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Regulation of Elastically Coupled Myosin IC Molecules
268a
Monday, February 13, 2017
of Kent, Canterbury, United Kingdom, 3Department of Physics and Mechanobiology Institute, National University of Singapore, Singapore, Singapore. Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, mechanically links the actin cytoskeleton to integrin-based cell-extracellular matrix adhesions at the plasma membrane. By quantifying the force-dependent rates of unfolding and refolding of the 13 alpha-helix bundles in the rod domain of talin, we show that talin can act as a force buffer, keeping the force in the talin mediated force-transmission pathway within a narrow range of 7-10 pN. This force-buffering function of talin is a result from a simple physical principle: increased tension during stretching of talin drives unfolding of its helix bundles, preventing tension accumulation. In contrast, during mechanical relaxation, the reduced tension drives refolding of domains, preventing tension from dropping too fast. This simple physical mechanism can be applied to any large force-bearing proteins serving as mechanical linkages in cells, and the buffered forcelevel depends on the mechanical stability of the domains inside the proteins. A tightly controlled tension range by such force-buffering proteins has an apparent advantage of allowing robust mechanosensitive interactions to take place. 1312-Pos Board B380 Vinculin forms a Catch Bond with F-Actin that Depends on Actin Filament Orientation Derek Huang, Nick Bax, Craig Buckley, William Weis, Alexander Dunn. Stanford University, Stanford, CA, USA. The actin-binding protein vinculin is thought to act as a key mechanical linker between the actin cytoskeleton and both cell-cell and cell-matrix adhesions. In migrating cells, vinculin is proposed to act as a molecular clutch that couples retrograde filamentous (F)-actin flow to integrin-based adhesions in order to transmit mechanical force to the extracellular matrix. Vinculin is also recruited to cell-cell junctions in a force-dependent manner, where it likely serves to reinforce intracellular junctions in response to increased mechanical loads. However, while vinculin’s ability to bind actin is central to its cellular functions, how mechanical load affects the vinculin/ F-actin bond has not, to our knowledge, been examined. Using a singlemolecule optical trap assay, we find that vinculin forms a force-dependent catch bond with F-actin, but with binding lifetimes that depend strongly on the direction of force application relative to the polarity of the actin filament. Specifically, we find that load oriented toward the pointed end of the actin filament results in the formation of a catch bond that is most stable at 8 pN, with an average lifetime of 12 s, while load oriented toward the filament’s barbed end results in transient binding events with ~10-fold shorter lifetimes. This directional dependence is consistent with the orientation of F-actin in the lamellae of migrating cells, in which retrograde flow exerts a force on adhesions oriented toward the pointed end of actin filaments. Our data indicate that vinculin acts as a force-dependent molecular clutch that automatically reinforces its linkage to F-actin in response to increases in load. Consistent with previous, cell biological studies, our observations likewise suggest a means by which the vinculin/F-actin bond may act to generate persistent directional cell migration by reinforcing directional cytoskeletal flow. 1313-Pos Board B381 Regulation of Elastically Coupled Myosin IC Molecules Florian Berger, A.J. Hudspeth. Laboratory of Sensory Neuroscience, The Rockefeller University, New York, NY, USA. The unconventional myosin Ic is thought to be the principal component of the motor that adjusts mechanical responsiveness during adaptation to prolonged stimuli by hair cells, the sensory receptors of the inner ear. To better understand myosin Ic’s function, we introduce a theoretical description based on myosin Ic’s chemomechanical cycle and consistent with recent single-molecule studies. Thermodynamic constraints imply that the nucleotide concentrations govern the dynamics of the cycle. We explicitly calculate the probabilities of myosin Ic’s occupying the different states of its cycle and show their dependence on the external force, nucleotide concentrations, and availability of actin. This analysis highlights that the strongly actin-bound states of myosin Ic are dominated by the ADP state for small forces and by the ATP state for large forces. Integrating this single-molecule cycle into a simplified ensemble description, we predict that the average number of bound myosin heads is regulated by the external force and by the nucleotide concentrations. We use our description to discuss possible mechanisms for calcium
to regulate a myosin ensemble with its implication for adaptation in hair cells. 1314-Pos Board B382 Trajectories of Gliding Filaments on Optically Patterned Myosin Surfaces Daniel Todd1, Vikrant Yadav1, Paul Ruijgrok2, Zev Bryant2,3, Jennifer Ross1. 1 Physics, University of Massachusetts Amherst, Amherst, MA, USA, 2 Bioengineering, Stanford University, Stanford, CA, USA, 3Structural Biology, Stanford University, Stanford, CA, USA. Interfaces are ubiquitous in biology. For a sub-cellular component moving inside a cell any change in its local environment across an interface whether chemical concentration, density, or other physical variables can produce novel dynamics. Recent advances in bioengineering allow us to control motor proteins’ velocities when prompted by an optical trigger. Using a photoactive LOV-domain in a myosin-XI construct and an optical diaphragm we can spatially pattern light to create interfaces across which speed of a gliding actin filament can differ as much as by a factor of two. We observe that when a gliding actin filament crosses an interface that has a discontinuous velocity jump, it buckles and changes its angle of orientation due to the velocity mismatch. We tracked the position and orientation of actin filaments using an in-house MATLAB tracking algorithm and measured that, for small angles of incidence, the angle of emergence increases linearly. If we increase the angle of incidence further we observe that the angle of emergence saturates. For actin filaments approaching the interface near-tangentially we observe total internal reflection as they fail to cross over the boundary. We have modeled our system using Cytosim software package and find excellent agreement with experimental data. These results have given insight into the dynamics of actinmyosin mechanics and have resulted in a better understanding of how to control direction changes in filaments and create dynamically programmable gliding trajectories. 1315-Pos Board B383 Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton Eric S. Schiffhauer1, Tianzhi Luo2, Krithika Mohan3, Xuyu Qian4, Vasudha Srivastava5, Pablo Iglesias6, Douglas N. Robinson1. 1 Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD, USA, 2 Department of Modern Mechanics, University of Science and Technology of China, Heifei, China, 3North Carolina State University, Raleigh, NC, USA, 4 Biomedical Engineering, Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA, 5Pharmaceutical Chemistry, University of California San Franscisco, San Fransisco, CA, USA, 6Electrical and Computer Engineering, Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA. Mechanoresponse, the ability of cells to sense and respond to mechanical stimuli, drives essential processes such as cell division, tissue morphogenesis, cell migration, and metastasis. However, the direct mechanism by which the components of the cortical cytoskeleton assemble and interact to exert force on the membrane in response to mechanical perturbations remains unclear. To determine the elements of the actin-cytoskeleton that sense and respond to applied stress, we perturbed cells by aspiration and defined the kinetics of fluorescent-protein accumulation to cell regions with unique strain environments. Non-muscle myosin II, a-actinin, and filamin accumulate to mechanically stressed regions in mammalian cells from diverse lineages. Using reaction-diffusion models of force-sensitive binding, we successfully predicted which mammalian a-actinin and filamin paralogs would be mechano-accumulative based on their actin-binding affinities. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of a-actinin and filamin catch-bonding behavior. We also uncovered cell-type specific control of myosin IIB mechanosensation, while no such control is apparent for myosins IIA and IIC, indicating a unique regulatory system for myosin IIB mechanoresponse. In fact, the relative assembly of myosin IIB into bipolar thick filaments can influence its mechanoresponse, primarily via a phosphorylation event at S1935. Cumulatively, these actin-binding proteins, and the molecular mechanisms driving their functions, represent critical components of the cellular response to physical perturbation that drive the force-dependent processes involved in cell shape change. 1316-Pos Board B384 Steady Dynein Activity Produces Dynamic Instability and Wavelike Oscillations in a 9-Doublet Finite Element Model of Flagella Tianchen Hu1, Susan K. Dutcher2, Philip V. Bayly1.
Monday, February 13, 2017
of Kent, Canterbury, United Kingdom, 3Department of Physics and Mechanobiology Institute, National University of Singapore, Singapore, Singapore. Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, mechanically links the actin cytoskeleton to integrin-based cell-extracellular matrix adhesions at the plasma membrane. By quantifying the force-dependent rates of unfolding and refolding of the 13 alpha-helix bundles in the rod domain of talin, we show that talin can act as a force buffer, keeping the force in the talin mediated force-transmission pathway within a narrow range of 7-10 pN. This force-buffering function of talin is a result from a simple physical principle: increased tension during stretching of talin drives unfolding of its helix bundles, preventing tension accumulation. In contrast, during mechanical relaxation, the reduced tension drives refolding of domains, preventing tension from dropping too fast. This simple physical mechanism can be applied to any large force-bearing proteins serving as mechanical linkages in cells, and the buffered forcelevel depends on the mechanical stability of the domains inside the proteins. A tightly controlled tension range by such force-buffering proteins has an apparent advantage of allowing robust mechanosensitive interactions to take place. 1312-Pos Board B380 Vinculin forms a Catch Bond with F-Actin that Depends on Actin Filament Orientation Derek Huang, Nick Bax, Craig Buckley, William Weis, Alexander Dunn. Stanford University, Stanford, CA, USA. The actin-binding protein vinculin is thought to act as a key mechanical linker between the actin cytoskeleton and both cell-cell and cell-matrix adhesions. In migrating cells, vinculin is proposed to act as a molecular clutch that couples retrograde filamentous (F)-actin flow to integrin-based adhesions in order to transmit mechanical force to the extracellular matrix. Vinculin is also recruited to cell-cell junctions in a force-dependent manner, where it likely serves to reinforce intracellular junctions in response to increased mechanical loads. However, while vinculin’s ability to bind actin is central to its cellular functions, how mechanical load affects the vinculin/ F-actin bond has not, to our knowledge, been examined. Using a singlemolecule optical trap assay, we find that vinculin forms a force-dependent catch bond with F-actin, but with binding lifetimes that depend strongly on the direction of force application relative to the polarity of the actin filament. Specifically, we find that load oriented toward the pointed end of the actin filament results in the formation of a catch bond that is most stable at 8 pN, with an average lifetime of 12 s, while load oriented toward the filament’s barbed end results in transient binding events with ~10-fold shorter lifetimes. This directional dependence is consistent with the orientation of F-actin in the lamellae of migrating cells, in which retrograde flow exerts a force on adhesions oriented toward the pointed end of actin filaments. Our data indicate that vinculin acts as a force-dependent molecular clutch that automatically reinforces its linkage to F-actin in response to increases in load. Consistent with previous, cell biological studies, our observations likewise suggest a means by which the vinculin/F-actin bond may act to generate persistent directional cell migration by reinforcing directional cytoskeletal flow. 1313-Pos Board B381 Regulation of Elastically Coupled Myosin IC Molecules Florian Berger, A.J. Hudspeth. Laboratory of Sensory Neuroscience, The Rockefeller University, New York, NY, USA. The unconventional myosin Ic is thought to be the principal component of the motor that adjusts mechanical responsiveness during adaptation to prolonged stimuli by hair cells, the sensory receptors of the inner ear. To better understand myosin Ic’s function, we introduce a theoretical description based on myosin Ic’s chemomechanical cycle and consistent with recent single-molecule studies. Thermodynamic constraints imply that the nucleotide concentrations govern the dynamics of the cycle. We explicitly calculate the probabilities of myosin Ic’s occupying the different states of its cycle and show their dependence on the external force, nucleotide concentrations, and availability of actin. This analysis highlights that the strongly actin-bound states of myosin Ic are dominated by the ADP state for small forces and by the ATP state for large forces. Integrating this single-molecule cycle into a simplified ensemble description, we predict that the average number of bound myosin heads is regulated by the external force and by the nucleotide concentrations. We use our description to discuss possible mechanisms for calcium
to regulate a myosin ensemble with its implication for adaptation in hair cells. 1314-Pos Board B382 Trajectories of Gliding Filaments on Optically Patterned Myosin Surfaces Daniel Todd1, Vikrant Yadav1, Paul Ruijgrok2, Zev Bryant2,3, Jennifer Ross1. 1 Physics, University of Massachusetts Amherst, Amherst, MA, USA, 2 Bioengineering, Stanford University, Stanford, CA, USA, 3Structural Biology, Stanford University, Stanford, CA, USA. Interfaces are ubiquitous in biology. For a sub-cellular component moving inside a cell any change in its local environment across an interface whether chemical concentration, density, or other physical variables can produce novel dynamics. Recent advances in bioengineering allow us to control motor proteins’ velocities when prompted by an optical trigger. Using a photoactive LOV-domain in a myosin-XI construct and an optical diaphragm we can spatially pattern light to create interfaces across which speed of a gliding actin filament can differ as much as by a factor of two. We observe that when a gliding actin filament crosses an interface that has a discontinuous velocity jump, it buckles and changes its angle of orientation due to the velocity mismatch. We tracked the position and orientation of actin filaments using an in-house MATLAB tracking algorithm and measured that, for small angles of incidence, the angle of emergence increases linearly. If we increase the angle of incidence further we observe that the angle of emergence saturates. For actin filaments approaching the interface near-tangentially we observe total internal reflection as they fail to cross over the boundary. We have modeled our system using Cytosim software package and find excellent agreement with experimental data. These results have given insight into the dynamics of actinmyosin mechanics and have resulted in a better understanding of how to control direction changes in filaments and create dynamically programmable gliding trajectories. 1315-Pos Board B383 Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton Eric S. Schiffhauer1, Tianzhi Luo2, Krithika Mohan3, Xuyu Qian4, Vasudha Srivastava5, Pablo Iglesias6, Douglas N. Robinson1. 1 Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD, USA, 2 Department of Modern Mechanics, University of Science and Technology of China, Heifei, China, 3North Carolina State University, Raleigh, NC, USA, 4 Biomedical Engineering, Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA, 5Pharmaceutical Chemistry, University of California San Franscisco, San Fransisco, CA, USA, 6Electrical and Computer Engineering, Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA. Mechanoresponse, the ability of cells to sense and respond to mechanical stimuli, drives essential processes such as cell division, tissue morphogenesis, cell migration, and metastasis. However, the direct mechanism by which the components of the cortical cytoskeleton assemble and interact to exert force on the membrane in response to mechanical perturbations remains unclear. To determine the elements of the actin-cytoskeleton that sense and respond to applied stress, we perturbed cells by aspiration and defined the kinetics of fluorescent-protein accumulation to cell regions with unique strain environments. Non-muscle myosin II, a-actinin, and filamin accumulate to mechanically stressed regions in mammalian cells from diverse lineages. Using reaction-diffusion models of force-sensitive binding, we successfully predicted which mammalian a-actinin and filamin paralogs would be mechano-accumulative based on their actin-binding affinities. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of a-actinin and filamin catch-bonding behavior. We also uncovered cell-type specific control of myosin IIB mechanosensation, while no such control is apparent for myosins IIA and IIC, indicating a unique regulatory system for myosin IIB mechanoresponse. In fact, the relative assembly of myosin IIB into bipolar thick filaments can influence its mechanoresponse, primarily via a phosphorylation event at S1935. Cumulatively, these actin-binding proteins, and the molecular mechanisms driving their functions, represent critical components of the cellular response to physical perturbation that drive the force-dependent processes involved in cell shape change. 1316-Pos Board B384 Steady Dynein Activity Produces Dynamic Instability and Wavelike Oscillations in a 9-Doublet Finite Element Model of Flagella Tianchen Hu1, Susan K. Dutcher2, Philip V. Bayly1.