Endogenous Force Transmission Between Epithelial Cells and a Role for α-Catenin

In epithelial tissues, epithelial cells adhere to each other as well as to the underlying extra-cellular matrix (ECM). E-cadherin-based intercellular junctions play an important role in tissue integrity. These junctions experience cell-generated mechanical forces via apparent adaptor proteins such as beta (β) catenin, alpha (α) catenin and vinculin. Abnormalities in these junctions may result in skin related diseases and cancers. Here, I devised methods to determine the endogenous intercellular force within cell pairs as well as in large epithelial islands. I further ascertained the factors that affect the level of inter-cellular tension. Experiments with pairs of epithelial cells exogenously expressing either of two altered E-cadherin constructs on top of endogenous E-cadherin showed that the inter-cellular tension for these cases was similar to wild type cells. This implied that the endogenous E-cadherin was dominant in setting the level of cell-cell contact tension. To analyze intercellular force transmission within large islands, I extended the traction force imbalance method to large micro-patterned islands. It was shown that colony level intercellular forces exerted at the midline by one half of the colony on the other were tensile in nature and showed significant anisotropy with respect to the midline orientation. Finally, to determine what molecular factors set the level of tension transmitted through single cell-cell contacts, I determined the inter-cellular tension in pairs of cells with perturbed α-catenin. I found that α-catenin knockout not only decreased inter-cellular tension but also the traction forces exerted at the cell-ECM interface. This may be due to roles outside of cell-cell junctions for α-catenin. However, α-catenin mutants with altered binding to vinculin binding did not show significant differences in inter-cellular tension compared to wild type cells. Thus, α-catenin is essential for normal levels of intercellular tension, but the forces transmitted at cell-cell contacts are not very sensitive to the level of vinculin at the cell-cell contact in cell pairs. This may be because vinculin can also be recruited to cell-cell contacts by molecules other than α-catenin, such as β-catenin. These results highlight some essential and non-essential molecular factors regulating cell-cell junctional tension level

Integrated Mathematical and Experimental Study of Cell Migration and Shape

Cell migration plays an essential role in many of physiological and pathological processes, including morphogenesis, inflammation, wound healing, and tumor metastasis. It is a complex process that involves multi-scale interactions between the cell and the extracellular matrix (ECM). Cells migrate through stromal ECM with native and cell-derived curvature at micron-meter scale are context-specific. How does the curvature of ECM mechanically change cell morphology and motility? Can the diverse migration behaviors from genetically identical cells be predictively using cell migrating data? We address these questions using an integrated computational and experimental approach: we developed three-dimensional biomechanical cell model and measured and analyzed a large number of cell migration images over time. Our findings suggest that 1. substrate curvature determines cell shape through contact and regulating protrusion dynamics; 2. effective cell migration is characterized with long cellular persistence time, low speed variation, spatial-temporally coordinated protrusion and contraction; 3. the cell shape variation space is low dimensional; and 4. migration behavior can be determined by a single image projected in the low dimensional cell shape variation space

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome

Microscale Measurements of Cell and Tissue Mechanics in Three Dimensions

Two-dimensional (2D) studies have revealed that mechanical forces drive cell migration and can feedback to regulate proliferation, differentiation and the synthesis/remodeling of extracellular matrix (ECM) proteins. Whether these observations can be translated to clinical settings or be utilized for tissue engineering will depend critically on our ability to translate these findings into physiologically relevant three-dimensional (3D) environments. The general goal of this dissertation has been to develop and apply new technologies capable of extending studies of cell and tissue mechanics into 3D environments. In the first project, we measured both shear and normal traction forces exerted by cells cultured on planar substrates. We observed that focal adhesions serve as pivots about which cells generate rotational moments. In the second project, we combined enzymatically degradable synthetic hydrogels with finite element models to measure the mechanical tractions exerted by cells fully encapsulated within 3D matrices. We found that cells reach out thin protrusions and pull back inward towards the cell body with the highest forces at the tip. Cellular extensions that were invading into the surrounding matrix displayed a strong inward force 10-15 microns behind the leading tip, suggesting that growing extensions may establish a contractile waypoint, before invading further. To study the forces cells exert during tissue remodeling, we utilized photolithograpy to generate arrays of microtissues consisting of cells encapsulated in 3D collagen matrices. Microcantilevers were used to constrain the remodeling of the collagen gel and to report the forces generated during this process. We used this technique to explore the effects of boundary stiffness and matrix density within model tendon and cardiac tissues. Finally, we combined this system with a Foerster radius energy transfer (FRET) based biosensor of fibronectin conformation to reveal how tissue geometry and cell-genereated tractions cooperate to pattern matrix conformation during tissue remodeling. Together, these studies highlight novel approaches to understand the nature of cell-ECM interactions in 3D matrices. Such mechanical insights will help us to understand how physical forces drive cell migration and behavior within physiologically relevant environments

Multiscale regulation of cellular mechanical properties

Thesis (Ph.D.)--Boston UniversityIn vivo, cells routinely experience mechanical stresses and strains in the form of circulatory pressure and flow, peristalsis ofthe gut, and airway inflation and deflation. Even on the microscale, all adherent cells apply contractile force to the extracellular matrix and to neighboring cells. Cells respond to these external forces both passively and actively. Passively, cells need to deform in a way that is tissue and function appropriate. Actively, cells use local mechanoreceptors present on their surface to trigger changes in global cell behavior. Dysregulation of cell responses to force are hallmarks of diseases such as atherosclerosis, asthma and cancer. Given the pluripotent role of cell mechanics in both normal cell behavior and disease, cell regulation of mechanical properties has become a major area of focus in biology. In this dissertation, we explore passive mechanical properties and active mechanical responses of cells on the subcellular, single cell and multicellular length scales. In Aim 1, we developed a new tool, called cell biomechanical imaging, for mapping intracellular stiffness and prestress. We have demonstrated a linear relationship between these two quantities, both at the whole cell and subcellular levels, which suggests prestress may be a unifying mechanism by which cells and tissues tune their mechanical properties. In Aim 2, we investigated how coordinated changes in cytoskeletal tension lead to cell reorientation. Previous research has shown that in response to strain applied through focal adhesions, the actin cytoskeleton promptly fluidizes and then slowly resolidifies. Using both experiments and a mathematical model, we found that repeated interplay ofthese phenomena was a driving force behind cytoskeletal reorganization during cell reorientation. It was previously hypothesized that the purpose of cytoskeletal remodeling in response to strain was to minimize changes in intracellular mechanical tension and maintain it at a preferred level. This feedback control mechanism, which balances forces between the cell and its microenvironment, is termed "tensional homeostasis." The dominant paradigm in vascular biology is that tensional homeostasis exists across multiple length and time scales. However, our results from Aim 2 challenged this idea; reoriented cells did not maintain steady levels of contractile force. In Aim 3, we investigated tensional homeostasis and its existence at multiple length scales. We found that cells do not have a preferred level of tension at the subcellular or single cell levels. However, in a cluster of confluent cells, contractile tension is maintained, the more so as cluster size increases. Together, the results of this dissertation emphasize the importance ofa multiscale approach to mechanobiology. Cells and tissue are hierarchically ordered systems that use mechanical stress (prestress) to tune their mechanical properties and responses across lengthscales. Thus, it is important to consider not just the behavior of separate components of each of these systems, but the behaviors that emerge when they interact with one another

Cellular and Cytoskeletal Responses of Myofibroblasts in Three Dimensional Culture to Mechanical Stretch

Myofibroblasts play important roles in wound healing and pathological organ remodeling, such as hypertensive cardiac fibrosis and promotion of metastasis. Differentiated myofibroblasts are characterized by increased production of extracellular matrix: ECM) proteins and by the development of α-smooth muscle actin: α-SMA) positive stress fibers that are connected to the ECM through focal adhesion assemblies. Moreover, mounting evidence suggests that development of myofibroblasts is profoundly influenced by the mechanical microenvironment, especially, by the structure, organization or stiffness of the ECM: Hinz and Gabbiani, 2003a). Myofibroblasts are likely signaled by mechanical changes in their environment transduced through their cytoskeletons, and the fundamental goal of this dissertation is to develop our understanding of this transduction, with the long term goal of guiding treatments that modulate the responses of myofibroblasts to their mechanical environment. Previous studies provide a tremendous amount of data for how cells adapt to mechanical stress in two dimensions: 2D). Many techniques exist for probing cyoskeletal responses in 2D. These range from pushing cells with a fluid to poking cells with an AFM probe to pulling cells using micropipette aspiration. However, myofibroblasts in a realistic tissue environment cannot be tested using these approaches. The challenge is that cells adopt very different dynamic morphologies in the network of fibers presented by a natural tissue, and cannot be pushed, poked, or pulled using these techniques inside a tissue. The only way to probe the cytoskeleton of such a cell naturally in a tissue is through its natural adhesions to the ECM. In this work, we developed a 3D tissue culture system to quantify the responses of contractile fibroblasts in an engineered tissue construct: ETC) to external mehanical stimuli. We try to answer the following questions. How does the actin cytoskeleton respond and adapt to mechanical stretch? How does this relate to whole-cell kinematics? How do cells interact with the extracellular environment in 3D? To address the first question, a suite of mechanical tests were performed on transfected myofibroblasts in ETCs. The transfection enabled the visualization of stress fiber dynamics in 3D, and the work showed for the first time that stress fibers can form in response to mechanical stretch. The work also quantified the diversity of cellular responses into three classes, as described in Chapter 2. To address the second question, we studied the nature of the retraction of filopodium-like processes of stained myofiboblasts in response to mechanical stretch. Results, described in Chapter 3, showed that retraction of these processes was independent of the magnitude of stretch and the length of the process. The time constants associated with process retractions were very slow compared to membrane retraction in other cells. The study of the last question confirmed that large focal adhesions existed in a 3D environment, early in remodeling, and that focal adhesion complexes evolve during ECM remodeling. The work in this dissertation helps establish a platform to study cellular mechanics and function in a 3D environment through both real-time imaging and force measurement simultaneously. While the work is at the level of basic biophysics, it provides a quantitative window into the mechanisms underlying myofibroblast-based pathologies

Role of boundary conditions in determining cell alignment in response to stretch

he ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotrans- duction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteo- blasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we used an experimental system to conduct a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direc- tion give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments (“strain avoidance”). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular align- ment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the ap- parently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels

Investigating the Effects of Biochemical and Biophysical Signals on Vascular Smooth Muscle Cell Differentiation

In blood vessel engineering, an optimal bioartifical scaffold can be characterized as a 3D tubular structure with high porosity for nutrient diffusion and enough mechanical strength to sustain in vivo dynamic environment. The luminal surface of the scaffold is supposed to have a continuous layer of endothelial cell that is ideally non-immunogenic and non-thrombogenic while the media layer of the construct is assigned for the ingrowth of vascular smooth muscle cell which can provide structural integrity and contractility. While reconstructing endothelial cell layer has been at the center of interest in most polymeric vascular replacements related research, growing VSMCs has had less attention due to the high risk of their excessive proliferation and unexpected phenotype shifts that can result in vessel restenosis. In addition, finding a reliable source of VSMC can be a formidable task. As such, we believe that if VSMCs can be modulated to remain quiescent and functional over time after they are obtained from an alternative source, they might eventually be considered to incorporate into artificial vascular substitute. To achieve this goal, first we investigated the potential of using stem cell to differentiate into functional VSMCs. Next, we designed a 3D culture construct to mimic blood vessel with distinct layers and analyzed the effect of combining different biochemical and biomechanical signals on modulating VSMCs behavior. Finally, we developed a biomechanical model that can incorporate the mechanical property of differentiated cell and distinct layers with geometrical information acquired from confocal images to predict cellular behavior under different conditions. The results of these studies provide insights from a basic science prospective about the potential of using stem cell to obtain functional VSMCs and the impact of environmental factors on VSMCs behavior. Researchers may use these results to optimize the culture condition of VSMCs in order to modulate its proliferation, phenotype and mechanical property. The model developed in this study might be used to predict cellular behavior under different culture environments without repetitive experiments

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

Design and Assembly Considerations in the Engineering of Vascular Tissue

Native vascular tissue functions are highly dependent on structural organization at the super-cellular, cellular, and sub-cellular spatial scales. We hypothesized that the structure-function relationship of vascular tissues in vivo can be leveraged to engineer vascular tissues in vitro by prescribing the shape of constituent cells and their assembly into organized three-dimensional structures. To this end, we first asked if vascular smooth muscle cell shape influences cellular contractility. We engineered human vascular smooth muscle cells to assume similar shapes to those in elastic and muscular arteries and then measured their contraction while stimulating with endothelin-1. We found that vascular smooth muscle cells with elongated shapes exhibited lower contractile strength but a greater percentage increase in contraction after endothelin-1 stimulation, suggesting that elongated vascular smooth muscle cell shape endows the muscular artery with greater dynamic contractile range. Next, we sought to assemble cells into tissues by employing a three-dimensional cellular patterning strategy based on the folding of porous, thin polymer films. We assembled different three-dimensional endothelial and vascular smooth muscle organizations by patterning two-dimensional poly(lactic-co-glycolic) acid and collagen thin films with cell suspensions at prescribed locations. The films were subsequently folded following Miura-ori geometry guidelines and the matrices were embedded subcutaneously in immunodeficient mice in order to assess the vascularization of the implanted constructs. We found that spatial organization that allowed endothelial and vascular smooth muscle cells to interact adjacent to each other laterally in the same folding plane created the densest vascularized network, suggesting that three-dimensional structural organization of vascular cells can influence the formation of vascularized networks. Taken together, our result shows that functional vascular tissues in vitro can be engineered by encoding structure cues in their design and assembly.Engineering and Applied Science