scholarly journals Microtubules form by progressively faster tubulin accretion, not by nucleation–elongation

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Luke M. Rice ◽  
Michelle Moritz ◽  
David A. Agard
Keyword(s):  
Conformational Transition ◽  
Energetic Cost ◽  
Actin Assembly ◽  
Microtubule Nucleation ◽  
Polymer Formation ◽  
Experimental Assembly ◽  
Microtubule Formation ◽  
Key Features ◽  
Complex Polymers ◽  
Assembly Pathways

Microtubules are dynamic polymers that play fundamental roles in all eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks have focused on variations of a nucleation–elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer then elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but they fail to account for how more complex polymers like hollow microtubules assemble. Here, we articulate a new model for microtubule formation that has three key features: (1) microtubules initiate via rectangular, sheet-like structures that grow faster the larger they become; (2) the dominant pathway proceeds via accretion, the stepwise addition of longitudinal or lateral layers; and (3) a “straightening penalty” to account for the energetic cost of tubulin’s curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation.

scholarly journals Microtubules form by progressively faster tubulin accretion, not by nucleation-elongation

2019 ◽  
Author(s):  
Luke M. Rice ◽  
Michelle Moritz ◽  
David A. Agard
Keyword(s):  
Conformational Transition ◽  
Energetic Cost ◽  
Actin Assembly ◽  
Microtubule Nucleation ◽  
Polymer Formation ◽  
Experimental Assembly ◽  
Microtubule Formation ◽  
Key Features ◽  
Complex Polymers ◽  
Assembly Pathways

AbstractMicrotubules are dynamic polymers that play fundamental roles in all eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks have focused on variations of a nucleation-elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer then elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but they fail to account for how more complex polymers like hollow microtubules assemble. Here we articulate a new model for microtubule formation that has three key features: i) microtubules initiate via rectangular, sheet-like structures which grow faster the larger they become; ii) the dominant pathway proceeds via accretion, stepwise addition of longitudinal or lateral layers; iii) a ‘straightening penalty’ to account for the energetic cost of tubulin’s curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation.

scholarly journals Antidepressants are modifiers of lipid bilayer properties

2019 ◽  
Vol 151 (3) ◽  
pp. 342-356 ◽  
Author(s):  
Ruchi Kapoor ◽  
Thasin A. Peyear ◽  
Roger E. Koeppe ◽  
Olaf S. Andersen
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Conformational Changes ◽  
Partition Coefficients ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Conformational Transition ◽  
Hydrophobic Interactions ◽  
Energetic Cost ◽  
Titration Calorimetry ◽  
Channel Activity

The two major classes of antidepressants, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), inhibit neurotransmitter reuptake at synapses. They also have off-target effects on proteins other than neurotransmitter transporters, which may contribute to both desired changes in brain function and the development of side effects. Many proteins modulated by antidepressants are bilayer spanning and coupled to the bilayer through hydrophobic interactions such that the conformational changes underlying their function will perturb the surrounding lipid bilayer, with an energetic cost (ΔGdef) that varies with changes in bilayer properties. Here, we test whether changes in ΔGdef caused by amphiphilic antidepressants partitioning into the bilayer are sufficient to alter membrane protein function. Using gramicidin A (gA) channels to probe whether TCAs and SSRIs alter the bilayer contribution to the free energy difference for the gramicidin monomer⇔dimer equilibrium (representing a well-defined conformational transition), we find that antidepressants alter gA channel activity with varying potency and no stereospecificity but with different effects on bilayer elasticity and intrinsic curvature. Measuring the antidepressant partition coefficients using isothermal titration calorimetry (ITC) or cLogP shows that the bilayer-modifying potency is predicted quite well by the ITC-determined partition coefficients, and channel activity is doubled at an antidepressant/lipid mole ratio of 0.02–0.07. These results suggest a mechanism by which antidepressants could alter the function of diverse membrane proteins by partitioning into cell membranes and thereby altering the bilayer contribution to the energetics of membrane protein conformational changes.

scholarly journals Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

2021 ◽  
Author(s):  
Andrea Enrico Prota ◽  
Daniel Lucena-Agell ◽  
Yuntao Ma ◽  
Juan Estevez-Gallego ◽  
Carlos Roca ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Core Structure ◽  
Side Chain ◽  
Binding Modes ◽  
Chemotherapeutic Drug ◽  
First Line ◽  
Baccatin Iii ◽  
Microtubule Stabilization ◽  
The Core ◽  
Microtubule Formation

Paclitaxel (Taxol) is a first-line chemotherapeutic drug that promotes the curved to straight conformational transition of tubulin, an activation step that is necessary for microtubule formation. Crystallization of Taxol bound to tubulin has been long elusive. We found that baccatin III, the core structure of paclitaxel which lacks the C13 side chain, readily co-crystallizes with curved tubulin. Tailor-made taxanes with alternative side chains also co-crystallized, allowing us to investigate their binding modes. Interestingly, these Taxol derived compounds lost their microtubule stabilizing activity and cytotoxicity but kept their full microtubule binding affinity, and all induced lattice expansion upon binding. Additional nuclear magnetic resonance studies propose that Taxol binds to a small fraction of straight tubulin present in solution. Our results suggest a mode of action of Taxol, where the core structure is responsible for the interacting energy while the bulky hydrophobic C13 side chain enables binding selectively to straight tubulin and promotes stabilization.

scholarly journals LSPR Biosensing Approach for the Detection of Microtubule Nucleation

Sensors ◽  
2019 ◽  
Vol 19 (6) ◽  
pp. 1436 ◽  
Author(s):  
Keisuke Hasegawa ◽  
Otabek Nazarov ◽  
Evan Porter
Keyword(s):  
Gold Nanoparticles ◽  
Refractive Index ◽  
Theoretical Model ◽  
Density Measurement ◽  
Localized Surface Plasmon ◽  
Proof Of Concept ◽  
Microtubule Nucleation ◽  
Cellular Processes ◽  
Microtubule Formation ◽  
Local Refractive Index

Microtubules are dynamic protein filaments that are involved in a number of cellular processes. Here, we report the development of a novel localized surface plasmon resonance (LSPR) biosensing approach for investigating one aspect of microtubule dynamics that is not well understood, namely, nucleation. Using a modified Mie theory with radially variable refractive index, we construct a theoretical model to describe the optical response of gold nanoparticles when microtubules form around them. The model predicts that the extinction maximum wavelength is sensitive to a change in the local refractive index induced by microtubule nucleation within a few tens of nanometers from the nanoparticle surface, but insensitive to a change in the refractive index outside this region caused by microtubule elongation. As a proof of concept to demonstrate that LSPR can be used for detecting microtubule nucleation experimentally, we induce spontaneous microtubule formation around gold nanoparticles by immobilizing tubulin subunits on the nanoparticles. We find that, consistent with the theoretical model, there is a redshift in the extinction maximum wavelength upon the formation of short microtubules around the nanoparticles, but no significant change in maximum wavelength when the microtubules are elongated. We also perform kinetic experiments and demonstrate that the maximum wavelength is sensitive to the microtubule nuclei assembly even when microtubules are too small to be detected from an optical density measurement.

scholarly journals Computer modelling of human alpha 1-antitrypsin reactive site loop behaviour under mild conditions.

1996 ◽  
Vol 43 (3) ◽  
pp. 467-474 ◽  
Author(s):  
H Kołoczek ◽  
G Jezierski ◽  
M Pasenkiewicz-Gierula
Keyword(s):  
Molecular Modelling ◽  
Active Site ◽  
Conformational Transition ◽  
Computer Modelling ◽  
Point Mutations ◽  
Salt Bridge ◽  
Reactive Site ◽  
Mild Conditions ◽  
Polymer Formation ◽  
Alpha 1 Antitrypsin

Human alpha 1-antitrypsin (alpha 1-PI) is a member of the serpin superfamily of proteins. The reactive site loop (RSL) of the serpin binds to the active site of its target proteinase. Deficiency of alpha 1-antitrypsin is associated with a spontaneous conformational transition in the molecule which leads to a polymer formation. Mild conditions (1 M guanidinium.HCl), temperature and point mutations within the RSL are the factors that induce polymerisation. Initiation of this process has been associated with the disruption of a salt bridge Glu342-->Lys290. In this paper the interaction of guanidinium ion with Glu342 and Lys290 as well as the effect of this interaction on the mobility of RSL is studied by molecular modelling.

scholarly journals A role for a micron-scale supramolecular myosin array in adherens junction cytoskeletal assembly

2021 ◽  
Author(s):  
Hui-Chia Yu-Kemp ◽  
Rachel A. Szymanski ◽  
Nicole C. Gadda ◽  
Madeline L. Lillich ◽  
Mark Peifer
Keyword(s):  
Epithelial Cells ◽  
Cell Shape ◽  
Shape Change ◽  
Cell Junctions ◽  
Actin Assembly ◽  
Cell Shape Change ◽  
Micrometer Scale ◽  
Assembly Pathways ◽  
E Cadherin ◽  
Cell Cell

AbstractEpithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms used to build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized and highly organized actomyosin cytoskeleton at the zonula adherens, and combined genetic and pharmacological approaches with super-resolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micrometer-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, while associated actin filaments remained disorganized. This suggested these myosin arrays might bundle actin at mature junctions. Consistent with this, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization, and prevented actin bundling and polarization. These results suggest a novel mechanism by which myosin self-assembly helps drive actin organization to facilitate cell shape change.SummaryWe explored mechanisms epithelial cells use to assemble supramolecular actomyosin structures at E-Cadherin-based cell-cell junctions. Our data suggest individual actin assembly pathways are not essential. Instead, microscopy and pharmacological inhibition suggest micrometer-scale supramolecular myosin arrays help bundle actin at mature junctions.

scholarly journals Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells

2008 ◽  
Vol 19 (5) ◽  
pp. 1873-1882 ◽  
Author(s):  
Liliana Torosantucci ◽  
Maria De Luca ◽  
Giulia Guarguaglini ◽  
Patrizia Lavia ◽  
Francesca Degrassi
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Spindle Assembly ◽  
Microtubule Nucleation ◽  
Leptomycin B ◽  
Data Support ◽  
Assembly Pathways ◽  
Export Signal ◽  
In Cells

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores.

scholarly journals The cryo-EM structure of a γ-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Erik Zupa ◽  
Anjun Zheng ◽  
Annett Neuner ◽  
Martin Würtz ◽  
Peng Liu ◽  
...  
Keyword(s):  
De Novo ◽  
Relative Positioning ◽  
Microtubule Nucleation ◽  
Cryo Electron Microscopy ◽  
Small Complex ◽  
Ring Complex ◽  
Regulation Mechanisms ◽  
Microtubule Formation ◽  
Nucleation Activity ◽  
Higher Eukaryotes

Abstract The nucleation of microtubules from αβ-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.

scholarly journals Stress fibers are generated by two distinct actin assembly mechanisms in motile cells

2006 ◽  
Vol 173 (3) ◽  
pp. 383-394 ◽  
Author(s):  
Pirta Hotulainen ◽  
Pekka Lappalainen
Keyword(s):  
Actin Polymerization ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Actin Assembly ◽  
Dissociation Kinetics ◽  
Fiber Assembly ◽  
Motile Cells ◽  
Live Cell Microscopy ◽  
Assembly Pathways ◽  
Cell Microscopy

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)–driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association–dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.

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