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Subcellular Spatial Control of Non-Muscle Myosin 2 Redistribution and Stress Fiber Strain by Molecular Tattoo
430a
Tuesday, February 14, 2017
(RGA-3/4) onto Actin filaments drives pulse termination. Using singlemolecule imaging, we further show that this mode of actin assembly drives the formation of structural units displaying a characteristically polarized architecture with (1) a gradient of myosin accumulation, myosin being recruited at the center of the pulses and (2) a transient anisotropic organization of the actin network, where filaments elongate from the instide of the pulse, barbed ends pointing outwards, as visualized by displacement of the actin-barbed-endtracking formins that processively elongate actin filaments. Based on these results, we propose here that pulsed contractions represent a mode of actomyosin assembly which structures the actomyosin network in functional modules to drive efficient cell contractility. 2112-Pos Board B432 Lifetime of Membrane-Cytoskeleton Bonds is Mediated by RHO-GTPases in a Cancer Cell Vivek Rajasekharan1, Varun K.A. Sreenivasan1, Jeffrey N. Myers2, Fred A. Pereira1, Brenda Farrell1. 1 Baylor College of Medicine, Houston, TX, USA, 2The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Cells especially cancer cells are capable of rapid cytoskeleton remodeling and often membrane reorganization in response to environmental cues. The propensity to remodel is reflected in part by the lifetime t of the membranecytoskeleton bonds at the edges of cells; t increases as their number density and on-rate increases. We measure t by monitoring the time-course of the membrane peeling force Fp at the edge of a cancer cell. Experiments are conducted at the near-equilibrium region (cf. irreversible), where an optical tweezers is used to apply a load with a handle which is bound to a slowly moving cell, and the handle displacement is detected at a resolution of 500 ms after averaging. Under constant load, Fp increases monotonically with time; at force fR and time tR this slope abruptly changes indicating membrane-cytoskeleton bond rupture. Repeating for many cells we find fR is not constant but increases with tR. We propose it represents the rupture of a cluster of bonds with larger clusters demonstrating greater rupture forces and lifetimes. This is in agreement with theory that calculates the lifetime of a cluster of bonds between two bodies (J.Chem.Phys.121:8997). Comparing experimental data with this theory, we find that molecular parameters are within expected ranges reported for biomolecular bonds in vitro withevidence that the bonds can re-bind. Cells treated with Rho-GTPase inhibitors possess membrane-cytoskeleton bonds with lower stiffness that show no rebinding on the timescale of the measurements (zero onrate). This is predictable since active Rho-GTPases form linkages between the membrane and actin-effector proteins of the cytoskeleton. This measurable change in bond properties provides a quantitative method for evaluating the role of Rho-GTPases in dynamic cytoskeleton remodeling. This is relevant as Rho-GTPases are upregulated in many human cancers including the HN31 cell line used in this study. 2113-Pos Board B433 Subcellular Spatial Control of Non-Muscle Myosin 2 Redistribution and Stress Fiber Strain by Molecular Tattoo Adam I. Horvath, Boglarka H. Varkuti, Miklos Kepiro, Gyorgy Hegyi, Mihaly Kovacs, Andras Malnasi-Csizmadia. Department of Biochemistry, MTA-ELTE Molecular Biophysics Research Group, Eotvos Lorand University, Budapest, Hungary. The cellular distribution of the motor protein non-muscle myosin 2 (NM2) leads to different forms of intracellular strain driving cell motility, cytokinesis and morphogenetic processes including axonal growth and retraction. However, it remains elusive how these cellular processes are governed by the dynamic changes in NM2 localization and supramolecular assembly. To address this problem, we determined the effect of different types of NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 redistribution via FRAP, applied also in combination with our recently developed optopharmacological tool, Molecular Tattoo, which allows subcellular confinement of drug effects via 2-photon induced photocrosslinking to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically accelerates NM2 redistribution and induces stress fiber dissolution, due to NM2 filament disassembly resulting from myosin light chain dephosphorylation. When NM2 was inhibited by para-nitroblebbistatin (pNBleb) or locally by tattooed azidoblebbistatin, in the stress fibers a significant acceleration and suppression of NM2 redistribution was detected at moderate and high inhibitor concentrations, respectively. The observed effects were local and specific for load-bearing peripheral stress fibers, implying the role of mechanical load in NM2 redistribution. Furthermore, in these tests stress fibers remained intact, contrary to that seen upon Rho-kinase inhibition. These
results highlight that variations in the localization and/or pharmacological mechanism of NM2 inhibition produce distinct effects on intracellular strain and cellular morphogenesis. Supported by Hungarian Research and Innovation Fund (VKSZ_14-1-20150052). 2114-Pos Board B434 An Potogenentic Toolkit for Reversible Labeling and Remote Manipulation of Cytoskeleton In Situ Qian Zhang1,2, Lian He1, Guolin Ma1, Yubin Zhou1,3. 1 Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA, 2Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, China, 3 Department of Medical Physiology, College of Medicine, Texas A&M University, Temple, TX, USA. Most of the current methodologies to visualize the cytoskeleton are irreversible and can only provide a static picture of cytoskeleton. These methods often involve the fixation of cells or permanently perturb the behaviors of cytoskeleton. To overcome this obstacle, we designed a set of optogenetic tools to reversibly label and manipulate cytoskeleton dynamics in living cells by harnessing the power of light. These genetically-encoded small optical tools were demonstrated to i) instantly and reversibly photo-label actin, microtubule and the plus-end of microtubule without perturbing their normal functions, ii) manipulate the dynamics of microtubule following stimulation with light emitting at varying wavelengths; and iii) drive the target protein or organelle towards either ends of MTs to recruit effectors and control the downstream signaling pathways. These noninvasive optical tools provide new opportunities to remotely monitor and perturb the cytoskeleton, and can also be applied in drug screening to identify potential adverse effects of drug candidates on cytoskeleton. (Supported by the National Institutes of Health (GM112003) and the Welch Foundation (BE-1913 to Y.Z.), and a China Scholarship Council award to Q.Z.) 2115-Pos Board B435 Mechanics and Dynamics of Cation-Induced Actin Bundles Nicholas Castaneda1, Tianyu Zheng2, Hector Rivera-Jacquez1, Qun Huo2, Hyeran Kang1. 1 NanoScience Technology Center, University of Central Florida, Orlando, FL, USA, 2NanoScience Technology Center, Department of Chemistry, University of Central Florida, Orlando, FL, USA. The assembly of actin filaments into bundles plays an essential role in mechanical strength and dynamic reorganization of cytoskeleton. Divalent counterions at high concentrations promote bundle formation through electrostatic attraction between charged filaments. Although it has been hypothesized that specific cation interactions may contribute to salt-induced bundling, molecular mechanisms of how salt modulates bundle assembly and mechanics are not well established. Here we determine the mechanical and dynamic properties of actin bundles at varying divalent cation concentrations. Using total internal reflection fluorescence (TIRF) microscopy, we measure the bending stiffness of actin bundles determined by persistence length analysis. We characterize real-time formation of bundles by dynamic light scattering intensity and direct visualization using TIRF microscopy. Our results show that divalent cations stiffen actin bundles and modulate time-dependent average bundle size. The work suggests that cation interactions serve a regulatory function in actin bundle mechanics and dynamics. 2116-Pos Board B436 CaMKII Control of Actin Cytoskeletal Dynamics Shahid M. Khan1, Justin E. Molloy2. 1 Macromolecular Dynamics, Molecular Biology Consortium, Berkeley, CA, USA, 2Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom. The calcium calmodulin dependent kinase (CaMKII) binds and organizes actin Binding is abolished by calcium calmodulin, triggering cytoskeletal remodeling with important implications for synaptic transmission in dendritic spines; CaMKII and actin-rich post-synaptic micro-compartments that expand within one minute upon synaptic stimulation to initiate long-term changes associated with learning. CaMKII organizes static F-actin bundles in-vitro, but fluorescence microscopy single particle tracking (SPT) of GFP-tagged CaMKII holoenzymes (GFP-CaMKII) in spines has not detected stimulus-dependent jumps in mobility consistent with un-bundling. However, changes in binding affinities, in response to osmotic and mechanical forces, might provoke diverse cytoskeletal CaMKII-actin architectures in vivo. To look for these, we initially used SPT in live cells and found both rat neuronal CaMKII isoforms bound RFP-actin labeled stress fibers weakly, comparable to G-actin, with strong
Tuesday, February 14, 2017
(RGA-3/4) onto Actin filaments drives pulse termination. Using singlemolecule imaging, we further show that this mode of actin assembly drives the formation of structural units displaying a characteristically polarized architecture with (1) a gradient of myosin accumulation, myosin being recruited at the center of the pulses and (2) a transient anisotropic organization of the actin network, where filaments elongate from the instide of the pulse, barbed ends pointing outwards, as visualized by displacement of the actin-barbed-endtracking formins that processively elongate actin filaments. Based on these results, we propose here that pulsed contractions represent a mode of actomyosin assembly which structures the actomyosin network in functional modules to drive efficient cell contractility. 2112-Pos Board B432 Lifetime of Membrane-Cytoskeleton Bonds is Mediated by RHO-GTPases in a Cancer Cell Vivek Rajasekharan1, Varun K.A. Sreenivasan1, Jeffrey N. Myers2, Fred A. Pereira1, Brenda Farrell1. 1 Baylor College of Medicine, Houston, TX, USA, 2The University of Texas MD Anderson Cancer Center, Houston, TX, USA. Cells especially cancer cells are capable of rapid cytoskeleton remodeling and often membrane reorganization in response to environmental cues. The propensity to remodel is reflected in part by the lifetime t of the membranecytoskeleton bonds at the edges of cells; t increases as their number density and on-rate increases. We measure t by monitoring the time-course of the membrane peeling force Fp at the edge of a cancer cell. Experiments are conducted at the near-equilibrium region (cf. irreversible), where an optical tweezers is used to apply a load with a handle which is bound to a slowly moving cell, and the handle displacement is detected at a resolution of 500 ms after averaging. Under constant load, Fp increases monotonically with time; at force fR and time tR this slope abruptly changes indicating membrane-cytoskeleton bond rupture. Repeating for many cells we find fR is not constant but increases with tR. We propose it represents the rupture of a cluster of bonds with larger clusters demonstrating greater rupture forces and lifetimes. This is in agreement with theory that calculates the lifetime of a cluster of bonds between two bodies (J.Chem.Phys.121:8997). Comparing experimental data with this theory, we find that molecular parameters are within expected ranges reported for biomolecular bonds in vitro withevidence that the bonds can re-bind. Cells treated with Rho-GTPase inhibitors possess membrane-cytoskeleton bonds with lower stiffness that show no rebinding on the timescale of the measurements (zero onrate). This is predictable since active Rho-GTPases form linkages between the membrane and actin-effector proteins of the cytoskeleton. This measurable change in bond properties provides a quantitative method for evaluating the role of Rho-GTPases in dynamic cytoskeleton remodeling. This is relevant as Rho-GTPases are upregulated in many human cancers including the HN31 cell line used in this study. 2113-Pos Board B433 Subcellular Spatial Control of Non-Muscle Myosin 2 Redistribution and Stress Fiber Strain by Molecular Tattoo Adam I. Horvath, Boglarka H. Varkuti, Miklos Kepiro, Gyorgy Hegyi, Mihaly Kovacs, Andras Malnasi-Csizmadia. Department of Biochemistry, MTA-ELTE Molecular Biophysics Research Group, Eotvos Lorand University, Budapest, Hungary. The cellular distribution of the motor protein non-muscle myosin 2 (NM2) leads to different forms of intracellular strain driving cell motility, cytokinesis and morphogenetic processes including axonal growth and retraction. However, it remains elusive how these cellular processes are governed by the dynamic changes in NM2 localization and supramolecular assembly. To address this problem, we determined the effect of different types of NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 redistribution via FRAP, applied also in combination with our recently developed optopharmacological tool, Molecular Tattoo, which allows subcellular confinement of drug effects via 2-photon induced photocrosslinking to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically accelerates NM2 redistribution and induces stress fiber dissolution, due to NM2 filament disassembly resulting from myosin light chain dephosphorylation. When NM2 was inhibited by para-nitroblebbistatin (pNBleb) or locally by tattooed azidoblebbistatin, in the stress fibers a significant acceleration and suppression of NM2 redistribution was detected at moderate and high inhibitor concentrations, respectively. The observed effects were local and specific for load-bearing peripheral stress fibers, implying the role of mechanical load in NM2 redistribution. Furthermore, in these tests stress fibers remained intact, contrary to that seen upon Rho-kinase inhibition. These
results highlight that variations in the localization and/or pharmacological mechanism of NM2 inhibition produce distinct effects on intracellular strain and cellular morphogenesis. Supported by Hungarian Research and Innovation Fund (VKSZ_14-1-20150052). 2114-Pos Board B434 An Potogenentic Toolkit for Reversible Labeling and Remote Manipulation of Cytoskeleton In Situ Qian Zhang1,2, Lian He1, Guolin Ma1, Yubin Zhou1,3. 1 Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA, 2Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, China, 3 Department of Medical Physiology, College of Medicine, Texas A&M University, Temple, TX, USA. Most of the current methodologies to visualize the cytoskeleton are irreversible and can only provide a static picture of cytoskeleton. These methods often involve the fixation of cells or permanently perturb the behaviors of cytoskeleton. To overcome this obstacle, we designed a set of optogenetic tools to reversibly label and manipulate cytoskeleton dynamics in living cells by harnessing the power of light. These genetically-encoded small optical tools were demonstrated to i) instantly and reversibly photo-label actin, microtubule and the plus-end of microtubule without perturbing their normal functions, ii) manipulate the dynamics of microtubule following stimulation with light emitting at varying wavelengths; and iii) drive the target protein or organelle towards either ends of MTs to recruit effectors and control the downstream signaling pathways. These noninvasive optical tools provide new opportunities to remotely monitor and perturb the cytoskeleton, and can also be applied in drug screening to identify potential adverse effects of drug candidates on cytoskeleton. (Supported by the National Institutes of Health (GM112003) and the Welch Foundation (BE-1913 to Y.Z.), and a China Scholarship Council award to Q.Z.) 2115-Pos Board B435 Mechanics and Dynamics of Cation-Induced Actin Bundles Nicholas Castaneda1, Tianyu Zheng2, Hector Rivera-Jacquez1, Qun Huo2, Hyeran Kang1. 1 NanoScience Technology Center, University of Central Florida, Orlando, FL, USA, 2NanoScience Technology Center, Department of Chemistry, University of Central Florida, Orlando, FL, USA. The assembly of actin filaments into bundles plays an essential role in mechanical strength and dynamic reorganization of cytoskeleton. Divalent counterions at high concentrations promote bundle formation through electrostatic attraction between charged filaments. Although it has been hypothesized that specific cation interactions may contribute to salt-induced bundling, molecular mechanisms of how salt modulates bundle assembly and mechanics are not well established. Here we determine the mechanical and dynamic properties of actin bundles at varying divalent cation concentrations. Using total internal reflection fluorescence (TIRF) microscopy, we measure the bending stiffness of actin bundles determined by persistence length analysis. We characterize real-time formation of bundles by dynamic light scattering intensity and direct visualization using TIRF microscopy. Our results show that divalent cations stiffen actin bundles and modulate time-dependent average bundle size. The work suggests that cation interactions serve a regulatory function in actin bundle mechanics and dynamics. 2116-Pos Board B436 CaMKII Control of Actin Cytoskeletal Dynamics Shahid M. Khan1, Justin E. Molloy2. 1 Macromolecular Dynamics, Molecular Biology Consortium, Berkeley, CA, USA, 2Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom. The calcium calmodulin dependent kinase (CaMKII) binds and organizes actin Binding is abolished by calcium calmodulin, triggering cytoskeletal remodeling with important implications for synaptic transmission in dendritic spines; CaMKII and actin-rich post-synaptic micro-compartments that expand within one minute upon synaptic stimulation to initiate long-term changes associated with learning. CaMKII organizes static F-actin bundles in-vitro, but fluorescence microscopy single particle tracking (SPT) of GFP-tagged CaMKII holoenzymes (GFP-CaMKII) in spines has not detected stimulus-dependent jumps in mobility consistent with un-bundling. However, changes in binding affinities, in response to osmotic and mechanical forces, might provoke diverse cytoskeletal CaMKII-actin architectures in vivo. To look for these, we initially used SPT in live cells and found both rat neuronal CaMKII isoforms bound RFP-actin labeled stress fibers weakly, comparable to G-actin, with strong