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Non-linear elasticity of growing actin networks
Track 10. Cellular and Molecular Mechanics 7006 Tu, 14:30-14:45 (P22) Non-linear elasticity of growing actin networks O. Chaudhuri, S. Parekh, D.A. Fletcher. Bioengineering Department, UC
Berkeley, Berkeley, CA, USA Actin networks provide eukaryotic cells with mechanical stability and generate forces for shape change and cell movement. The mechanical properties of bulk actin networks, which serve as a model system for studying semiflexible polymers, have been extensively studied using both macroscopic and microscopic rheology techniques. Non-linear elasticity observed in entangled or cross-linked actin networks has been attributed to entropic contributions from stretching of individual filaments. However, actin networks in cells are rarely composed of randomly oriented filaments. Growing actin networks, such as those assembled at the leading edge of crawling cells, form highly organized networks with filament orientation biased towards the membrane, raising the question of how network anisotropy influences elasticity. Here we present recent mechanical property measurements of growing actin networks to investigate the role of network architecture on mechanical properties. Using a differential force microscopy technique based on an atomic force microscope (AFM), we directly quantified the mechanical properties of growing actin networks in vitro under controlled loads. The force microscopy technique uses a dual-cantilever configuration to minimize the influence of sample drift so that network displacements can be measured continuously over long times. Our results reveal that growing actin networks exhibit different non-linear elasticity with increasing load than random networks, indicating that filament orientation can play a significant role in altering mechanical properties. Our results are only partially explained by existing models of actin networks and pose new challenges for theoretical prediction of actin network mechanics. 7264 Tu, 14:45-15:00 (P22) Reversible ratchet in closed systems A. Grillo 1, S. Federico 2, R.A. Haddou 2, G. Giaquinta 1, W. Herzog 2.
1Department of Physical and Chemical Methodologies for Engineering, University of Catania, Italy, 2Human Performance Laboratory, The University ef Calgary, Canada Many biological systems allow for transport phenomena in the absence of macroscopic driving forces. This is the case for reversible ratchets [1,2], i.e. systems consisting of a Brownian particle which is able to rectify noise to induce unidirectional motion. On the basis of Streater's model [3], we studied the interaction of a Brownian particle and a heat-particle inside the unit cell of a one-dimensional lattice, under the influence of an adiabatic potential which depends on time through a collection of parameters [2]. When thermodynamic equilibrium is attained, the probability distribution of the Brownian particle coincides with the Gibbs distribution, and temperature approaches a constant value. Under these conditions, and suitably chosen boundary conditions, we proved that entropy remains constant in time, while the integral of energy along a closed path in the space of parameters can be set equal to zero by varying temperature with parameters according to a prescribed law. We determined such a law by requiring the variation of temperature to compensate for the work exerted on the system by the variation of the adiabatic potential along a nonisothermal cycle in the space of parameters. Thus, although the total energy of the system depends on time, we conclude that, if the two subsystems made by the Brownian and the heat-particle, respectively, are properly combined, we can still have a reversible ratchet in a closed system. References [1] Magnasco M.O. Physical Review Letters 1993; 71: 1477. [2] Parrondo J.M.R. Physical Review E 1988; 57: 7297. [3] Streater R.F. Journal of Statistical Physics 1997; 88: 447. 5128 Tu, 15:00-15:15 (P22) Myosin II inserts into lipid membranes V. Schewkunow, W.H. Goldmann. Friedrich-Alexander-University ef
Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Erlangen, Germany The motor protein myosin II is an integral part of the cytoskeleton and is involved in cytoskeletal prestress generation and contraction, motility, and other complex mechanical behaviors observed in living cells. For efficient spatial coordination of such complex mechanical functions in response to extracellular stimuli, the association of myosin II with the cell membrane has long been suspected but has not yet been demonstrated. In this study, we used differential scanning calorimetry to test the hypothesis that myosin II inserts into negatively charged phospholipid membranes. Lipid vesicles were made of DMPG/DMPC (molar ratio 1:1) at 5mg/ml in the presence of different myosin II concentrations between 3.4~tM and 10.2~tM. Vesicles in the absence of myosin II exhibited a main phase transition at -22.6°C. With increasing concentrations of myosin II, the thermotropic properties of the lipid vesicle changed, and the phase transitions occurred
10.4. Cytoskeletal, Nuclear, and Membrane Rheology
$235
at increasingly higher temperatures from 22.6°C ~ 23.2°C, at the highest myosin concentration. In addition, the enthalpy change, AH, during phase transition decreased with increasing myosin II concentration. Such changes are indicative of protein-lipid interactions/insertions. We hypothesize that myosin II binds to acidic phospholipids first through electrostatic interaction using the basic side groups of positive residues and then partially penetrates into the bilayer to form an anchor through the flexible, amphipathic helix of myosin II. Our results demonstrate that myosin II can insert into lipid membranes. This suggests that complex mechanical functions may be regulated through the insertion and functional modulation of myosin II in the cell membrane. To explore this possibility, we are currently using the stopped-flow method to determine the binding affinity between lipid-anchored myosin II and actin. 7388 Tu, 15:15-15:30 (P22) Protrusion forces driving rapidly translocating cells M. Goegler, C. Brunner, A. Ehrlicher, B. Kohlstrunk, J. K~s. Institute for Soft
Matter Physics, University of Leipzig, Germany Cell motility is a fundamental process of many phenomena in nature, such as immune response, wound healing, and metastasis. Mechanisms of force generation for cell migration have been described in various hypotheses requiring actin polymerization and/or molecular motors, but quantitative force measurements to date have focused on traction forces. Here we present a direct measurement of the forward force generated at the leading edge of the lamellipodium and at the cell body of a translocating fish keratocyte. We positioned an elastic spring, the cantilever of a scanning force microscope (SFM), in front of a moving cell, which pushed the cantilever out of its path. The forward force was calculated using the detected lateral deflection and the vertical deflection of the cantilever in an "elastic wedge model", which considers cellular deformation. We measured forward forces between 1-8nN without visibly affecting the cells. At stronger opposing forces up to at least 15nN the lamellipodium of the cell retracted locally whereas the overall movement of the cell remained unaffected. Stall forces for keratocytes were measured in the range of 30-40 nN. Measurements with steadily increasing applied force were carried out to determine a load dependence behaviour for the lamellipodium and the cell as a whole. During these experiments the cells were visualized by interference reflection microscopy. We investigated the effect of the actin capper cytochalasin D in force measurements to elucidate the importance of actin polymerization in cellular protrusion.
10.4. Cytoskeletal, Nuclear, and Membrane Rheology 6928 We, 08:15-08:30 (P28) Measuring protein conformational changes from single molecules up to cells - a proteomic method C. Johnson, D.E. Discher. Biophysical Engineering Lab, University of
Pennsylvania; Philadelphia, PA, USA Conformational changes in proteins under stress are believed to be key to many processes in cell elasticity and mechanotransduction, including how tissue cells feel the stiffness of their matrix. Starting with forced unfolding of single proteins by AFM, we describe a relatively simple scheme for revealing conformational changes in proteins and that can be applied to whole cells and perhaps even tissues. The method proves sensitive to point mutations that cause disease, can be used to pin-point regions of molecules undergoing conformational change, and can identify proteins that undergo large conformational changes such as occur in cells on elastic substrates. 5568 We, 08:30-08:45 (P28) Phase behaviour and micro-mechanical properties of crosslinked actin-networks O. Lieleg, A.R. Bausch. Technische Universit~t M~nchen, Germany Cell shape, mechanics and motility are mainly determined by crosslinked actinnetworks. Despite their importance, the mechanical function of crosslinking molecules is not well understood. As in living cells many different actin crosslinking molecules are used simultaneously, it is necessary to study their effect in in vitro systems. Here two structural related crosslinking molecules are compared: ,J,-actinin and I-plastin. Their effect on the structure and mechanics of in vitro actin networks is investigated. Actin networks crosslinked by ,J,-actinin or I-plastin show pronounced differences in their elastic properties as a function of the crosslinker-to-actin-ratio. Interestingly, these differences are observed although both crosslinking molecules use the same calmodulin-homologous domain for actin binding. For both systems at least three distinct phases of actin-networks with different viscoelastic properties are observed. By rheological and optical methods these are related to the microscopic structure of the networks. The occurrence of mixed networks containing bundles embedded in an isotropic network indicates that a sharp distinction between crosslinking
Berkeley, Berkeley, CA, USA Actin networks provide eukaryotic cells with mechanical stability and generate forces for shape change and cell movement. The mechanical properties of bulk actin networks, which serve as a model system for studying semiflexible polymers, have been extensively studied using both macroscopic and microscopic rheology techniques. Non-linear elasticity observed in entangled or cross-linked actin networks has been attributed to entropic contributions from stretching of individual filaments. However, actin networks in cells are rarely composed of randomly oriented filaments. Growing actin networks, such as those assembled at the leading edge of crawling cells, form highly organized networks with filament orientation biased towards the membrane, raising the question of how network anisotropy influences elasticity. Here we present recent mechanical property measurements of growing actin networks to investigate the role of network architecture on mechanical properties. Using a differential force microscopy technique based on an atomic force microscope (AFM), we directly quantified the mechanical properties of growing actin networks in vitro under controlled loads. The force microscopy technique uses a dual-cantilever configuration to minimize the influence of sample drift so that network displacements can be measured continuously over long times. Our results reveal that growing actin networks exhibit different non-linear elasticity with increasing load than random networks, indicating that filament orientation can play a significant role in altering mechanical properties. Our results are only partially explained by existing models of actin networks and pose new challenges for theoretical prediction of actin network mechanics. 7264 Tu, 14:45-15:00 (P22) Reversible ratchet in closed systems A. Grillo 1, S. Federico 2, R.A. Haddou 2, G. Giaquinta 1, W. Herzog 2.
1Department of Physical and Chemical Methodologies for Engineering, University of Catania, Italy, 2Human Performance Laboratory, The University ef Calgary, Canada Many biological systems allow for transport phenomena in the absence of macroscopic driving forces. This is the case for reversible ratchets [1,2], i.e. systems consisting of a Brownian particle which is able to rectify noise to induce unidirectional motion. On the basis of Streater's model [3], we studied the interaction of a Brownian particle and a heat-particle inside the unit cell of a one-dimensional lattice, under the influence of an adiabatic potential which depends on time through a collection of parameters [2]. When thermodynamic equilibrium is attained, the probability distribution of the Brownian particle coincides with the Gibbs distribution, and temperature approaches a constant value. Under these conditions, and suitably chosen boundary conditions, we proved that entropy remains constant in time, while the integral of energy along a closed path in the space of parameters can be set equal to zero by varying temperature with parameters according to a prescribed law. We determined such a law by requiring the variation of temperature to compensate for the work exerted on the system by the variation of the adiabatic potential along a nonisothermal cycle in the space of parameters. Thus, although the total energy of the system depends on time, we conclude that, if the two subsystems made by the Brownian and the heat-particle, respectively, are properly combined, we can still have a reversible ratchet in a closed system. References [1] Magnasco M.O. Physical Review Letters 1993; 71: 1477. [2] Parrondo J.M.R. Physical Review E 1988; 57: 7297. [3] Streater R.F. Journal of Statistical Physics 1997; 88: 447. 5128 Tu, 15:00-15:15 (P22) Myosin II inserts into lipid membranes V. Schewkunow, W.H. Goldmann. Friedrich-Alexander-University ef
Erlangen-Nuremberg, Center for Medical Physics and Technology, Biophysics Group, Erlangen, Germany The motor protein myosin II is an integral part of the cytoskeleton and is involved in cytoskeletal prestress generation and contraction, motility, and other complex mechanical behaviors observed in living cells. For efficient spatial coordination of such complex mechanical functions in response to extracellular stimuli, the association of myosin II with the cell membrane has long been suspected but has not yet been demonstrated. In this study, we used differential scanning calorimetry to test the hypothesis that myosin II inserts into negatively charged phospholipid membranes. Lipid vesicles were made of DMPG/DMPC (molar ratio 1:1) at 5mg/ml in the presence of different myosin II concentrations between 3.4~tM and 10.2~tM. Vesicles in the absence of myosin II exhibited a main phase transition at -22.6°C. With increasing concentrations of myosin II, the thermotropic properties of the lipid vesicle changed, and the phase transitions occurred
10.4. Cytoskeletal, Nuclear, and Membrane Rheology
$235
at increasingly higher temperatures from 22.6°C ~ 23.2°C, at the highest myosin concentration. In addition, the enthalpy change, AH, during phase transition decreased with increasing myosin II concentration. Such changes are indicative of protein-lipid interactions/insertions. We hypothesize that myosin II binds to acidic phospholipids first through electrostatic interaction using the basic side groups of positive residues and then partially penetrates into the bilayer to form an anchor through the flexible, amphipathic helix of myosin II. Our results demonstrate that myosin II can insert into lipid membranes. This suggests that complex mechanical functions may be regulated through the insertion and functional modulation of myosin II in the cell membrane. To explore this possibility, we are currently using the stopped-flow method to determine the binding affinity between lipid-anchored myosin II and actin. 7388 Tu, 15:15-15:30 (P22) Protrusion forces driving rapidly translocating cells M. Goegler, C. Brunner, A. Ehrlicher, B. Kohlstrunk, J. K~s. Institute for Soft
Matter Physics, University of Leipzig, Germany Cell motility is a fundamental process of many phenomena in nature, such as immune response, wound healing, and metastasis. Mechanisms of force generation for cell migration have been described in various hypotheses requiring actin polymerization and/or molecular motors, but quantitative force measurements to date have focused on traction forces. Here we present a direct measurement of the forward force generated at the leading edge of the lamellipodium and at the cell body of a translocating fish keratocyte. We positioned an elastic spring, the cantilever of a scanning force microscope (SFM), in front of a moving cell, which pushed the cantilever out of its path. The forward force was calculated using the detected lateral deflection and the vertical deflection of the cantilever in an "elastic wedge model", which considers cellular deformation. We measured forward forces between 1-8nN without visibly affecting the cells. At stronger opposing forces up to at least 15nN the lamellipodium of the cell retracted locally whereas the overall movement of the cell remained unaffected. Stall forces for keratocytes were measured in the range of 30-40 nN. Measurements with steadily increasing applied force were carried out to determine a load dependence behaviour for the lamellipodium and the cell as a whole. During these experiments the cells were visualized by interference reflection microscopy. We investigated the effect of the actin capper cytochalasin D in force measurements to elucidate the importance of actin polymerization in cellular protrusion.
10.4. Cytoskeletal, Nuclear, and Membrane Rheology 6928 We, 08:15-08:30 (P28) Measuring protein conformational changes from single molecules up to cells - a proteomic method C. Johnson, D.E. Discher. Biophysical Engineering Lab, University of
Pennsylvania; Philadelphia, PA, USA Conformational changes in proteins under stress are believed to be key to many processes in cell elasticity and mechanotransduction, including how tissue cells feel the stiffness of their matrix. Starting with forced unfolding of single proteins by AFM, we describe a relatively simple scheme for revealing conformational changes in proteins and that can be applied to whole cells and perhaps even tissues. The method proves sensitive to point mutations that cause disease, can be used to pin-point regions of molecules undergoing conformational change, and can identify proteins that undergo large conformational changes such as occur in cells on elastic substrates. 5568 We, 08:30-08:45 (P28) Phase behaviour and micro-mechanical properties of crosslinked actin-networks O. Lieleg, A.R. Bausch. Technische Universit~t M~nchen, Germany Cell shape, mechanics and motility are mainly determined by crosslinked actinnetworks. Despite their importance, the mechanical function of crosslinking molecules is not well understood. As in living cells many different actin crosslinking molecules are used simultaneously, it is necessary to study their effect in in vitro systems. Here two structural related crosslinking molecules are compared: ,J,-actinin and I-plastin. Their effect on the structure and mechanics of in vitro actin networks is investigated. Actin networks crosslinked by ,J,-actinin or I-plastin show pronounced differences in their elastic properties as a function of the crosslinker-to-actin-ratio. Interestingly, these differences are observed although both crosslinking molecules use the same calmodulin-homologous domain for actin binding. For both systems at least three distinct phases of actin-networks with different viscoelastic properties are observed. By rheological and optical methods these are related to the microscopic structure of the networks. The occurrence of mixed networks containing bundles embedded in an isotropic network indicates that a sharp distinction between crosslinking