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Real-time microdamage detection during micromechanical testing of trabecular bone
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Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
Oral Presentations
distant focal adhesions are thought to be a principal organelle involved in the mechanical signaling pathway, its qualitative and quantitative contributions are still poorly understood. Here, we evaluated tension level in single stress fibers of vascular endothelial cells and smooth muscle cells to understand how intracellular forces are balanced or transmitted in the cytoskeletal network. First, tensile properties of stress fibers were determined in vitro using a tensile testing system we developed [1]. Preexisting strain level in the basal stress fiber network was then examined with fluorescence microscopy by removing cytoplasmic constituents except for materials physically attaching to the substrate (i.e., stress fiber-focal adhesion complexities) and measuring stress-releaseinduced deformation of the stress fibers. The results showed that the single stress fibers had a preexisting tension o f - 1 0 n N on average. The order of magnitude of the pre-tension was similar with that of traction force generated by adherent cells at single adhesion sites to keep cell integrity, suggesting that the pre-tension was balanced in tensed SF network in the cells. Better understanding of such force balance/transmission in subcellular structural components including the nucleus [2] will be crucial to relate unidirectional external mechanical stimuli to intracellularly-localized biochemical responses.
properties of red blood cells and also may function to transmit forces from the cytoskeleton to ion transporters to mechanically regulate ion fluxes across the plasma membrane [2]. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of TRP channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles [2]. We report that tandem ankyrin repeats exhibit unusual tertiary structure-based elasticity and behave as a linear and fully reversible spring in single molecule measurements by atomic force microscopy (AFM). We also observe an unanticipated ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we demonstrate that one of the most common amino acid motifs displays spring properties that could be important in cellular mechanics, mechanotransduction and in design of nanodevices [3].
References
5374
[1] S. Deguchi, T. Ohashi and M. Sato. Tensile properties of single stress fiber isolated form cultured vascular smooth muscle cells. Journal of Biomechanics, in press. [2] S. Deguchi, K. Maeda, T. Ohashi and M. Sato. Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle. Journal of Biomechanics 2005; 38: 1751-1759. 4547 Fr, 11:30-11:45 (P51 ) Quantitative evaluation o f strain field in the lamella region o f cellular fragments from fish keratocytes K.O. Okeyo, '~ Shitagawa, T. Adachi & M. Hojo. Department efMechanical Engineering & Science, Kyoto University, Kyoto, Japan Mechanical factors regulate many cellular functions such as cell adhesion and cytoskeletal organization [1]. Although a lot of work has been done on cell motility, much of it is concentrated on the molecular and physiological processes involved, with little work reported on the contribution by mechanical factors such as stress and strain in the actin structure. We therefore carried out an analysis of the strain field in the lamella region of cellular fragments of fish keratocytes in order to correlate internal mechanical state to the dynamics of actin structure. Keratocytes are known to exhibit a rapid and highly efficient mode of movement, and cellular fragments of these cells, which are nothing more than an actin machinery enclosed by a membrane, retain the property of directional motility and can move with remarkable speed and persistence [2]. Since they are devoid of both the nucleus and most organelles, these fragments provide a simplified system ideal for the analysis of actin structure dynamics in keratocytes. We applied Fluorescent Speckle Microscopy (FSM) to detect retrograde flow of actin structure in the lamella region of the cellular fragments and further applied Particle Image Velocimetry (PIV) to analyze the displacement field induced by the flow. We found out that a net compressive strain exists in the lamella region. We hypothesize that the existence of compressive strain in this region may favor depolymerization of actin filaments thereby regulating directional motility in keratocytes. Thus, our work is a first step toward understanding the role of internal mechanical strain in the depolymerization process.
References
[1] Michaely P., et al. EMBO J. 2002; 21 : 6387~396. [2] Howard J., Bechstedt S. Curr. Biol. 2004; 14: 224-226. [3] Lee G., et al. Nature 2006; in press. Fr, 12:00-12:15 (P51)
Molecular biomechanics o f muscle contraction: a time-resolved x-ray
diffraction study S.Y. Bershitsky 1, N. Koubassova 2, M.A. Ferenczi 3, A.K. Tsaturyan 2. 1Institute of Immunology and Physiology, Ural Branch of RAS, Yekaterinburg, Russia, 2Department of Biomechanics, Institute of Mechanics, Moscow University, Russia, 3Biomedical Sciences Division, Imperial College London, UK Muscle force results from cyclic interaction of globular heads of myosin molecules with actin filaments. During this interaction free energy of ATP hydrolysis is converted into mechanical work. To correlate molecular movement with macroscopic muscle mechanics fast length steps or temperature jumps were applied to contracting permeabilized fibres from rabbit skeletal muscle. Tension responses and 2D x-ray diffraction pattern were recorded with the time resolution up to 0.1 ms using a synchrotron x-ray beam (16.1 at the SRS, Daresbury, UK or ID02 at the ESRF, Grenoble, France). We found that apart from an axial tilt of myosin heads or their 'neck' domains, force generation in muscle is accompanied by an azimuthal 'roll and lock' transition of the heads. A kinetic-structural model explaining these observations predicted some new phenomena. Now the validity of some these predictions was checked experimentally. We confirmed that the 'locking' of myosin heads on actin is strain-dependent and estimated the contribution of viscoelastic behaviour of pre-force-generating myosin heads to mechanical and structural properties of actively contracting muscle fibres. We also used x-ray diffraction to measure deformation of the actin filaments in muscle fibres and found that binding of myosin heads itself induces their elongation and twisting additional to elastic extensibility. Now a mechanical model of a sarcomere that accounts for these effects is developed. Simulation of tension relaxation experiments using this model shows that actin deformation is essential for correct interpretation of their results. References
Ferenczi M.A., Bershitsky S.Y., Koubassova N.A., Siththanandan V., Helsby W.I., Panine P., Roessle M., Narayanan T., Tsaturyan A.K. (2005). Structure 13: 131141. Tsaturyan A.K., Koubassova N.A., Ferenczi M.A., Narayanan T., Roessle M., Bershitsky S.Y. (2005). Biophys. J. 88: 1902-1910.
References
[1] Sato K., Adachi T., Matsuo M., Tomita Y. Quantitative evaluation of threshold fiber strain that induces reorganization of the cytoskeletal actin fiber stucture in osteoblastic cells. Journal of Biomechanics 2005; 38: 1895-1901. [2] Verkhovsky B.A., Svitkina M.T., Borisy G.G. Self-polarization and directional motility of cytoplasm. Current Biology 1999; 9: 11-20. 5459 Fr, 11:45-12:00 (P51) Mechanical properties o f ankyrin repeats examined with single-molecule force s p e c t r o s c o p y G. Lee 1, K. Abdi 2, Y. Jiang 1, P. Michaely 3, V. Bennett 2, P.E. Marszalek 1. 1Department of Mechanical Engineering and Materials Science and Center for Biologically Inspired Materials and Material Systems, Duke University, Durham, NC, USA, 2Howard Hughes Medical Institute and Department of Cell Biology, Duke University Medical Center, Durham, NC, USA, 3Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA Ankyrin repeats are an amino acid motif believed to function in protein recognition and are present in tandem copies in over 4,700 diverse proteins. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats [1] suggests the possibility of spring-like behavior of the putative superhelix. Thus, ankyrin-R repeats may affect the mechanical
5538 Fr, 12:15-12:30 (P51) Real-time microdamage detection during micromechanical testing o f trabecular bone P.J. Thurner, B. Erickson, R. Jungmann, S. Lam, J.C. Weaver, G. Schitter, G.E. Fantner, D.E. Morse, P.K. Hansma. University ef California, Santa Barbara, USA To assess local deformations during mechanical loading of trabecular bone, we recently combined mechanical testing of trabecular bone with high-speed photography. In the presented study we investigated two types of human and bovine samples: 5 5 4mm pieces of vertebral trabecular bone and single trabeculae from femur and tibia. Pieces were tested in compression, whereas single trabeculae were tested in three-point-bending. In the pieces we found large local deformations even at small apparent strains. Strained trabeculae were seen to whiten, similar to stress-whitening in polymers. Scanning electron microscopy (SEM) showed that whitening is due to microdamage, including microcracks with lengths up to 200 microns. Microcracks were bridged by mineralized collagen fibrils. Using immunolabeling and SEM we see that noncollagenous proteins (NCPs) are abundant on fractured surfaces and on ligaments bridging microcracks, pointing to their putative mechanical importance. We recently showed that immersing bone samples in NaF prior to mechanical testing decouples mineral crystals and collagen fibrils without changing their weight and volume fraction. NaF treatment not only reduces
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
Oral Presentations
distant focal adhesions are thought to be a principal organelle involved in the mechanical signaling pathway, its qualitative and quantitative contributions are still poorly understood. Here, we evaluated tension level in single stress fibers of vascular endothelial cells and smooth muscle cells to understand how intracellular forces are balanced or transmitted in the cytoskeletal network. First, tensile properties of stress fibers were determined in vitro using a tensile testing system we developed [1]. Preexisting strain level in the basal stress fiber network was then examined with fluorescence microscopy by removing cytoplasmic constituents except for materials physically attaching to the substrate (i.e., stress fiber-focal adhesion complexities) and measuring stress-releaseinduced deformation of the stress fibers. The results showed that the single stress fibers had a preexisting tension o f - 1 0 n N on average. The order of magnitude of the pre-tension was similar with that of traction force generated by adherent cells at single adhesion sites to keep cell integrity, suggesting that the pre-tension was balanced in tensed SF network in the cells. Better understanding of such force balance/transmission in subcellular structural components including the nucleus [2] will be crucial to relate unidirectional external mechanical stimuli to intracellularly-localized biochemical responses.
properties of red blood cells and also may function to transmit forces from the cytoskeleton to ion transporters to mechanically regulate ion fluxes across the plasma membrane [2]. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of TRP channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles [2]. We report that tandem ankyrin repeats exhibit unusual tertiary structure-based elasticity and behave as a linear and fully reversible spring in single molecule measurements by atomic force microscopy (AFM). We also observe an unanticipated ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we demonstrate that one of the most common amino acid motifs displays spring properties that could be important in cellular mechanics, mechanotransduction and in design of nanodevices [3].
References
5374
[1] S. Deguchi, T. Ohashi and M. Sato. Tensile properties of single stress fiber isolated form cultured vascular smooth muscle cells. Journal of Biomechanics, in press. [2] S. Deguchi, K. Maeda, T. Ohashi and M. Sato. Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle. Journal of Biomechanics 2005; 38: 1751-1759. 4547 Fr, 11:30-11:45 (P51 ) Quantitative evaluation o f strain field in the lamella region o f cellular fragments from fish keratocytes K.O. Okeyo, '~ Shitagawa, T. Adachi & M. Hojo. Department efMechanical Engineering & Science, Kyoto University, Kyoto, Japan Mechanical factors regulate many cellular functions such as cell adhesion and cytoskeletal organization [1]. Although a lot of work has been done on cell motility, much of it is concentrated on the molecular and physiological processes involved, with little work reported on the contribution by mechanical factors such as stress and strain in the actin structure. We therefore carried out an analysis of the strain field in the lamella region of cellular fragments of fish keratocytes in order to correlate internal mechanical state to the dynamics of actin structure. Keratocytes are known to exhibit a rapid and highly efficient mode of movement, and cellular fragments of these cells, which are nothing more than an actin machinery enclosed by a membrane, retain the property of directional motility and can move with remarkable speed and persistence [2]. Since they are devoid of both the nucleus and most organelles, these fragments provide a simplified system ideal for the analysis of actin structure dynamics in keratocytes. We applied Fluorescent Speckle Microscopy (FSM) to detect retrograde flow of actin structure in the lamella region of the cellular fragments and further applied Particle Image Velocimetry (PIV) to analyze the displacement field induced by the flow. We found out that a net compressive strain exists in the lamella region. We hypothesize that the existence of compressive strain in this region may favor depolymerization of actin filaments thereby regulating directional motility in keratocytes. Thus, our work is a first step toward understanding the role of internal mechanical strain in the depolymerization process.
References
[1] Michaely P., et al. EMBO J. 2002; 21 : 6387~396. [2] Howard J., Bechstedt S. Curr. Biol. 2004; 14: 224-226. [3] Lee G., et al. Nature 2006; in press. Fr, 12:00-12:15 (P51)
Molecular biomechanics o f muscle contraction: a time-resolved x-ray
diffraction study S.Y. Bershitsky 1, N. Koubassova 2, M.A. Ferenczi 3, A.K. Tsaturyan 2. 1Institute of Immunology and Physiology, Ural Branch of RAS, Yekaterinburg, Russia, 2Department of Biomechanics, Institute of Mechanics, Moscow University, Russia, 3Biomedical Sciences Division, Imperial College London, UK Muscle force results from cyclic interaction of globular heads of myosin molecules with actin filaments. During this interaction free energy of ATP hydrolysis is converted into mechanical work. To correlate molecular movement with macroscopic muscle mechanics fast length steps or temperature jumps were applied to contracting permeabilized fibres from rabbit skeletal muscle. Tension responses and 2D x-ray diffraction pattern were recorded with the time resolution up to 0.1 ms using a synchrotron x-ray beam (16.1 at the SRS, Daresbury, UK or ID02 at the ESRF, Grenoble, France). We found that apart from an axial tilt of myosin heads or their 'neck' domains, force generation in muscle is accompanied by an azimuthal 'roll and lock' transition of the heads. A kinetic-structural model explaining these observations predicted some new phenomena. Now the validity of some these predictions was checked experimentally. We confirmed that the 'locking' of myosin heads on actin is strain-dependent and estimated the contribution of viscoelastic behaviour of pre-force-generating myosin heads to mechanical and structural properties of actively contracting muscle fibres. We also used x-ray diffraction to measure deformation of the actin filaments in muscle fibres and found that binding of myosin heads itself induces their elongation and twisting additional to elastic extensibility. Now a mechanical model of a sarcomere that accounts for these effects is developed. Simulation of tension relaxation experiments using this model shows that actin deformation is essential for correct interpretation of their results. References
Ferenczi M.A., Bershitsky S.Y., Koubassova N.A., Siththanandan V., Helsby W.I., Panine P., Roessle M., Narayanan T., Tsaturyan A.K. (2005). Structure 13: 131141. Tsaturyan A.K., Koubassova N.A., Ferenczi M.A., Narayanan T., Roessle M., Bershitsky S.Y. (2005). Biophys. J. 88: 1902-1910.
References
[1] Sato K., Adachi T., Matsuo M., Tomita Y. Quantitative evaluation of threshold fiber strain that induces reorganization of the cytoskeletal actin fiber stucture in osteoblastic cells. Journal of Biomechanics 2005; 38: 1895-1901. [2] Verkhovsky B.A., Svitkina M.T., Borisy G.G. Self-polarization and directional motility of cytoplasm. Current Biology 1999; 9: 11-20. 5459 Fr, 11:45-12:00 (P51) Mechanical properties o f ankyrin repeats examined with single-molecule force s p e c t r o s c o p y G. Lee 1, K. Abdi 2, Y. Jiang 1, P. Michaely 3, V. Bennett 2, P.E. Marszalek 1. 1Department of Mechanical Engineering and Materials Science and Center for Biologically Inspired Materials and Material Systems, Duke University, Durham, NC, USA, 2Howard Hughes Medical Institute and Department of Cell Biology, Duke University Medical Center, Durham, NC, USA, 3Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA Ankyrin repeats are an amino acid motif believed to function in protein recognition and are present in tandem copies in over 4,700 diverse proteins. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats [1] suggests the possibility of spring-like behavior of the putative superhelix. Thus, ankyrin-R repeats may affect the mechanical
5538 Fr, 12:15-12:30 (P51) Real-time microdamage detection during micromechanical testing o f trabecular bone P.J. Thurner, B. Erickson, R. Jungmann, S. Lam, J.C. Weaver, G. Schitter, G.E. Fantner, D.E. Morse, P.K. Hansma. University ef California, Santa Barbara, USA To assess local deformations during mechanical loading of trabecular bone, we recently combined mechanical testing of trabecular bone with high-speed photography. In the presented study we investigated two types of human and bovine samples: 5 5 4mm pieces of vertebral trabecular bone and single trabeculae from femur and tibia. Pieces were tested in compression, whereas single trabeculae were tested in three-point-bending. In the pieces we found large local deformations even at small apparent strains. Strained trabeculae were seen to whiten, similar to stress-whitening in polymers. Scanning electron microscopy (SEM) showed that whitening is due to microdamage, including microcracks with lengths up to 200 microns. Microcracks were bridged by mineralized collagen fibrils. Using immunolabeling and SEM we see that noncollagenous proteins (NCPs) are abundant on fractured surfaces and on ligaments bridging microcracks, pointing to their putative mechanical importance. We recently showed that immersing bone samples in NaF prior to mechanical testing decouples mineral crystals and collagen fibrils without changing their weight and volume fraction. NaF treatment not only reduces