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Effects of Lattice Segmentation on Microtubule Mechanics
Sunday, February 28, 2016 the mean squared displacements over time to determine the type of motility they display compared to multiple models of particle transport. 660-Pos Board B440 Effect of Active Kinesin Motor Density on Microtubules During SelfAssembly of Spools Amanda Tan1, Dail Chapman2, Linda Hirst1, Jing Xu1. 1 UC Merced, Merced, CA, USA, 2UC Irvine, Irvine, CA, USA. Active self-assembly systems are energy driven and can organize into various structures. Microtubules and their associated motor proteins, such as kinesin, are widely used to study active self-assembly of higher order structures, such as linear bundles and spools. Microtubules are polymers composed of tubulin that are found in the cytoskeleton. Kinesin motors convert ATP into energy through hydrolysis and walk along microtubules. Microtubules functionalized with biotin and streptavidin bind together and form bundles and spools when gliding. The spools are able to maintain their shape and continue to rotate in the presence of ATP. We use gliding assays to investigate the effect of the active motor density on microtubules during spool formation. By tuning the velocity of gliding microtubules, we can effectively tune the kinesin density on microtubules. There was no significant change in average spool circumference and no reduction in spool density over a 10-fold reduction in microtubule gliding velocity. We find spool characteristics are robust against active kinesin density on microtubules. 661-Pos Board B441 Post-Translational Modification in Microtubule Arrays Exhibits Spatial Patterns that can act as a Signal to Tightly Localize Motor-Driven Cargo Abdon Iniguez1, Jun F. Allard2,3. 1 Center for Complex Biological Systems, University of California, Irvine, Irvine, CA, USA, 2Department of Mathematics, University of California, Irvine, Irvine, CA, USA, 3Department of Physics and Astronomy, University of California, Irvine, Irvine, CA, USA. Microtubule (MT) ‘‘age’’ has been interpreted through nucleotide state, lattice defects, and post-translational modification (PTM) such as acetylation and detyrosination. All three cases have been recently shown to have functionallyimportant effects on the dynamics of MT arrays, which can present spatial and temporal heterogeneity. While mathematical models for MT array densities are well-established, we present equations describing MT age, defined here as the mean time since the MT’s building blocks (tubulin) were polymerized from their soluble dimer state. These equations can recapitulate the observation that the oldest (most acetylated) tubulin in axons is near the middle of axons during neuronal development. Furthermore, PTMs influence motor kinetics up to approximately 3-fold for off-rates and velocities. Our simulations demonstrate that this relatively weak dependence of motor kinetics is sufficient to target motor cargo to a specific location along the array. This localization is tightly peaked in a way that magnifies the relatively small signal of PTM spatial heterogeneity. Thus, MT age can produce long-range spatial patterning without feedbacks or diffusing signals. 662-Pos Board B442 Label-Free Imaging of Microtubules with Subnanometer Precision using Interferometric Scattering Microscopy Joanna Andrecka1, Jaime Ortega-Arroyo1, Robert Cross2, Philipp Kukura1. 1 University of Oxford, Oxford, United Kingdom, 2University of Warwick, Warwick, United Kingdom. Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we show that individual, unlabelled microtubules bound to a cover glass generate sufficient contrast in interferometric scattering microscopy (iSCAT) to be tracked with sub-nm precision. We reveal 8 nm steps at 1000 frames per second imaging rate in a glidin assay as expected for a single kinesin molecule moving a microtubule, a result which cannot be achieved with fluorescence detection at this imaging speed. At higher motor densities, we frequently observe fractional steps on the order of 4 and even 2 nm, most likely resulting from several motors acting on a single MT. Finally, we demonstrate label-free imaging of a single MT shrinking over 30 min after removal of free tubulin and taxol. In general, our methodology provides a new means to image and study microtubules and serves as a platform for new generation methods to study biological processes involving biological filaments without the limitations and perturbations imposed by fluorescence labelling.
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663-Pos Board B443 Deformability of Microtubules: An Atomistic Computational Study Ondrej Kucera1, Daniel Havelka1, Marco A. Deriu2, Michal Cifra1. 1 Institute of Photonics and Electronics ASCR, Prague, Czech Republic, 2 University of Applied Sciences and Arts of Southern Switzerland, Manno, Switzerland. Thanks to their material properties, microtubules can play complex variety of biological functions. Experimental studies have shown that mechanical characteristics of microtubules are length-dependent and anisotropic, however, the origin of this feature has not been traced down to the level of tubulin sequence yet. Here we use high-resolution elastic network model to show local deformations patterns in microtubule structure up to the level of individual amino acid residues. The mechanical strain within the molecular structure of a microtubule is, according to our results, localized to interdimer contacts with energetic preference of deformation in longitudinal contacts. These findings are in agreement with reported mechanical anisotropy of microtubules, i.e. higher Young’s modulus in axial direction compared to radial direction. Our results contribute to understanding the origin of deformability of microtubules on the molecular level, which is important for prospective targeting of microtubules in medical therapeutic strategies. 664-Pos Board B444 Effects of Lattice Segmentation on Microtubule Mechanics Scott A. Erickson1, Naoto Isozaki2, Jennifer Ross3, Taviare Hawkins1. 1 Department of Physics, University of Wisconsin - La Crosse, La Crosse, WI, USA, 2Department of Microengineering, Kyoto University, Kyoto, Japan, 3 Department of Physics, University of Massachusetts - Amherst, Amherst, MA, USA. Multiple cell functions including motility, intracellular transport, and mitosis depend on microtubules. Particularly during mitosis, microtubules must be long and rigid, a quality characterized by persistence length (Lp). Our previous work has shown that the stabilizer GMPCPP increases rigidity (Lp = 1.85 5 0.5 mm) over Taxol alone (Lp = 0.65 5 0.1 mm). When under the influence of both GMPCPP and Taxol, the effect of GMPCPP dominates and the microtubule is more rigid (Lp = 1.95 5 0.7 mm). We investigate microtubules with variable stiffness along their length made from GMPCPP and Taxol segments. Segmented microtubules are polymerized by growing Taxol segments from a GMPCPP seed or annealing segments after each are polymerized separately. Annealing creates defects in the lattice structure, while growth from a seed reduces defects. Two experimental techniques are used to determine persistence length of: (1) individual, freely fluctuating microtubules and (2) microtubules with one fixed end. The use of two methods allows for a more accurate interpretation of the effects of segmentation on microtubule rigidity. We report the initial findings of this study. 665-Pos Board B445 Is Microtubule Rigidity Proportional to Protofilament Number? Brandon J. Harris. Physics, University of Wisconsin-La Crosse, La Crosse, WI, USA. Microtubules are cytoskeletal filaments that participate in key cellular processes by providing structural support, intracellular transportation, and cell division. Despite having been measured for the past 20 years, there are still open questions regarding the mechanical stiffness of microtubules and their persistence length, Lp. The persistence length is a measure of stiffness, proportional to the flexural rigidity, EI, defined as the elastic modulus and the second moment of area. In vitro, GMPCPP (a slowly hydrolyzable form of GTP) microtubules produce 14 protofilaments, taxol-stabilized microtubules produce 12-13 protofilaments, and microtubules polymerized in high-sodium (580 mM NaCl) concentration produce 9-10 protofilaments and lattice shifts resulting in numerous ‘‘seams.’’ Persistence lengths of single microtubule filaments were measured. Microtubules were formed in respective buffers and stabilized with the chemotherapeutic drug Taxol. After formation, microtubules were confined to oscillate within a thin, 2-D chamber (% 3 mm). Fourier mode analysis was used to fit images of fluorescently labeled microtubules. From each individual fit, a respective persistence length was determined. Bootstrapping statistics was then applied to produce a more accurate measure of filament rigidity. Our prior measurements for GMPCPP microtubules have lognormal distributions with average Lp of 1.8 5 0.5 mm and 1.9 5 0.7 mm (with taxol) respectively. Similarly, Taxol stabilized microtubules have a lognormal distribution but with a smaller Lp of 0.65 5 0.1 mm. Preliminary results show high-sodium polymerized microtubules with an Lp of 0.67 5 0.1 mm.
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663-Pos Board B443 Deformability of Microtubules: An Atomistic Computational Study Ondrej Kucera1, Daniel Havelka1, Marco A. Deriu2, Michal Cifra1. 1 Institute of Photonics and Electronics ASCR, Prague, Czech Republic, 2 University of Applied Sciences and Arts of Southern Switzerland, Manno, Switzerland. Thanks to their material properties, microtubules can play complex variety of biological functions. Experimental studies have shown that mechanical characteristics of microtubules are length-dependent and anisotropic, however, the origin of this feature has not been traced down to the level of tubulin sequence yet. Here we use high-resolution elastic network model to show local deformations patterns in microtubule structure up to the level of individual amino acid residues. The mechanical strain within the molecular structure of a microtubule is, according to our results, localized to interdimer contacts with energetic preference of deformation in longitudinal contacts. These findings are in agreement with reported mechanical anisotropy of microtubules, i.e. higher Young’s modulus in axial direction compared to radial direction. Our results contribute to understanding the origin of deformability of microtubules on the molecular level, which is important for prospective targeting of microtubules in medical therapeutic strategies. 664-Pos Board B444 Effects of Lattice Segmentation on Microtubule Mechanics Scott A. Erickson1, Naoto Isozaki2, Jennifer Ross3, Taviare Hawkins1. 1 Department of Physics, University of Wisconsin - La Crosse, La Crosse, WI, USA, 2Department of Microengineering, Kyoto University, Kyoto, Japan, 3 Department of Physics, University of Massachusetts - Amherst, Amherst, MA, USA. Multiple cell functions including motility, intracellular transport, and mitosis depend on microtubules. Particularly during mitosis, microtubules must be long and rigid, a quality characterized by persistence length (Lp). Our previous work has shown that the stabilizer GMPCPP increases rigidity (Lp = 1.85 5 0.5 mm) over Taxol alone (Lp = 0.65 5 0.1 mm). When under the influence of both GMPCPP and Taxol, the effect of GMPCPP dominates and the microtubule is more rigid (Lp = 1.95 5 0.7 mm). We investigate microtubules with variable stiffness along their length made from GMPCPP and Taxol segments. Segmented microtubules are polymerized by growing Taxol segments from a GMPCPP seed or annealing segments after each are polymerized separately. Annealing creates defects in the lattice structure, while growth from a seed reduces defects. Two experimental techniques are used to determine persistence length of: (1) individual, freely fluctuating microtubules and (2) microtubules with one fixed end. The use of two methods allows for a more accurate interpretation of the effects of segmentation on microtubule rigidity. We report the initial findings of this study. 665-Pos Board B445 Is Microtubule Rigidity Proportional to Protofilament Number? Brandon J. Harris. Physics, University of Wisconsin-La Crosse, La Crosse, WI, USA. Microtubules are cytoskeletal filaments that participate in key cellular processes by providing structural support, intracellular transportation, and cell division. Despite having been measured for the past 20 years, there are still open questions regarding the mechanical stiffness of microtubules and their persistence length, Lp. The persistence length is a measure of stiffness, proportional to the flexural rigidity, EI, defined as the elastic modulus and the second moment of area. In vitro, GMPCPP (a slowly hydrolyzable form of GTP) microtubules produce 14 protofilaments, taxol-stabilized microtubules produce 12-13 protofilaments, and microtubules polymerized in high-sodium (580 mM NaCl) concentration produce 9-10 protofilaments and lattice shifts resulting in numerous ‘‘seams.’’ Persistence lengths of single microtubule filaments were measured. Microtubules were formed in respective buffers and stabilized with the chemotherapeutic drug Taxol. After formation, microtubules were confined to oscillate within a thin, 2-D chamber (% 3 mm). Fourier mode analysis was used to fit images of fluorescently labeled microtubules. From each individual fit, a respective persistence length was determined. Bootstrapping statistics was then applied to produce a more accurate measure of filament rigidity. Our prior measurements for GMPCPP microtubules have lognormal distributions with average Lp of 1.8 5 0.5 mm and 1.9 5 0.7 mm (with taxol) respectively. Similarly, Taxol stabilized microtubules have a lognormal distribution but with a smaller Lp of 0.65 5 0.1 mm. Preliminary results show high-sodium polymerized microtubules with an Lp of 0.67 5 0.1 mm.