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Experimental and Computational Methodologies to Measure Intercellular Forces during Tissue Development
270a
Monday, February 13, 2017
diagnosis of the disease is difficult, mainly in the chronic asymptomatic stages. There are only two anti-parasitic treatments for the acute onset. During infection, parasites adhere to specific host tissues, and the flagellum seems to be an important structure involved in motility and cellular infection. This structure is constantly subjected to mechanical stress due to the presence of intense shear from the circulatory system (trypomastigotes) in the vertebrate host and the digestive system (epimastigotes) of the vector. Here, the behavior of the epimastigote’s flagellum under the influence of numerous shear stresses was analyzed using a parallel plate flow chamber. The flagellum exhibited a plastic-viscoelastic deformation under high shear. The dynamics of extension and contraction of the flagellum were also characterized. The elongation of this structure under flow is consistent with previous observations done in assays with leukocytes and bacteria, and supports the idea that extensible linkers are important for adhesion under flow. 1322-Pos Board B390 Coordination of Sequential Action in Asymmetrical Hexameric ATPase by Arginine Finger Zhengyi Zhao, Peixuan Guo. College of Pharmacy, College of Medicine, The Ohio State University, Columbus, OH, USA. Biomotors are involved in countless vital active processes including muscle motion, heart beating, lung breathing, DNA replication, cell division and viral DNA packaging. In this report, the sequential action of the ATPase ring in the dsDNA packaging motor of phi29 is revealed to be regulated by an arginine finger. Arginine finger is shown to extend from one ATPase subunit to the adjacent one for a noncovalent dimer formation, and is involved in ATP binding, hydrolysis, and DNA translocation. Dimer formation is observed when arginine mutants were mixed with wild-types, which can offer their arginine to promote the inter-subunit interaction. Ultracentrifugation and in vitro virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures, based on the results that the isolated dimers alone were deficient in DNA translocation, but the addition of monomer could restore the activity. Moreover, ATP binding or hydrolysis induce two rounds of conformational entropy changes of the ATPase with high or low DNA affinity. Taken together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The formation of one dimer and four monomers inside the hexamer lead to an asymmetrical configuration of the hexameric ATPase complex. Such organization is supported by structural evidences of many other ATPase systems. All the results above provide clues for why the hexameric phi29 ATPase was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM). Since the bridging by the arginine finger renders two adjacent ATPase subunits closer than other subunits, thus, the asymmetrical hexamer would appear as a pseudo-pentamer by cryo-EM, a technology that acquires the average of numerous images. 1323-Pos Board B391 Probing Cellular Mechanostat Dynamics Tomas Andersen. Liphy, Grenoble, France. The cellular mechanostat relates to the regulation of dynamical forces that maintain the mechanic equilibrium in cells and cellular networks. It is the capacity of a cell to sense mechanical stimuli, preserving the memory of it and responding accordingly by setting or adjusting mechanical ‘switches’, however timescales (1). A key limitant element in the understanding of the integration of these force regulation in the cell mechanical sensing is the difficulty of coupling a deviation from an internal ‘‘tensional cellular homeostasis’’, with the active force measurement of its returning to equilibrium. Using optogenetics combined with time resolved TFM on micropatterns as a strategy; we probed the time scales at which mechanostat works by measuring the dynamics of cellular forces. This analysis was done within the same cell thus preventing intercellular variability. Among the great strengths of optogenetics we find the possibility of performing precise transient and spatially signaling disruptions (2). This technique allowed us therefore to disturb the cellular mechanical equilibrium in a temporally controlled way. As a result, the force cellular profile showed a clear response to the light perturbations which enabled the analysis of the mechanostat time scales. This work intends to increase our understanding of the tensional and mechanical homeostasis of the cell. We will also discuss the relation of the optical perturbation of cell’s contractile machinery with the dynamic of actin cytoskeletal structures.
1324-Pos Board B392 Experimental and Computational Methodologies to Measure Intercellular Forces during Tissue Development Ernesto Criado-Hidalgo, Yi-Ting Yeh, Ricardo Serrano, Juan Carlos, del Alamo, Juan Lasheras. Mechanical and Aerospace Engineering, University of California, San Diego, San Diego, CA, USA. During two and three-dimensional tissue development, cell-cell confinement, cell-cell traction forces, and the resulting intracellular tension play a key role in regulating cell proliferation, differentiation, migration, and apoptosis. Yet, the molecular mechanisms that underlie the biochemical response of living cells to these mechanical forces are largely elusive, in part due to the lack of suitable methods to measure accurately the intercellular forces in developing living tissues with high spatial and temporal resolution. We have developed a novel experimental and computational methodology to measure the spatial and temporal evolution of the intercellular forces during tissue development while simultaneously monitoring the dynamics of the resulting proliferation, differentiation, and migration of the cells within the tissue using a small, slender, elastic PDMS microrod. The microfabricated rod is functionalized and embedded in the tissue as we track its shape deformations over time. As tissue forms around the rod, the cells in the developing tissue exert stresses producing bending deformations along the length of the microrod. By tracking these deformations we can backtrack a solution to the primary forces exerted by the cells. Preliminary experimental results on wild type HaCaT, MDCK and HeLa cells show that the method has enough sensitivity to measure single cell-to-cell forces. A validation strategy for this methodology is presented and tested in 1D and 2D scenarios. Traction forces are obtained solving the classical solution of the elastostatic equation from the deformations obtained from fluorescent beads on polyacrylamide substrates. Direct measurements of the intercellular stresses are then compared with calculations of the monolayer stresses obtained solving the Kirchhoff-Love thin plate theory equations. A quantification of the limit tension that the developing tissue is able to sustain to maintain tissue integrity is also presented. 1325-Pos Board B393 Investigation of the Reliability of AFM Nanoindentation-Derived Measurements of Cell Mechanics Matthew Dragovich1, Jared Feindt1, Daniel Altman1, Cassandra Christman2, Nathan DeRaymond3, Ibrahim Hashmi1, Adama Shaw4, Katie Wu5, Serge Ayinou1, Felipe Torres1, Frank Zhang1, Hannah Dailey1. 1 Mechanical Engineering & Mechanics, Lehigh University, Bethlehem, PA, USA, 2Bioengineering Program, Lehigh University, Bethlehem, PA, USA, 3 IDEAS Program, Lehigh University, Bethlehem, PA, USA, 4Biochemistry Program, Lehigh University, Bethlehem, PA, USA, 5Department of Mathematics, Lehigh University, Bethlehem, PA, USA. Introduction: Atomic force microscopy (AFM) is an experimental technique for measuring the mechanical properties of cells and other soft materials. Despite its widespread adoption as a biophysical assay, no universal standards have been adopted for the technique. The purpose of this study was to assess the accuracy and repeatability of AFM-derived cell stiffness measurements. Materials and Methods: A) Experiments: A series of experiments were conducted on ASPC-1 cells to compare the following conditions: conical vs. spherical AFM tips, nuclear vs. peripheral indentation locations, four actuation speeds (0.94, 1.88, 3.76, and 7.52 mm/s), and three indentation forces (100, 300, and 1000 pN). Apparent cell stiffness was calculated using classical contact mechanics with the Hertz model for the spherical tip and Sneddon model for the conical tip. B) Finite Element Models: Idealized 2D and 3D cell body geometries were created based on geometric analysis of 3D confocal scan images of the ASPC-1 cells. A series of models were created to vary cell height and mechanical properties. Apparent cell stiffness was then inferred from indentation depth using the same contact mechanics formulae used in the experiments. C) Literature Review and Statistical Regression: Over 70 publications containing cell elasticity estimates derived from AFM measurements were reviewed and their methods categorized to record: indentation depth, actuation speed, cell type (epithelial/endothelial), cancer (yes/no), indentation location (periphery/nucleus), and tip type (sphere/cone). An analysis of covariance (ANCOVA) regression model was developed to identify which factors significantly influence apparent cell stiffness across the body of published studies. Results and Conclusion: A) Experiments: Across experimental conditions, cells appeared to be stiffer when probed with conical tips compared to spherical tips. There were no differences in apparent cell stiffness based on AFM actuation speed or indentation force. B) Finite Element Models: Across all modeling scenarios, conical tips produced higher
Monday, February 13, 2017
diagnosis of the disease is difficult, mainly in the chronic asymptomatic stages. There are only two anti-parasitic treatments for the acute onset. During infection, parasites adhere to specific host tissues, and the flagellum seems to be an important structure involved in motility and cellular infection. This structure is constantly subjected to mechanical stress due to the presence of intense shear from the circulatory system (trypomastigotes) in the vertebrate host and the digestive system (epimastigotes) of the vector. Here, the behavior of the epimastigote’s flagellum under the influence of numerous shear stresses was analyzed using a parallel plate flow chamber. The flagellum exhibited a plastic-viscoelastic deformation under high shear. The dynamics of extension and contraction of the flagellum were also characterized. The elongation of this structure under flow is consistent with previous observations done in assays with leukocytes and bacteria, and supports the idea that extensible linkers are important for adhesion under flow. 1322-Pos Board B390 Coordination of Sequential Action in Asymmetrical Hexameric ATPase by Arginine Finger Zhengyi Zhao, Peixuan Guo. College of Pharmacy, College of Medicine, The Ohio State University, Columbus, OH, USA. Biomotors are involved in countless vital active processes including muscle motion, heart beating, lung breathing, DNA replication, cell division and viral DNA packaging. In this report, the sequential action of the ATPase ring in the dsDNA packaging motor of phi29 is revealed to be regulated by an arginine finger. Arginine finger is shown to extend from one ATPase subunit to the adjacent one for a noncovalent dimer formation, and is involved in ATP binding, hydrolysis, and DNA translocation. Dimer formation is observed when arginine mutants were mixed with wild-types, which can offer their arginine to promote the inter-subunit interaction. Ultracentrifugation and in vitro virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures, based on the results that the isolated dimers alone were deficient in DNA translocation, but the addition of monomer could restore the activity. Moreover, ATP binding or hydrolysis induce two rounds of conformational entropy changes of the ATPase with high or low DNA affinity. Taken together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The formation of one dimer and four monomers inside the hexamer lead to an asymmetrical configuration of the hexameric ATPase complex. Such organization is supported by structural evidences of many other ATPase systems. All the results above provide clues for why the hexameric phi29 ATPase was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM). Since the bridging by the arginine finger renders two adjacent ATPase subunits closer than other subunits, thus, the asymmetrical hexamer would appear as a pseudo-pentamer by cryo-EM, a technology that acquires the average of numerous images. 1323-Pos Board B391 Probing Cellular Mechanostat Dynamics Tomas Andersen. Liphy, Grenoble, France. The cellular mechanostat relates to the regulation of dynamical forces that maintain the mechanic equilibrium in cells and cellular networks. It is the capacity of a cell to sense mechanical stimuli, preserving the memory of it and responding accordingly by setting or adjusting mechanical ‘switches’, however timescales (1). A key limitant element in the understanding of the integration of these force regulation in the cell mechanical sensing is the difficulty of coupling a deviation from an internal ‘‘tensional cellular homeostasis’’, with the active force measurement of its returning to equilibrium. Using optogenetics combined with time resolved TFM on micropatterns as a strategy; we probed the time scales at which mechanostat works by measuring the dynamics of cellular forces. This analysis was done within the same cell thus preventing intercellular variability. Among the great strengths of optogenetics we find the possibility of performing precise transient and spatially signaling disruptions (2). This technique allowed us therefore to disturb the cellular mechanical equilibrium in a temporally controlled way. As a result, the force cellular profile showed a clear response to the light perturbations which enabled the analysis of the mechanostat time scales. This work intends to increase our understanding of the tensional and mechanical homeostasis of the cell. We will also discuss the relation of the optical perturbation of cell’s contractile machinery with the dynamic of actin cytoskeletal structures.
1324-Pos Board B392 Experimental and Computational Methodologies to Measure Intercellular Forces during Tissue Development Ernesto Criado-Hidalgo, Yi-Ting Yeh, Ricardo Serrano, Juan Carlos, del Alamo, Juan Lasheras. Mechanical and Aerospace Engineering, University of California, San Diego, San Diego, CA, USA. During two and three-dimensional tissue development, cell-cell confinement, cell-cell traction forces, and the resulting intracellular tension play a key role in regulating cell proliferation, differentiation, migration, and apoptosis. Yet, the molecular mechanisms that underlie the biochemical response of living cells to these mechanical forces are largely elusive, in part due to the lack of suitable methods to measure accurately the intercellular forces in developing living tissues with high spatial and temporal resolution. We have developed a novel experimental and computational methodology to measure the spatial and temporal evolution of the intercellular forces during tissue development while simultaneously monitoring the dynamics of the resulting proliferation, differentiation, and migration of the cells within the tissue using a small, slender, elastic PDMS microrod. The microfabricated rod is functionalized and embedded in the tissue as we track its shape deformations over time. As tissue forms around the rod, the cells in the developing tissue exert stresses producing bending deformations along the length of the microrod. By tracking these deformations we can backtrack a solution to the primary forces exerted by the cells. Preliminary experimental results on wild type HaCaT, MDCK and HeLa cells show that the method has enough sensitivity to measure single cell-to-cell forces. A validation strategy for this methodology is presented and tested in 1D and 2D scenarios. Traction forces are obtained solving the classical solution of the elastostatic equation from the deformations obtained from fluorescent beads on polyacrylamide substrates. Direct measurements of the intercellular stresses are then compared with calculations of the monolayer stresses obtained solving the Kirchhoff-Love thin plate theory equations. A quantification of the limit tension that the developing tissue is able to sustain to maintain tissue integrity is also presented. 1325-Pos Board B393 Investigation of the Reliability of AFM Nanoindentation-Derived Measurements of Cell Mechanics Matthew Dragovich1, Jared Feindt1, Daniel Altman1, Cassandra Christman2, Nathan DeRaymond3, Ibrahim Hashmi1, Adama Shaw4, Katie Wu5, Serge Ayinou1, Felipe Torres1, Frank Zhang1, Hannah Dailey1. 1 Mechanical Engineering & Mechanics, Lehigh University, Bethlehem, PA, USA, 2Bioengineering Program, Lehigh University, Bethlehem, PA, USA, 3 IDEAS Program, Lehigh University, Bethlehem, PA, USA, 4Biochemistry Program, Lehigh University, Bethlehem, PA, USA, 5Department of Mathematics, Lehigh University, Bethlehem, PA, USA. Introduction: Atomic force microscopy (AFM) is an experimental technique for measuring the mechanical properties of cells and other soft materials. Despite its widespread adoption as a biophysical assay, no universal standards have been adopted for the technique. The purpose of this study was to assess the accuracy and repeatability of AFM-derived cell stiffness measurements. Materials and Methods: A) Experiments: A series of experiments were conducted on ASPC-1 cells to compare the following conditions: conical vs. spherical AFM tips, nuclear vs. peripheral indentation locations, four actuation speeds (0.94, 1.88, 3.76, and 7.52 mm/s), and three indentation forces (100, 300, and 1000 pN). Apparent cell stiffness was calculated using classical contact mechanics with the Hertz model for the spherical tip and Sneddon model for the conical tip. B) Finite Element Models: Idealized 2D and 3D cell body geometries were created based on geometric analysis of 3D confocal scan images of the ASPC-1 cells. A series of models were created to vary cell height and mechanical properties. Apparent cell stiffness was then inferred from indentation depth using the same contact mechanics formulae used in the experiments. C) Literature Review and Statistical Regression: Over 70 publications containing cell elasticity estimates derived from AFM measurements were reviewed and their methods categorized to record: indentation depth, actuation speed, cell type (epithelial/endothelial), cancer (yes/no), indentation location (periphery/nucleus), and tip type (sphere/cone). An analysis of covariance (ANCOVA) regression model was developed to identify which factors significantly influence apparent cell stiffness across the body of published studies. Results and Conclusion: A) Experiments: Across experimental conditions, cells appeared to be stiffer when probed with conical tips compared to spherical tips. There were no differences in apparent cell stiffness based on AFM actuation speed or indentation force. B) Finite Element Models: Across all modeling scenarios, conical tips produced higher