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

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

University of San Diego

The interface between two fluids is roughened by thermally excited capillary waves. By using colloid-polymer systems which exhibit liquid-gas phase separation, the time and length scales of capillary waves become accessible to optical microscopy methods. Here, we study such a system using bright-field optical microscopy combined with a novel extension of differential dynamic microscopy. With differential dynamic microscopy, we analyze images in order to determine the decay time of interfacial fluctuations spanning wavevectors from 0.1 to 1 μm-1. We find capillary velocities on the order of 0.1 μm s-1 that depend on the sample composition in expected ways and that match values from the literature. This work demonstrates the first application of differential dynamic microscopy to the study of interfacial dynamics.

Measuring capillary wave dynamics using differential dynamic microscopy.

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking.

The cytoskeleton, a complex network of protein filaments and crosslinking proteins, dictates diverse cellular processes ranging from division to cargo transport. Yet, the role the cytoskeleton plays in the intracellular transport of DNA and other macromolecules remains poorly understood. Here, using single-molecule conformational tracking, we measure the transport and conformational dynamics of linear and relaxed circular (ring) DNA in composite networks of actin and microtubules with variable types of crosslinking. While both linear and ring DNA undergo anomalous, non-Gaussian, and non-ergodic subdiffusion, the detailed dynamics are controlled by both DNA topology (linear vs. ring) and crosslinking motif. Ring DNA swells, exhibiting heterogeneous subdiffusion controlled via threading by cytoskeleton filaments, while linear DNA compacts, exhibiting transport via caging and hopping. Importantly, while the crosslinking motif has little effect on ring DNA, linear DNA in networks with actin-microtubule crosslinking is significantly less ergodic and shows more heterogeneous transport than with actin-actin or microtubule-microtubule crosslinking.

Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

Correction for 'Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks' by Jonathan Garamella et al., Soft Matter, 2020, DOI: 10.1039/d0sm00544d.

Correction: Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks.

The cytoskeleton is a dynamic network of proteins, including actin, microtubules, and their associated motor proteins, that enables essential cellular processes such as motility, division, and growth. While actomyosin networks are extensively studied, how interactions between actin and microtubules, ubiquitous in the cytoskeleton, influence actomyosin activity remains an open question. Here, we create a network of co-entangled actin and microtubules driven by myosin II. We combine dynamic differential microscopy, particle image velocimetry, and particle tracking to show that both actin and microtubules undergo ballistic contraction with unexpectedly indistinguishable characteristics. This contractility is distinct from faster disordered motion and rupturing that active actin networks exhibit. Our results suggest that microtubules enable self-organized myosin-driven contraction by providing flexural rigidity and enhanced connectivity to actin networks. Beyond the immediate relevance to cytoskeletal dynamics, our results shed light on the design of active materials that can be precisely tuned by the network composition.

Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics.

Microcapsules allow for the controlled containment, transport, and release of cargoes ranging from pharmaceuticals to fragrances. Given the interest from a variety of industries in microcapsules and other core-shell structures, a multitude of fabrication strategies exist. Here, we report on a method relying on a mixture of temperature-responsive microgel particles, poly(-isopropylacrylamide) (pNIPAM), and a polymer which undergo fluid-fluid phase separation. At room temperature this mixture separates into colloid-rich (liquid) and colloid-poor (gas) fluids. By heating the sample above a critical temperature where the microgel particles shrink dramatically and develop a more deeply attractive interparticle potential, the droplets of the colloid-rich phase become gel-like. As the temperature is lowered back to room temperature, these droplets of gelled colloidal particles reliquefy and phase separation within the droplet occurs. This phase separation leads to colloid-poor droplets within the colloid-rich droplets surrounded by a continuous colloid-poor phase. The gas/liquid/gas all-aqueous double emulsion lasts only a few minutes before a majority of the inner droplets escape. However, the colloid-rich shell of the core-shell droplets can solidify with the addition of salt. That this method creates core-shell structures with a shell composed of stimuli-sensitive microgel colloidal particles using only aqueous components makes it attractive for encapsulating biological materials and making capsules that respond to changes in, for example, temperature, salt concentration, or pH.

Core-shell droplets and microcapsules formed through liquid-liquid phase separation of a colloid-polymer mixture.

The cytoskeleton is a model active matter system that controls processes as diverse as cell motility and mechanosensing. While both active actomyosin dynamics and actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay is lacking. Here, we couple microscale experiments with mechanistic modeling to elucidate how connectivity, rigidity, and force-generation affect emergent material properties in composite networks of actin, tubulin, and myosin. We use multi-spectral imaging, time-resolved differential dynamic microscopy and spatial image autocorrelation to show that ballistic contraction occurs in composites with sufficient flexibility and motor density, but that a critical fraction of microtubules is necessary to sustain controlled dynamics. The active double-network models we develop, which recapitulate our experimental findings, reveal that while percolated actomyosin networks are essential for contraction, only composites with comparable actin and microtubule densities can simultaneously resist mechanical stresses while supporting substantial restructuring. The comprehensive phase map we present not only provides important insight into the different routes the cytoskeleton can use to alter its dynamics and structure, but also serves as a much-needed blueprint for designing cytoskeleton-inspired materials that couple tunability with resilience and adaptability for diverse applications ranging from wound healing to soft robotics.

Article	Year
Measuring capillary wave dynamics using differential dynamic microscopy. Soft matter\| PubMed ID: 31465080	2019
Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking. Science advances\| PubMed ID: 31853502	2019
Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks. Soft matter\| PubMed ID: 32555863	2020
Correction: Anomalous and heterogeneous DNA transport in biomimetic cytoskeleton networks. Soft matter\| PubMed ID: 32639492	2020
Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics. Science advances\| PubMed ID: 33547082	2021
Core-shell droplets and microcapsules formed through liquid-liquid phase separation of a colloid-polymer mixture. Soft matter\| PubMed ID: 34550150	2021
Active cytoskeletal composites display emergent tunable contractility and restructuring. Soft matter\| PubMed ID: 34792082	2021

Ryan J. McGorty

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