roundish nuclear configuration in cells adhering to the nichoid grid, and a spread configuration in cells adhering to the flat substrate surrounding the grid. We then measured the diffusion through the nuclear envelope of an inert green-fluorescent protein, by fluorescence recovery after photobleaching (FRAP). Finally, we compared the diffusion times predicted by the numerical model for roundish vs. spread cells, with the measured times. Our data show that cell stretching modulates the characteristic time needed for the nuclear import of a small inert molecule, GFP, and the model predicts a faster import of diffusive molecules in the spread compared to roundish cells.Peer ReviewedPostprint (published version

The Effect of Biomechanical and Biochemical Factors on Endothelial Cells: Relevance to Atherosclerosis

Microscale technologies create great opportunities for biologists to unveil cellular or molecular mechanisms of complex biological processes. Advanced measuring techniques, like atomic force microscope (AFM), allow detecting and controlling biological samples at high spatial and temporal resolution. Further integration with microsystems, such as microfluidic platforms, gives the ability to get detailed insight into basic biological phenomena. Highly integrated microdevices show great promise for biomedical research and potential clinical applications. It is hypothesized that biomechanical factors play a significant role in the development of vascular diseases like atherosclerosis. To explore effects of biomechanical and biochemical stimuli on endothelial cells (ECs), AFM, which allows measurements of living cells, was utilized. Due to the heterogeneity of cells, standard characterization methods for mechanical properties of cells are still lacking. Therefore, a new quantitative method was developed for evaluation of cell elasticity correlating with cell morphology in this study. Moreover, cells are intrinsically viscoelastic materials revealed by stress relaxation measurements. A mechanically distinct bilayer model was proposed to discover the mechanical behaviour of cell components. Based on the elasticity characterization method and the stress relaxation model, the effect of cholesterol content on the mechanical response of ECs was examined, focusing on the behaviour of plasma membrane. To mimic physiological conditions more closely for in vitro settings, a mask-free, highly integrated, low cost and time effective method was developed to rapidly fabricate a prototype of microfluidic cell culture system (MCCS). To better understand cell-cell interaction in circulatory systems like MCCS, a theoretical study of evaluating intercellular forces was also performed. Based on MCCS and microvalve technique, a novel bio-inspired and cell-based system was developed to simulate the formation of atherosclerosis plaque. Biomechanical properties of ECs, hemodynamic effects, cell rolling and adhesion events were investigated under this pathological model. The devices can be leveraged for potential applicability to biological research and clinical tests such as drug screening. This research project has led to a better understanding of the underlying mechanisms of atherosclerosis and mechanical behaviours of ECs, as well as the development of AFM-based models that will be useful in determining cellular mechanical properties

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism

Endothelium and subendothelial matrix mechanics modulate cancer cell transendothelial migration

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push–pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.Peer ReviewedPostprint (published version