Dataset Information

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

ABSTRACT: The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

SUBMITTER: Sasanpour M

PROVIDER: S-EPMC10290881 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

Sasanpour Mehrzad M Achiriloaie Daisy H DH Lee Gloria G Leech Gregor G Hendija Maya M Lindsay K Alice KA Ross Jennifer L JL McGorty Ryan J RJ Robertson-Anderson Rae M RM

Journal of visualized experiments : JoVE 20220825 186

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to eng ...[more]

PMID: 36094259

Similar Datasets

Project description:We use optical tweezers microrheology and fluorescence microscopy to characterize the nonlinear mesoscale mechanics and mobility of in vitro co-entangled actin-microtubule composites. We create a suite of randomly oriented, well-mixed networks of actin and microtubules by co-polymerizing varying ratios of actin and tubulin in situ. To perturb each composite far from equilibrium, we use optical tweezers to displace an embedded microsphere a distance greater than the lengths of the filaments at a speed much faster than their intrinsic relaxation rates. We simultaneously measure the force the filaments exert on the bead and the subsequent force relaxation. We find that the presence of a large fraction of microtubules (>0.7) is needed to substantially increase the measured force, which is accompanied by large heterogeneities in force response. Actin minimizes these heterogeneities by reducing the mesh size of the composites and supporting microtubules against buckling. Composites also undergo a sharp transition from strain softening to stiffening when the fraction of microtubules (ϕT) exceeds 0.5, which we show arises from faster poroelastic relaxation and suppressed actin bending fluctuations. The force after bead displacement relaxes via power-law decay after an initial period of minimal relaxation. The short-time relaxation profiles (t < 0.06 s) arise from poroelastic and bending contributions, whereas the long-time power-law relaxation is indicative of filaments reptating out of deformed entanglement constraints. The scaling exponents for the long-time relaxation exhibit a nonmonotonic dependence on ϕT, reaching a maximum for equimolar composites (ϕT = 0.5), suggesting that reptation is fastest in ϕT = 0.5 composites. Corresponding mobility measurements of steady-state actin and microtubules show that both filaments are indeed the most mobile in ϕT = 0.5 composites. This nonmonotonic dependence of mobility on ϕT demonstrates the important interplay between mesh size and filament rigidity in polymer networks and highlights the surprising emergent properties that can arise in composites.

Dataset Information

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

Publications

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets