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Stress Propagation through Biological Lipid-Bilayers Revealed by Atomistic and Coarse-Grained Simulations
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Sunday, February 12, 2017
the effect of incorporating protein on the mechanical properties of lipid bilayers we have reconstituted the Escherichia coli transporter lactose permease (LacY), a transmembrane protein representative of the major facilitator superfamily, into synthetic lipid vesicles. We observed directly the fluctuation of the lipid bilayer in these protein reconstituted GUVs, with sizes in the order of micrometers, by phase contrast microscopy. This allowed the determination of the bending rigidity, which characterizes the ability of membranes to bend under low stress, by fluctuation analysis. Changes in the bending rigidity parameter allowed us to get better insights into the effect of lipids and protein on the mechanical properties of GUVs in a quantitative fashion. 390-Pos Board B155 Shear Stress Stimulated MSC Activities: Direct Changes of Membrane Tension or Cytoskeletal Stress? Mohammad Mehdi Maneshi1, Frederick Sachs2, Susan Zonglu Hua1,2. 1 Mechanical and Aerospace Engineering, University at Buffalo, Buffalo, NY, USA, 2Physiology & Biophysics, University at Buffalo, Buffalo, NY, USA. Fluid shear stress induced membrane transport such as Ca2þ influx and the activation of ion channels has been widely reported. The shear stress can be mediated by a direct change in bilipid membrane tension and/or by a change in cytoskeletal stress via binding proteins that link channels to actin, but how the shear stress is coupled to ion channels is unclear. Using narrow shear pulse stimuli generated by a pressure servo in a microfluidic chamber, we measured the changes of membrane tension and cytoskeletal protein stress simultaneously in astrocytes. The membrane tension was reported using molecular motor probes (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ) and cytoskeletal tension reported by genetically encoded force probe actinin-cpst-FRET. Our results show that the changes of membrane tension are highly localized and the gradient is relevant to the flow directions. A shear stress pulse (23 dyn/cm2, 400 ms duration) caused a rapid increase in membrane tension at the front edge of the cell with respect to the flow and a decrease in tension (compression) at the distal edge. The rise time was less than 30 ms, and the tension dropped to the initial state within ~30 ms post stimulus, showing a typical elastic behavior. In contrast, the same shear pulse generated profound and long-lasting tension in cytoskeletal cross-linking proteins a-actinin at the front edge of the cell, and the tension persisted for the entire experimental duration of 60 s. In situ Ca2þ imaging showed that the initial Ca2þ influx was strongly correlated with the region having high cytoskeletal tension, but weakly linked to the bilayer tension. The results suggest the cytoskeletal tension plays primary role in shear stress activated Ca2þ influx. This work was funded by NINDS. 391-Pos Board B156 Stress Propagation through Biological Lipid-Bilayers Revealed by Atomistic and Coarse-Grained Simulations Camilo Aponte-Santamaria1,2, Frauke Gr€ater1,2. 1 Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, 2Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany. Membrane tension has been shown to play various critical roles in cell signaling. We here asked if and how pulses of local stress dynamically propagate through membranes, and propose this mechanism as a novel way of quickly propagating signals along the membrane. In both atomistic and coarse-grained MARTINI molecular dynamics simulations of biological lipid-bilayers, we observed short stress pulses to very efficiently propagate laterally at a velocity of the order of km/s, in close agreement with the expected speed of sound. The temperature dependence of pulse propagation shows tendencies very comparable to analogous experiments [1,2,3], with insightful differences between the atomistic and coarse-grained simulations. Remarkably, the propagation of the lateral stress was damped at length scales in the ~100 nm range. Our data supports the notion of lateral stress propagation through membranes as a potential ultrafast way of short-range signal propagation in biology [4]. [1] W. Schrader, et al. J. Phys. Chem. B. 106:6581-6586 (2002) [2] S. Shrivastava S and MF Schneider. J. R. Soc. Interface 11:20140098 (2014). [3] J. Kappler and R. Netz. EPL. 112: 19002 (2015) [4] C. Aponte-Santamarı´a and F. Gr€ater. In preparation
392-Pos Board B157 Interdependence between Collective Thermal Fluctuations and Elastic and Viscous Properties in Model Lipid Bilayers Michihiro Nagao1,2, Elizabeth G. Kelley1, Rana Ashkar3, Robert Bradbury1,2, Paul D. Butler1,4. 1 NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD, USA, 2Center for Exploration of Energy and Matter, Indiana University, Bloomington, IN, USA, 3Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4 Department of Chemical and Bimolecular Engineering, University of Delaware, Newark, DE, USA. Lipid membranes undergo an array of conformational and dynamic transitions, ranging from individual lipid motions to undulations of micron-sized patches of the membrane. However, the dynamics at intermediate length scales are largely unexplored due to experimental challenges in accessing the appropriate length and time scales. Over the past several years our group has used neutron spin echo spectroscopy (NSE) to provide unique insights into these elusive dynamics in model lipid bilayers, measuring collective bending and thickness fluctuations. These thermally induced collective membrane fluctuations are controlled by elastic and viscous properties of the membranes. It has long been known that the bending fluctuations are characterized by the bending modulus, k, of the membranes and the motion is damped by the viscosity of solvent, h. By contrast, according to a recent theory proposed by Bingham, Smye and Olmsted, the collective thickness fluctuations are characterized by the bilayer area compressibility modulus, KA, which is damped by the membrane and solvent viscosities, m and h, respectively. Therefore, by measuring these two collective membrane fluctuations the membrane’s elastic and viscous parameters can be evaluated. Here we use this novel method to determine these characteristic parameters of lipid bilayers from neutron scattering data for a couple of simple saturated phosphatidylcholine bilayers. The estimated values are k ~ 1019 J, KA ~ 0.3 to 0.4 N/m, and m ~ 10 nPa s m, which are all consistent with literature values. 393-Pos Board B158 Hydration-Mediated Elastic Deformations in Biological Membranes Trivikram R. Molugu1, Soohyun K. Lee1, Xiaolin Xu2, Rami Musharrafieh1, K.J. Mallikarjunaiah1, Constantin Job1, Michael Brown1,2. 1 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA. Lipid membranes are excellent examples of biological soft matter [1]. Many functions of biomembranes involve collective phenomena with motional timescales spanning several decades (1012 s to s). For liquid-crystalline membranes atomistic interactions often explain bulk material properties in relation to key biological functions. Solid-state 2H NMR spectroscopy provides such information by simultaneously probing structure and dynamics [2]. Here we examine the effect of hydration on the liquid-crystalline properties of membranes using NMR relaxation methods. We performed 2H NMR longitudinal (R1Z) and transverse quadrupolar-echo decay (R2QE) experiments on DMPCd54 bilayers, to study membrane-lipid dynamics. Plots of the R1Z rates versus squared segmental order parameters (SCD2) follow an empirical square-law showing the emergence of collective lipid dynamics [3]. Such a functional behavior characterizes 3-D order-director fluctuations due to the onset of membrane elasticity over mesoscopic dimensions [3]. The R2QE rates also showed similar results. At high hydration there is an R2QE enhancement of the functional square-law for the segments deeper in the bilayer. Additional contributions from slower dynamics involving water-mediated membrane deformation are evident over mesoscopic length scales on the order of bilayer thickness. Such membrane deformations are also evident from bilayer structural parameters calculated using a statistical mean-torque model [4]. In addition, the square-law confinement must be due to water penetration into the hydrophobic interior of the bilayer. The slow dynamics at high hydration must be a consequence of modulation of membrane elastic properties. The QCPMG frequency dispersions provide quantitative viscoelastic properties of the liquid-crystalline membranes. Such studies on model membranes give insights into lipid rafts and membrane compositions relevant for biomembrane functions. [1] A. Leftin et al. (2014) BJ 107, 2274 [2] K.J. Mallikarjuniah et al. (2011) BJ 100, 98. [3] T.R. Molugu et al. Chem. Phys.Lipids. (2016) [4] A. Leftin et al. (2014) eMagRes 4, 199.
Sunday, February 12, 2017
the effect of incorporating protein on the mechanical properties of lipid bilayers we have reconstituted the Escherichia coli transporter lactose permease (LacY), a transmembrane protein representative of the major facilitator superfamily, into synthetic lipid vesicles. We observed directly the fluctuation of the lipid bilayer in these protein reconstituted GUVs, with sizes in the order of micrometers, by phase contrast microscopy. This allowed the determination of the bending rigidity, which characterizes the ability of membranes to bend under low stress, by fluctuation analysis. Changes in the bending rigidity parameter allowed us to get better insights into the effect of lipids and protein on the mechanical properties of GUVs in a quantitative fashion. 390-Pos Board B155 Shear Stress Stimulated MSC Activities: Direct Changes of Membrane Tension or Cytoskeletal Stress? Mohammad Mehdi Maneshi1, Frederick Sachs2, Susan Zonglu Hua1,2. 1 Mechanical and Aerospace Engineering, University at Buffalo, Buffalo, NY, USA, 2Physiology & Biophysics, University at Buffalo, Buffalo, NY, USA. Fluid shear stress induced membrane transport such as Ca2þ influx and the activation of ion channels has been widely reported. The shear stress can be mediated by a direct change in bilipid membrane tension and/or by a change in cytoskeletal stress via binding proteins that link channels to actin, but how the shear stress is coupled to ion channels is unclear. Using narrow shear pulse stimuli generated by a pressure servo in a microfluidic chamber, we measured the changes of membrane tension and cytoskeletal protein stress simultaneously in astrocytes. The membrane tension was reported using molecular motor probes (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ) and cytoskeletal tension reported by genetically encoded force probe actinin-cpst-FRET. Our results show that the changes of membrane tension are highly localized and the gradient is relevant to the flow directions. A shear stress pulse (23 dyn/cm2, 400 ms duration) caused a rapid increase in membrane tension at the front edge of the cell with respect to the flow and a decrease in tension (compression) at the distal edge. The rise time was less than 30 ms, and the tension dropped to the initial state within ~30 ms post stimulus, showing a typical elastic behavior. In contrast, the same shear pulse generated profound and long-lasting tension in cytoskeletal cross-linking proteins a-actinin at the front edge of the cell, and the tension persisted for the entire experimental duration of 60 s. In situ Ca2þ imaging showed that the initial Ca2þ influx was strongly correlated with the region having high cytoskeletal tension, but weakly linked to the bilayer tension. The results suggest the cytoskeletal tension plays primary role in shear stress activated Ca2þ influx. This work was funded by NINDS. 391-Pos Board B156 Stress Propagation through Biological Lipid-Bilayers Revealed by Atomistic and Coarse-Grained Simulations Camilo Aponte-Santamaria1,2, Frauke Gr€ater1,2. 1 Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, 2Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany. Membrane tension has been shown to play various critical roles in cell signaling. We here asked if and how pulses of local stress dynamically propagate through membranes, and propose this mechanism as a novel way of quickly propagating signals along the membrane. In both atomistic and coarse-grained MARTINI molecular dynamics simulations of biological lipid-bilayers, we observed short stress pulses to very efficiently propagate laterally at a velocity of the order of km/s, in close agreement with the expected speed of sound. The temperature dependence of pulse propagation shows tendencies very comparable to analogous experiments [1,2,3], with insightful differences between the atomistic and coarse-grained simulations. Remarkably, the propagation of the lateral stress was damped at length scales in the ~100 nm range. Our data supports the notion of lateral stress propagation through membranes as a potential ultrafast way of short-range signal propagation in biology [4]. [1] W. Schrader, et al. J. Phys. Chem. B. 106:6581-6586 (2002) [2] S. Shrivastava S and MF Schneider. J. R. Soc. Interface 11:20140098 (2014). [3] J. Kappler and R. Netz. EPL. 112: 19002 (2015) [4] C. Aponte-Santamarı´a and F. Gr€ater. In preparation
392-Pos Board B157 Interdependence between Collective Thermal Fluctuations and Elastic and Viscous Properties in Model Lipid Bilayers Michihiro Nagao1,2, Elizabeth G. Kelley1, Rana Ashkar3, Robert Bradbury1,2, Paul D. Butler1,4. 1 NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD, USA, 2Center for Exploration of Energy and Matter, Indiana University, Bloomington, IN, USA, 3Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4 Department of Chemical and Bimolecular Engineering, University of Delaware, Newark, DE, USA. Lipid membranes undergo an array of conformational and dynamic transitions, ranging from individual lipid motions to undulations of micron-sized patches of the membrane. However, the dynamics at intermediate length scales are largely unexplored due to experimental challenges in accessing the appropriate length and time scales. Over the past several years our group has used neutron spin echo spectroscopy (NSE) to provide unique insights into these elusive dynamics in model lipid bilayers, measuring collective bending and thickness fluctuations. These thermally induced collective membrane fluctuations are controlled by elastic and viscous properties of the membranes. It has long been known that the bending fluctuations are characterized by the bending modulus, k, of the membranes and the motion is damped by the viscosity of solvent, h. By contrast, according to a recent theory proposed by Bingham, Smye and Olmsted, the collective thickness fluctuations are characterized by the bilayer area compressibility modulus, KA, which is damped by the membrane and solvent viscosities, m and h, respectively. Therefore, by measuring these two collective membrane fluctuations the membrane’s elastic and viscous parameters can be evaluated. Here we use this novel method to determine these characteristic parameters of lipid bilayers from neutron scattering data for a couple of simple saturated phosphatidylcholine bilayers. The estimated values are k ~ 1019 J, KA ~ 0.3 to 0.4 N/m, and m ~ 10 nPa s m, which are all consistent with literature values. 393-Pos Board B158 Hydration-Mediated Elastic Deformations in Biological Membranes Trivikram R. Molugu1, Soohyun K. Lee1, Xiaolin Xu2, Rami Musharrafieh1, K.J. Mallikarjunaiah1, Constantin Job1, Michael Brown1,2. 1 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA. Lipid membranes are excellent examples of biological soft matter [1]. Many functions of biomembranes involve collective phenomena with motional timescales spanning several decades (1012 s to s). For liquid-crystalline membranes atomistic interactions often explain bulk material properties in relation to key biological functions. Solid-state 2H NMR spectroscopy provides such information by simultaneously probing structure and dynamics [2]. Here we examine the effect of hydration on the liquid-crystalline properties of membranes using NMR relaxation methods. We performed 2H NMR longitudinal (R1Z) and transverse quadrupolar-echo decay (R2QE) experiments on DMPCd54 bilayers, to study membrane-lipid dynamics. Plots of the R1Z rates versus squared segmental order parameters (SCD2) follow an empirical square-law showing the emergence of collective lipid dynamics [3]. Such a functional behavior characterizes 3-D order-director fluctuations due to the onset of membrane elasticity over mesoscopic dimensions [3]. The R2QE rates also showed similar results. At high hydration there is an R2QE enhancement of the functional square-law for the segments deeper in the bilayer. Additional contributions from slower dynamics involving water-mediated membrane deformation are evident over mesoscopic length scales on the order of bilayer thickness. Such membrane deformations are also evident from bilayer structural parameters calculated using a statistical mean-torque model [4]. In addition, the square-law confinement must be due to water penetration into the hydrophobic interior of the bilayer. The slow dynamics at high hydration must be a consequence of modulation of membrane elastic properties. The QCPMG frequency dispersions provide quantitative viscoelastic properties of the liquid-crystalline membranes. Such studies on model membranes give insights into lipid rafts and membrane compositions relevant for biomembrane functions. [1] A. Leftin et al. (2014) BJ 107, 2274 [2] K.J. Mallikarjuniah et al. (2011) BJ 100, 98. [3] T.R. Molugu et al. Chem. Phys.Lipids. (2016) [4] A. Leftin et al. (2014) eMagRes 4, 199.