Feb 14, 2017 - Jerome Irianto1, Christina Y. Chen3, Manorama Tewari1, Andrea Liu2, .... 2Department of Pathology, Robert Wood Johnson Medical School,.
Tuesday, February 14, 2017
Cell Mechanics, Mechanosensing and Motility III 2126-Pos Board B446 As the Beating Heart Stiffens in Development, So Does the Nuclear Lamina Sangkyun Cho1, Stephanie Majkut2, Amal Abbas1, Ken Vogel1, Jerome Irianto1, Christina Y. Chen3, Manorama Tewari1, Andrea Liu2, Benjamin Prosser3, Dennis E. Discher1. 1 University of Pennsylvania, Philadelphia, PA, USA, 2Department of Physics & Astronomy, University of Pennsylvania, Philadelphia, PA, USA, 3 Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. In the first organ that develops - the embryonic heart - coordinated beating of cardiomyocytes is driven by contractile striations that couple to collagenous extracellular matrix (ECM). However, it is unclear what impact beating has on the large nucleus, especially on the levels of lamin-A/C (LMNA), which is a major nuclear structural protein and disease gene. While Lmna-knockout mice exhibit ‘developmental defects of the heart’ and die shortly after birth, we first show by quantitative mass spectrometry (MS) that lamin-A/C is expressed very early on in development, with levels paralleling the development of tissue stiffness and tension in normal embryonic hearts. Lamin-B expression is comparatively constant, such that lamin-A:B stoichiometry scales with stiffness very similarly to that reported for normal adult tissues, which vary in stiffness by several orders of magnitude [Swift, Science 2013]. Within intact embryonic hearts and its 3D arrangement of cardiomyocytes, nuclei are seen to beat in synchrony with tissue. ‘Nuclear beating’ also occurs in vitro on collagen-I coated gels of controlled stiffness, and exhibits an optimum on gels that match the stiffness of embryonic hearts. Lamin-A:B in the embryonic cardiomyocytes exhibit an optimum at somewhat higher matrix stiffness that corresponds surprisingly well to the striation order of cytoplasmic myofibrils (rather than peripheral pre-myofibrils). These observations are consistent with the Theory of Coherent Beating within single cells that correctly predicts matrix-dependent contraction. Nuclear lamin-A thus seems to be an embryonic sensor of coherent stress that mechanically couples to surrounding myofibrils and, in turn, to the collagenous matrix. 2127-Pos Board B447 Understanding the Role of Stiffness in Pathological Cardiac Fibroblast Signaling Tova Christensen1, Kristi Anseth2, Leslie Leinwand1. 1 Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA, 2Chemical Engineering, University of Colorado, Boulder, CO, USA. Cardiac fibrosis is characterized by extracellular matrix buildup and tissue stiffening. Fibroblasts in the heart respond to pathological cues by activating to proliferate, upregulate expression of alpha smooth muscle actin, synthesize extracellular matrix protein, and secrete biochemical cues to surrounding cells. The cues that trigger this transformation remain incompletely resolved; however, stiffness appears to play a critical role in this process. In vitro experiments using hydrogels with different stiffnesses show that fibroblasts activate when cultured on stiff hydrogels while they remain unactivated on soft hydrogels as they do in the diseased and healthy heart, respectively. Understanding fibroblast activation through mechanosensitive signaling will provide new therapeutic targets for fibrosis treatment. 2128-Pos Board B448 Mechanobiology of the Ligament to Bone Insertion Aisa Biria1, Shreyas Mandre2, Madhusudhan Venkadesan1. 1 Department of Mechanical Engineering and Materials Science, Yale University, New Haven, CT, USA, 2School of Engineering, Brown University, Providence, RI, USA. The interface between materials with mismatched elastic moduli is failure-prone due to singular stress concentrations that could develop at the interface. Yet most ligaments tear at mid-substance and not at the ligament-bone interface. Current physiological understanding of these interfaces centers around the graded material properties of the ligament as it nears the bone, with increasing mineralization of the ligament. Open questions remain on which features of the gradation, or more generally of the ligament-bone interface, significantly improve the robustness and eliminate the stress singularity. To address these questions, we develop a 2D elasticity model of the interface and analyze it using a combination of numerical simulations and asymptotics. Increasing size of the gradation zone yields diminishing returns once the zone gets longer than the width of the ligament. Moreover, we apply a previous result in elasticity and show that the frequently-observed fanned geometry of ligaments near the interface can also eliminate the singularity. Previously, the functional advantage of the fanned geometry was thought simply to increase the cross-sectional area. However, our analysis reveals a different and more significant functional benefit to this geom-
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etry. The contribution of this work is to identify scaling laws for the region with graded material properties and a fanned geometry. Based on the observation that neonatal ligament tears often occur at the ligament-bone interface, while injuries in adults primarily occur mid-substance, we propose that the graded interface develops in response to the stress state experienced at dissimilar material interfaces. 2129-Pos Board B449 Electron Microscopy of the Complex Formed by Heavy Meromyosin and C-Protein Charlotte Scarff1, Alba Fuentes Balaguer1, Donald Winkelmann2, John Trinick1. 1 School of Molecular and Cellular Biology, Leeds University, Leeds, United Kingdom, 2Department of Pathology, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA. Most studies of regulation of vertebrate striated muscle contraction have focussed on activation of the thin filament via calcium binding to the troponin-tropomyosin complex. There is accumulating evidence, however, that thick filament activation is an additional regulatory mechanism. Under relaxing conditions, the two heads of a myosin can interact to form an inactive asymmetric off-state, called the interacting-heads motif (IHM). The off-state inhibits cross-bridge formation and cycling by blocking actin binding in one head and ATPase activity in the other. Docking of the IHM onto the thick filament backbone results in the super-relaxed state (SRX) observed in muscle fibres, where the myosin ATPase is strongly inhibited. What regulates formation and disruption of the IHM is largely uncharacterised. Myosin binding protein C, also known as C-protein, is a key sarcomere component, bound at sites spaced by 43 nm in the cross-bridge regions of thick filaments. The arrangement, mode of action, and interactions of C-protein with myosin are poorly understood. In the heart, phosphorylation of C-protein increases the rate of contraction and its ablation disrupts the SRX. To investigate the interaction of human cardiac C-protein with b-cardiac heavy meromyosin (HMM), we have imaged the complex by electron microscopy and single particle averaging. Cardiac HMM in the IHM motif has the characteristic blocked and inhibited head conformations. The fraction of HMM in the IHM motif is increased when dephosphorylated C-protein is bound, suggesting it stabilises the IHM. Characterization of the complex should provide insight into the regulatory and structural roles of the cardiac C-protein/myosin interaction in heart. 2130-Pos Board B450 Mechanosensitive Adhesion Explains Stepping Motility in Amoeboid Cells Calina A. Copos1, Robert D. Guy1, Sam Walcott1, Juan Carlos del Alamo2, Alex Mogilner3. 1 Applied Mathematics, University of California Davis, Davis, CA, USA, 2 Mechanical and Aerospace Engineering, University of California San Diego, San Diego, CA, USA, 3Courant Institute and Department of Biology, New York University, New York, NY, USA. Cell movement is required in many physiological and pathological processes such as immune system response and cancer metastasis. The movement of the single-cell amoeba Dictyostelium discoideum, is characterized by cycles of morphological expansion and contraction and highly coordinated mechanical forces on the substrate by means of transient cell-substrate adhesions. Despite recent intense studies, the mechanisms of rapid shape changes and how they lead to motility of amoeboid cells is still an open question. The existing paradigm for the migration of Dictyostelium has been proposed to be the result of complex biochemical processes coupled to biomechanics. Here, we develop a model to study the interplay of cellular mechanics, cell-substrate interaction, and the resulting migration. The novelty of this work is that we demonstrate that a simple mechanical-only model can explain how amoeboid motility is achieved and robustly maintained to produce the complex and highly-coordinated features of amoeboid motility. We explore several models for cell-substrate adhesion and conclude that cyclic expansions and contractions in cell length together with the experimentally measured spatio-temporal dynamics of traction forces are emergent features of a mechano-sensitive interaction with the substrate. Experiments of wild-type and mutant strains of Dictyostelium suggest different types of motility behavior. Our model shows how the different motility modes arise based on surface properties but also how cells mediate the frequency of the motility cycle in order to continue migrating with the same stride length. 2131-Pos Board B451 A Computational Framework to Accurately Predict Enthalpy and Configurational Entropy Landscapes of Multivalent Interactions of Cell Mimetics Aravind R. Rammohan1, Matthew Mckenzie1, Ravi Radhakrishnan2, Natesan Ramakrishnan2. 1 Corning Inc., Corning, NY, USA, 2UPENN, Philadephia, PA, USA.
