- Email: [email protected]
Simulation of the Effect of Confinement in Actin Ring Formation
126a
Sunday, February 28, 2016
homodimer with one actin-binding domain (ABD) at each end. The ABD is composed of two calponin homology domains, CH1 and CH2, that can exist in either open or closed conformation, which most likely determines their affinity for actin. It has been suggested that the open conformation is more favorable for actin binding but it is not yet clear how molecular interactions of the open ABD are different from the closed conformation. We compared ABD association to actin in both closed and open states and revealed the key differences between the two using all-atomic molecular dynamics simulations. Furhtermore, the k255E mutation in human a-actinin4 is involved in a kidney disease and located at the interface between CH1 and CH2. It was shown that this mutation alters mechanical properties of the cytoskeleton presumably by increasing the affinity between a-actinin and actin. We investigated the effect of this mutaiton on the the ABD conformation and showed the molecular mechanism by which the K255E mutation changes the binding between a-actinin and actin. Our results provide a valuable insight into developing novel therapeutics for the kidney disease cause by the K255E mutation. 637-Pos Board B417 3D Model of Fission Yeast Contractile Ring Assembly: Effects of Cytokinetic Node Interaction with the Cell Membrane and Myosin Motors Tamara C. Bidone, Dimitrios Vavylonis. Lehigh University, Bethlehem, PA, USA. Cytokinetic ring assembly in model organism fission yeast is a dynamic process, involving condensation of a network of actin filaments and myosin motors bound to the cell membrane through cortical nodes. A 3D computational model of cytokinetic ring assembly based on the ‘‘search, capture, pull and release’’ mechanism illustrates how the combined activities of myosin motors, filament crosslinkers and actin filament turnover can lead to robust ring formation [Bidone et al. Biophys. J, 2014]. We extended the model to study the importance of the physical properties of node movement along the cell membrane and of myosin recruitment to nodes. Experiments by D. Zhang (Temasek Life Sciences) show that tethering of the cortical endoplasmic reticulum (ER) to the plasma membrane modulates the speed of node condensation and the degree of node clumping. We were able to capture the trend observed in these experiments by changes in the node drag coefficient and initial node distribution in simulations. The model predicted that reducing crosslinking activities in ER tethering mutants with faster node speed enhances the probability of actomyosin clumping. We developed a model of how tilted and/or misplaced rings assemble in cells that lack the node structural component anillin-like Mid1 and thus fail to recruit myosin II to nodes independently of actin. If actin-dependent binding of diffusive myosin to the cortex is incorporated into the model, it generates progressively elongating cortical actomyosin strands with fluctuating actin bundles at the tails. These stands often close into a ring, similar to observations by the group of J.Q. Wu (The Ohio State University). 638-Pos Board B418 Simulation of the Effect of Confinement in Actin Ring Formation Maral Adeli Koudehi, Haosu Tang, Dimitrios Vavylonis. Lehigh University, Bethlehem, PA, USA. Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers and motor proteins. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. We simulate cross-linking implicitly as an attractive short-range potential between filament beads. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We observed and quantified other forms of networks for larger radii. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.
639-Pos Board B419 Role of Cross-Linkers in Yeast Branched Actin Networks: Linking Biochemistry and Mechanics Jessica Planade1, Audrey Guillotin2, Alphe´e Michelot2, Olivia du Roure1, Julien Heuvingh1. 1 Physique et Me´canique des Milieux He´te´roge`nes, UMR CNRS 7636/ PSL – ESPCI ParisTech/ Sorbonne Universite´ – UPMC/Sorbonne Paris Cite´ - UDD, Paris, France, 2Laboratoire de Physiologie Cellulaire, IRTSV, CNRS/CEA/ INRA/UJF, Grenoble, France. The actin cytoskeleton is an assembly of organized polymer structures. In cells, actin contributes to their internal organization, their rigidity, and their ability to exert forces. The properties of the actin networks are regulated by multiple families of actin binding proteins (ABPs). In this work, we focus on Arp2/3branched networks, which are implicated in a variety of cellular functions such as motility or endocytosis. We propose to examine the relationship between the mechanical properties of actin networks and the biochemical composition of these gels. While atomic force microscopy and micropipette techniques have been successfully used to probe the mechanics of actin gels in vitro, their main drawback for our purpose is the limited amount of measurements per experiment. To overcome this limitation, we use instead a quantitative high-throughput system of magnetic colloids (Pujol et al., 2012). Actin networks are assembled around the colloidal particles from sets of purified proteins (bottom-up approach) or from yeast protein extracts. The advantage of these extracts is that proteins can be genetically removed one-by-one, in order to test for their functions in a near-physiological environment (top-down approach) (Michelot and Drubin, 2014). In this first study, we focus on the impact of crosslinkers, which create attachment points between neighboring filaments. The absence of two crosslinkers Sac6 (fimbrin) and Scp1 (calponin) softens and modifies the long-term evolution of actin gels assembled from extracts. Indeed, networks’ structural integrity is not recovered after load. In agreement with previous data, the addition of purified fimbrin in the bottom-up approach increases the elastic modulus of actin gels in a sigmoidal dose-dependent manner. Moreover, the system seems to evolve from little plasticity and non-linear behavior under load to a more plastic and less non-linear response. 640-Pos Board B420 Self-Organization of Actomyosin Networks Attached to Artificial Membranes Markus Scho¨n1, Corinna Kramer1, Helen Noeding2, Ingo Mey1, Andreas Janshoff2, Claudia Steinem1. 1 Institute of Organic and Biomolecular Chemistry, University Go¨ttingen, Go¨ttingen, Germany, 2Institute of Physical Chemistry, University Go¨ttingen, Go¨ttingen, Germany. The shape and mechanical stability of cells are highly dependent on their cytoskeleton. Actin is one of the main proteins which contributes to this biological network. The mechanical movements of the cell rely on the interaction of F-actin with several other proteins. One of these proteins is myosin II, a molecular motor protein. By hydrolysing ATP myosin is able to walk along F-actin or to induce tension on these filaments. In our model system F-actin networks are attached to pore spanning lipid bilayers (PSLBs) via electrostatical interactions or the linker protein ezrin which mimics the biological situation. Ezrin has a PIP2 binding site located at the N-terminus and a F-actin binding site at the C-terminus and is responsible for the linkage of F-actin to PIP2 present in the PSLB. Several actin binding proteins and cross-linkers are introduced during the polymerization of actin filaments. Besides the visual self-organization the mechanical properties of different F-actin networks are examined. Atomic force microscopy is used to determine the lateral membrane tension of the PSLB dependent on different actomyosin networks. The viscoelastic properties of the network will be recorded by passive microrheology using the mean square displacement of a polymer beads Brownian movement. 641-Pos Board B421 Differences in the Spatial Distribution of Actin in the Left and Right Ventricles of Healthy Human Hearts Janhavi Nagwekar1, Divya Duggal1, Ryan Rich2, Sangram Raut1, zygmunt gryczynski1, Julian Borejdo1. 1 Cell Biology, University of North Texas Health Science Center (UNTHSC), Fort Worth, TX, USA, 2Cell Biology, Texas Wesleyan University, Fort Worth, TX, USA.
Sunday, February 28, 2016
homodimer with one actin-binding domain (ABD) at each end. The ABD is composed of two calponin homology domains, CH1 and CH2, that can exist in either open or closed conformation, which most likely determines their affinity for actin. It has been suggested that the open conformation is more favorable for actin binding but it is not yet clear how molecular interactions of the open ABD are different from the closed conformation. We compared ABD association to actin in both closed and open states and revealed the key differences between the two using all-atomic molecular dynamics simulations. Furhtermore, the k255E mutation in human a-actinin4 is involved in a kidney disease and located at the interface between CH1 and CH2. It was shown that this mutation alters mechanical properties of the cytoskeleton presumably by increasing the affinity between a-actinin and actin. We investigated the effect of this mutaiton on the the ABD conformation and showed the molecular mechanism by which the K255E mutation changes the binding between a-actinin and actin. Our results provide a valuable insight into developing novel therapeutics for the kidney disease cause by the K255E mutation. 637-Pos Board B417 3D Model of Fission Yeast Contractile Ring Assembly: Effects of Cytokinetic Node Interaction with the Cell Membrane and Myosin Motors Tamara C. Bidone, Dimitrios Vavylonis. Lehigh University, Bethlehem, PA, USA. Cytokinetic ring assembly in model organism fission yeast is a dynamic process, involving condensation of a network of actin filaments and myosin motors bound to the cell membrane through cortical nodes. A 3D computational model of cytokinetic ring assembly based on the ‘‘search, capture, pull and release’’ mechanism illustrates how the combined activities of myosin motors, filament crosslinkers and actin filament turnover can lead to robust ring formation [Bidone et al. Biophys. J, 2014]. We extended the model to study the importance of the physical properties of node movement along the cell membrane and of myosin recruitment to nodes. Experiments by D. Zhang (Temasek Life Sciences) show that tethering of the cortical endoplasmic reticulum (ER) to the plasma membrane modulates the speed of node condensation and the degree of node clumping. We were able to capture the trend observed in these experiments by changes in the node drag coefficient and initial node distribution in simulations. The model predicted that reducing crosslinking activities in ER tethering mutants with faster node speed enhances the probability of actomyosin clumping. We developed a model of how tilted and/or misplaced rings assemble in cells that lack the node structural component anillin-like Mid1 and thus fail to recruit myosin II to nodes independently of actin. If actin-dependent binding of diffusive myosin to the cortex is incorporated into the model, it generates progressively elongating cortical actomyosin strands with fluctuating actin bundles at the tails. These stands often close into a ring, similar to observations by the group of J.Q. Wu (The Ohio State University). 638-Pos Board B418 Simulation of the Effect of Confinement in Actin Ring Formation Maral Adeli Koudehi, Haosu Tang, Dimitrios Vavylonis. Lehigh University, Bethlehem, PA, USA. Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers and motor proteins. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. We simulate cross-linking implicitly as an attractive short-range potential between filament beads. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We observed and quantified other forms of networks for larger radii. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.
639-Pos Board B419 Role of Cross-Linkers in Yeast Branched Actin Networks: Linking Biochemistry and Mechanics Jessica Planade1, Audrey Guillotin2, Alphe´e Michelot2, Olivia du Roure1, Julien Heuvingh1. 1 Physique et Me´canique des Milieux He´te´roge`nes, UMR CNRS 7636/ PSL – ESPCI ParisTech/ Sorbonne Universite´ – UPMC/Sorbonne Paris Cite´ - UDD, Paris, France, 2Laboratoire de Physiologie Cellulaire, IRTSV, CNRS/CEA/ INRA/UJF, Grenoble, France. The actin cytoskeleton is an assembly of organized polymer structures. In cells, actin contributes to their internal organization, their rigidity, and their ability to exert forces. The properties of the actin networks are regulated by multiple families of actin binding proteins (ABPs). In this work, we focus on Arp2/3branched networks, which are implicated in a variety of cellular functions such as motility or endocytosis. We propose to examine the relationship between the mechanical properties of actin networks and the biochemical composition of these gels. While atomic force microscopy and micropipette techniques have been successfully used to probe the mechanics of actin gels in vitro, their main drawback for our purpose is the limited amount of measurements per experiment. To overcome this limitation, we use instead a quantitative high-throughput system of magnetic colloids (Pujol et al., 2012). Actin networks are assembled around the colloidal particles from sets of purified proteins (bottom-up approach) or from yeast protein extracts. The advantage of these extracts is that proteins can be genetically removed one-by-one, in order to test for their functions in a near-physiological environment (top-down approach) (Michelot and Drubin, 2014). In this first study, we focus on the impact of crosslinkers, which create attachment points between neighboring filaments. The absence of two crosslinkers Sac6 (fimbrin) and Scp1 (calponin) softens and modifies the long-term evolution of actin gels assembled from extracts. Indeed, networks’ structural integrity is not recovered after load. In agreement with previous data, the addition of purified fimbrin in the bottom-up approach increases the elastic modulus of actin gels in a sigmoidal dose-dependent manner. Moreover, the system seems to evolve from little plasticity and non-linear behavior under load to a more plastic and less non-linear response. 640-Pos Board B420 Self-Organization of Actomyosin Networks Attached to Artificial Membranes Markus Scho¨n1, Corinna Kramer1, Helen Noeding2, Ingo Mey1, Andreas Janshoff2, Claudia Steinem1. 1 Institute of Organic and Biomolecular Chemistry, University Go¨ttingen, Go¨ttingen, Germany, 2Institute of Physical Chemistry, University Go¨ttingen, Go¨ttingen, Germany. The shape and mechanical stability of cells are highly dependent on their cytoskeleton. Actin is one of the main proteins which contributes to this biological network. The mechanical movements of the cell rely on the interaction of F-actin with several other proteins. One of these proteins is myosin II, a molecular motor protein. By hydrolysing ATP myosin is able to walk along F-actin or to induce tension on these filaments. In our model system F-actin networks are attached to pore spanning lipid bilayers (PSLBs) via electrostatical interactions or the linker protein ezrin which mimics the biological situation. Ezrin has a PIP2 binding site located at the N-terminus and a F-actin binding site at the C-terminus and is responsible for the linkage of F-actin to PIP2 present in the PSLB. Several actin binding proteins and cross-linkers are introduced during the polymerization of actin filaments. Besides the visual self-organization the mechanical properties of different F-actin networks are examined. Atomic force microscopy is used to determine the lateral membrane tension of the PSLB dependent on different actomyosin networks. The viscoelastic properties of the network will be recorded by passive microrheology using the mean square displacement of a polymer beads Brownian movement. 641-Pos Board B421 Differences in the Spatial Distribution of Actin in the Left and Right Ventricles of Healthy Human Hearts Janhavi Nagwekar1, Divya Duggal1, Ryan Rich2, Sangram Raut1, zygmunt gryczynski1, Julian Borejdo1. 1 Cell Biology, University of North Texas Health Science Center (UNTHSC), Fort Worth, TX, USA, 2Cell Biology, Texas Wesleyan University, Fort Worth, TX, USA.