Understanding the role of transport velocity in biomotor-powered microtubule spool assembly

Tan, Amanda; Chapman, Dail; Hirst, Linda S.; Xu, Jing

doi:10.1039/c6ra19094d

Cited by 3 publications

(8 citation statements)

References 30 publications

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“…Because ATP concentration controls the kinesin step-rate at the molecular level, we expect the topological entropy and Lyapunov exponent to increase with increasing ATP concentration. When single microtubules glide on a kinesin-decorated glass surface, their gliding velocity as a function of ATP concentration is generally well described on a local level by Michaelis-Menten kinetics [42][43][44]. Similarly, we observed that the average v rms (Methods: PIV velocity analysis) for our attached beads also follows Michaelis-Menten kinetics under ATP variation, Fig.…”

Section: Bi-v)supporting

confidence: 67%

Topological chaos in active nematics

et al. 2019

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Active nematics are out-of-equilibrium fluids composed of rod-like subunits, which can generate large-scale, self-driven flows. We examine a microtubule-kinesin-based active nematic confined to two-dimensions, exhibiting chaotic flows with moving topological defects. Applying tools from chaos theory, we investigate self-driven advection and mixing on different length scales. Local fluid stretching is quantified by the Lyapunov exponent. Global mixing is quantified by the topological entropy, calculated from both defect braiding and curve extension rates. We find excellent agreement between these independent measures of chaos, demonstrating that the extensile stretching between microtubules directly translates into macroscopic braiding of positive defects. Remarkably, increasing extensile activity (via ATP concentration) does not increase the dimensionless topological entropy. This study represents the first application of chaotic advection to the emerging field of active nematics and the first time that the collective motion of an ensemble of defects has been quantified (via topological entropy) in a liquid crystal.

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Section: Bi-v)supporting

confidence: 67%

Topological chaos in active nematics

et al. 2019

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“…The tubulin solution was then incubated in a 37 °C bath for 12 hours to allow for polymerization after which microtubules are then stored at room temperature in a dark box. Recombinant penta-histidine-tagged kinesin protein was purified from e coli as previously described 19,20 . All other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Flow cells 19 for the experiments are constructed by placing two thin strips of tape on a glass slide. Glass was sonicated in Acetone, Methanol, Ethanol, and Nanopure water for 1 hour each and dried with N 2 gas.…”

Section: Methodsmentioning

confidence: 99%

“…Microtubules were then given 10 minutes to adhere to the remaining motors bound on surface. Lastly a motility mix (PEM80 supplemented with 1 mM ATP, 1 mM DTT, 10 uM taxol, 0.22 mg/ml glucose oxidase, 0.04 mg/ml catalase, 3.68 mg/ml glucose, 2 mM phosphocreatine and 70 μg/mL creatine phosphokinase) 19 was added to provide a regenerative ATP source and reduce bleaching.…”

Section: Methodsmentioning

confidence: 99%

“…Applying the membrane to a flat substrate allows us to take advantage of more convenient imaging possibilities and the use of a quasi-infinite bath of motors. The experimental gliding assay can be used to easily investigate transport over large distances 19 , as opposed to bead-cargo methods 20,21 in which the motion of individual cargo-coupled beads is analyzed, but the surface of the bead cannot be imaged. Since biological kinesin cargos (e.g.…”

Section: Introductionmentioning

confidence: 99%

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Membrane mediated motor kinetics in microtubule gliding assays

Lopes

Quint

Chapman

et al. 2019

Sci Rep

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Motor-based transport mechanisms are critical for a wide range of eukaryotic cell functions, including the transport of vesicle cargos over long distances. Our understanding of the factors that control and regulate motors when bound to a lipid substrate is however incomplete. We used microtubule gliding assays on a lipid bilayer substrate to investigate the role of membrane diffusion in kinesin-1 on/off binding kinetics and thereby transport velocity. Fluorescence imaging experiments demonstrate motor clustering on single microtubules due to membrane diffusion in the absence of ATP, followed by rapid ATP-induced dissociation during gliding. Our experimental data combined with analytical modeling show that the on/off binding kinetics of the motors are impacted by diffusion and, as a consequence, both the effective binding and unbinding rates for motors are much lower than the expected bare rates. Our results suggest that motor diffusion in the membrane can play a significant role in transport by impacting motor kinetics and can therefore function as a regulator of intracellular transport dynamics.

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Active nematic order and dynamic lane formation of microtubules driven by membrane-bound diffusing motors

Memarian

Lopes

Schwarzendahl

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Dynamic lane formation and long-range active nematic alignment are reported using a geometry in which kinesin motors are directly coupled to a lipid bilayer, allowing for in-plane motor diffusion during microtubule gliding. We use fluorescence microscopy to image protein distributions in and below the dense two-dimensional microtubule layer, revealing evidence of diffusion-enabled kinesin restructuring within the fluid membrane substrate as microtubules collectively glide above. We find that the lipid membrane acts to promote filament–filament alignment within the gliding layer, enhancing the formation of a globally aligned active nematic state. We also report the emergence of an intermediate, locally ordered state in which apolar dynamic lanes of nematically aligned microtubules migrate across the substrate. To understand this emergent behavior, we implement a continuum model obtained from coarse graining a collection of self-propelled rods, with propulsion set by the local motor kinetics. Tuning the microtubule and kinesin concentrations as well as active propulsion in these simulations reveals that increasing motor activity promotes dynamic nematic lane formation. Simulations and experiments show that, following fluid bilayer substrate mediated spatial motor restructuring, the total motor concentration becomes enriched below the microtubule lanes that they drive, with the feedback leading to more dynamic lanes. Our results have implications for membrane-coupled active nematics in vivo as well as for engineering dynamic and reconfigurable materials where the structural elements and power sources can dynamically colocalize, enabling efficient mechanical work.

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Understanding the role of transport velocity in biomotor-powered microtubule spool assembly

Cited by 3 publications

References 30 publications

Topological chaos in active nematics

Topological chaos in active nematics

Membrane mediated motor kinetics in microtubule gliding assays

Active nematic order and dynamic lane formation of microtubules driven by membrane-bound diffusing motors

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