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Open Access Publications from the University of California

Scholarly Works (16 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

The collective transmission of forces in multicellular colonies plays an important role in biological processes such as development, endothelial function and wound healing. The quantification of intracellular forces by monolayer stress microscopy (MSM) has improved our understanding of these phenomena, and provides great promise for the functional screening of contractile tissues in the context of drug development. However, current MSM methods present a number of limitations: they assume that the monolayer only deforms laterally, introduce boundary artifacts, and are restricted to low-throughput applications. This dissertation addresses these limitations and provides the first high-throughput quantification of three-dimensional (3D) intracellular stresses in applications relevant to biology and biotechnology, ranging from leukocyte extravasation to high-throughput functional screening of cardiomyocytes.

We introduce a 3D-MSM method that incorporates for the first time monolayer bending. 3D-MSM measures the 3D traction stresses exerted by the cells on a flexible substrate and calculates intracellular stresses by imposing equilibrium of forces and moments in the monolayer. We present 3D-MSM measurements in micropatterned cell islands of different sizes and shapes. We show that intracellular stresses caused by lateral deformation are transmitted across long distances, whereas bending-induced stresses remain confined to 1-2 cell lengths from bending sites. We hypothesize that the localized effect of bending-induced stresses may be important in processes such as cellular extravasation, which are accompanied by large 3D deflections of the endothelium and require localized permeability changes. Overall, these results suggest that bending and lateral stresses can propagate mechanical signals at different length scales, and reveal that the transmission of forces across cell junctions is more three-dimensional and complex than previously believed.

We also present a novel high-throughput functional screening assay that quantifies cardiomyocyte force generation and stiffness changes during the cardiac cycle. The method jointly measures the intracellular stresses within cardiomyocyte monolayers and their elastic modulus. The main novelty of this method is that it utilizes the forces generated by the cells themselves to probe their material properties and is, thus, fully non-invasive. We present the proof-of-concept application of our method with dose-response measurements of a series of benchmark compounds tested on monolayers of hiPSC cardiomyocytes.

    Cover page: Monolayer Stress Microscopy: Developing 3D, Elastographic, and High-Throughput Capabilities
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2017)
    The aim of this work is to quantify the spatio-temporal dynamics of flow-driven amoeboid locomotion in small (~100 µm) fragments of the true slime mold Physarum polycephalum. In this model organism, cellular contraction drives intracellular flows, and these flows transport the chemical signals that regulate contraction in the first place. As a consequence of these non-linear interactions, a diversity of migratory behaviors can be observed in migrating Physarum fragments. To study these dynamics, we measure the spatio-temporal distributions of the velocities of the endoplasm and ectoplasm of each migrating fragment, the traction stresses it generates on the substratum, and the concentration of free intracellular calcium. Using these unprecedented experimental data, we classify migrating Physarum fragments according to their dynamics, finding that they often exhibit spontaneously coordinated waves of flow, contractility and chemical signaling. We show that Physarum fragments exhibiting symmetric spatio-temporal patterns of endoplasmic flow migrate significantly slower than fragments with asymmetric patterns. In addition, our joint measurements of ectoplasm velocity and traction stress at the substratum suggest that forward motion of the ectoplasm is enabled by a succession of stick-slip transitions, which we conjecture are also organized in the form of waves. Combining our experiments with a simplified convection-diffusion model, we show that the convective transport of calcium ions may be key for establishing and maintaining the spatiotemporal patterns of calcium concentration that regulate the generation of contractile forces.
      Cover page: Self-organized mechano-chemical dynamics in amoeboid locomotion of Physarum fragments
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2015)
      This work examines the relationship between spatio-temporal coordination of intracellular flow and traction stress and the speed of amoeboid locomotion of microplasmodia of Physarum polycephalum. We simultaneously perform particle image velocimetry and traction stress microscopy to measure the velocity of cytoplasmic flow and the stresses applied to the substrate by migrating Physarum microamoebae. In parallel, we develop a mathematical model of a motile cell which includes forces from the viscous cytosol, a poro-elastic, contractile cytoskeleton and adhesive interactions with the substrate. Our experiments show that flow and traction stress exhibit back-to-front-directed waves with a distinct phase difference. The model demonstrates that the direction and speed of locomotion are determined by this coordination between contraction, flow and adhesion. Using the model, we identify forms of coordination that generate model predictions consistent with experiments. We demonstrate that this coordination produces near optimal migration speed and is insensitive to heterogeneity in substrate adhesiveness. While it is generally thought that amoeboid motility is robust to changes in extracellular geometry and the nature of extracellular adhesion, our results demonstrate that coordination of adhesive forces is essential to producing robust migration.
        Cover page: Coordination of contractility, adhesion and flow in migrating Physarum amoebae
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2016)
        Streams of migratory cells are initiated by the formation of tandem pairs of cells connected head to tail to which other cells subsequently adhere. The mechanisms regulating the transition from single to streaming cell migration remain elusive, although several molecules have been suggested to be involved. In this work, we investigate the mechanics of the locomotion ofDictyosteliumtandem pairs by analyzing the spatiotemporal evolution of their traction adhesions (TAs). We find that in migrating wild-type tandem pairs, each cell exerts traction forces on stationary sites (∼80% of the time), and the trailing cell reuses the location of the TAs of the leading cell. Both leading and trailing cells form contractile dipoles and synchronize the formation of new frontal TAs with ∼54-s time delay. Cells not expressing the lectin discoidin I or moving on discoidin I-coated substrata form fewer tandems, but the trailing cell still reuses the locations of the TAs of the leading cell, suggesting that discoidin I is not responsible for a possible chemically driven synchronization process. The migration dynamics of the tandems indicate that their TAs' reuse results from the mechanical synchronization of the leading and trailing cells' protrusions and retractions (motility cycles) aided by the cell-cell adhesions.
          Cover page: Cooperative cell motility during tandem locomotion of amoeboid cells.
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2013)
          Microscale and nanoscale surface topography changes can influence cell functions, including morphology. Although in vitro responses to static topography are novel, cells in vivo constantly remodel topography. To better understand how cells respond to changes in topography over time, we developed a soft polyacrylamide hydrogel with magnetic nickel microwires randomly oriented in the surface of the material. Varying the magnetic field around the microwires reversibly induced their alignment with the direction of the field, causing the smooth hydrogel surface to develop small wrinkles; changes in surface roughness, ΔRRMS , ranged from 0.05 to 0.70 μm and could be oscillated without hydrogel creep. Vascular smooth muscle cell morphology was assessed when exposed to acute and dynamic topography changes. Area and shape changes occurred when an acute topographical change was imposed for substrates exceeding roughness of 0.2 μm, but longer-term oscillating topography did not produce significant changes in morphology irrespective of wire stiffness. These data imply that cells may be able to use topography changes to transmit signals as they respond immediately to changes in roughness.
            Cover page: Dynamic and reversible surface topography influences cell morphology
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2017)
            Cells employing amoeboid motility exhibit repetitive cycles of rapid expansion and contraction and apply coordinated traction forces to their environment. Although aspects of this process are well studied, it is unclear how the cell controls the coordination of cell length changes with adhesion to the surface. Here, we develop a simple model to mechanistically explain the emergence of periodic changes in length and spatiotemporal dynamics of traction forces measured in chemotaxing unicellular amoeba, Dictyostelium discoideum. In contrast to the biochemical mechanisms that have been implicated in the coordination of some cellular processes, we show that many features of amoeboid locomotion emerge from a simple mechanochemical model. The mechanism for interaction with the environment in Dictyostelium is unknown and thus, we explore different cell-environment interaction models to reveal that mechanosensitive adhesions are necessary to reproduce the spatiotemporal adhesion patterns. In this modeling framework, we find that the other motility modes, such as smooth gliding, arise naturally with variations in the physical properties of the surface. Thus, our work highlights the prominent role of biomechanics in determining the emergent features of amoeboid locomotion.
              Cover page: Mechanosensitive Adhesion Explains Stepping Motility in Amoeboid Cells
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2016)
              Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.
                Cover page: Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread.
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2017)
                Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.
                  Cover page: Two-Layer Elastographic 3-D Traction Force Microscopy
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2016)
                  Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds.
                    Cover page: Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2019)
                    We present a method to leverage the high fidelity of computed tomography (CT) to quantify regional left ventricular function using topography variation of the endocardium as a surrogate measure of strain. 4DCT images of 10 normal and 10 abnormal subjects, acquired with standard clinical protocols, are used. The topography of the endocardium is characterized by its regional values of fractal dimension ( FD ), computed using a box-counting algorithm developed in-house. The average FD in each of the 16 American Heart Association segments is calculated for each subject as a function of time over the cardiac cycle. The normal subjects show a peak systolic percentage change in FD of 5.9%±2% in all free-wall segments, whereas the abnormal cohort experiences a change of 2%±1.2% ( p<0.00001 ). Septal segments, being smooth, do not undergo large changes in FD . Additionally, a principal component analysis is performed on the temporal profiles of FD to highlight the possibility for unsupervised classification of normal and abnormal function. The method developed is free from manual contouring and does not require any feature tracking or registration algorithms. The FD values in the free-wall segments correlated well with radial strain and with endocardial regional shortening measurements.
                      Cover page: Regional dynamics of fractal dimension of the left ventricular endocardium from cine computed tomography images
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