reconstituting-characterizing-actin-microtubule-composites-with

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 ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Syracuse University

Liquid crystals (LCs) are easily influenced by external interactions, particularly at interfaces. When rod-like LC molecules are confined to spherical droplets, they experience a competition between interfacial tension and elastic deformations. The configuration of LCs inside a droplet can be controlled using surfactants that influence the interfacial orientation of the LC molecules in the oil-phase of an oil in water emulsion. Here, we used the surfactant sodium dodecyl sulfate (SDS) to manipulate the orientation of 5CB molecules in a polydisperse emulsion and examined the configuration of the droplets as a function of SDS concentration. We triggered pronounced morphological transitions by altering the SDS concentration while observing an individual LC droplet held in place using an optical tweezer. We compared the experimental configuration changes to predictions from simulations. We observed a hysteresis in the SDS concentration that induced the morphological transition from radial to bipolar and back as well as a fluctuations in the configuration during the transition.

Direct Observation of Liquid Crystal Droplet Configurational Transitions using Optical Tweezers.

Microtubule (MT)-associated proteins regulate the dynamic behavior of MTs during cellular processes. MT severing enzymes are the associated proteins which destabilize MTs by removing subunits from the lattice. One model for how severing enzymes remove tubulin dimers from the MT lattice is by unfolding its subunits through pulling on the carboxy-terminal tails of tubulin dimers. This model stems from the fact that severing enzymes are AAA+ unfoldases. To test this mechanism, we apply pulling forces on the carboxy-terminal regions of MT subunits using coarse grained molecular simulations. In our simulations, we used different MT lattices and concentrations of severing enzymes. We compare our simulation results with data from in vitro severing assays and find that the experimental data is best fit by a model of cooperative removal of protofilament fragments by severing enzymes, which depends on the severing enzyme concentration and placement on the MT lattice.

Molecular investigations into the unfoldase action of severing enzymes on microtubules.

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.

Microtubules are an essential physical building block of cellular systems. They are organized using specific crosslinkers, motors, and influencers of nucleation and growth. With the addition of antiparallel crosslinkers, microtubule self-organization patterns go through a transition from fanlike structures to homogeneous tactoid condensates in vitro. Tactoids are reminiscent of biological mitotic spindles, the cell division machinery. To create these organizations, we previously used polymer crowding agents. Here we study how altering the properties of the crowders, such as type, size, and molecular weight, affects microtubule organization. Comparing simulations with experiments, we observe a scaling law associated with the fanlike patterns in the absence of crosslinkers. Tactoids formed in the presence of crosslinkers show variable length, depending on the crowders. We correlate the subtle differences to filament contour length changes, affected by nucleation and growth rate changes induced by the polymers in solution. Using quantitative image analysis, we deduce that the tactoids differ from traditional liquid crystal organization, as they are limited in width irrespective of crowders and surfaces, and behave as solidlike condensates.

Crowder and surface effects on self-organization of microtubules.

The dynamic morphology and mechanics of the cytoskeleton is determined by interacting networks of semiflexible actin filaments and rigid microtubules. Active rearrangement of networks of actin and microtubules can not only be driven by motor proteins but by changes to ionic conditions. For example, high concentrations of multivalent ions can induce bundling and crosslinking of both filaments. Yet, how cytoskeleton networks respond in real-time to changing ion concentrations, and how actin-microtubule interactions impact network response to these changing conditions remains unknown. Here, we use microfluidic perfusion chambers and two-color confocal fluorescence microscopy to show that increasing magnesium ions trigger contraction of both actin and actin-microtubule networks. Specifically, we use microfluidics to vary the Mg concentration between 2 and 20 mM while simultaneously visualizing the triggered changes to the overall network size. We find that as Mg concentration increases both actin and actin-microtubule networks undergo bulk contraction, which we measure as the shrinking width of each network. However, surprisingly, lowering the Mgconcentration back to 2 mM does not stop or reverse the contraction but rather causes both networks to contract further. Further, actin networks begin to contract at lower Mg concentrations and shorter times than actin-microtubule networks. In fact, actin-microtubule networks only undergo substantial contraction once the Mg concentration begins to lower from 20 mM back to 2 mM. Our intriguing findings shed new light on how varying environmental conditions can dynamically tune the morphology of cytoskeleton networks and trigger active contraction without the use of motor proteins.

Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics.

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.

Active cytoskeletal composites display emergent tunable contractility and restructuring.

Liquid crystals are able to transform a local molecular interaction into a macroscopic change of state, making them a valuable "smart" material. Here, we investigate a novel polymeric amphiphile as a candidate for molecular triggering of liquid crystal droplets in aqueous background. Using microscopy equipped with crossed polarizers and optical tweezers, we find that the monomeric amphiphile is able to trigger both a fast phase change and then a subsequent transition from nematic to isotropic. We next include sodium dodecyl sulfate (SDS), a standard surfactant, with the novel amphiphilic molecules to test phase transitioning when both were present. As seen previously, we find that the activity of SDS at the surface can result in configuration changes with hysteresis. We find that the presence of the polymeric amphiphile reverses the hysteresis previously observed during such transitions. This work demonstrates a variety of phase and configuration changes of liquid crystals that can be controlled by multiple exogenous chemical triggers.

Controlling Liquid Crystal Configuration and Phase Using Multiple Molecular Triggers.

The composite cytoskeleton, comprising interacting networks of semiflexible actin and rigid microtubules, generates forces and restructures by using motor proteins such as myosins to enable key processes including cell motility and mitosis. Yet, how motor-driven activity alters the mechanics of cytoskeleton composites remains an open challenge. Here, we perform optical tweezers microrheology and confocal imaging of composites with varying actin-tubulin molar percentages (25-75, 50-50, and 75-25), driven by light-activated myosin II motors, to show that motor activity increases the elastic plateau modulus by over 2 orders of magnitude by active restructuring of both actin and microtubules that persists for hours after motor activation has ceased. Nonlinear microrheology measurements show that motor-driven restructuring increases the force response and stiffness and suppresses actin bending. The 50-50 composite exhibits the most dramatic mechanical response to motor activity due to the synergistic effects of added stiffness from the microtubules and sufficient motor substrate for pronounced activity.

ArticleTotal : 2	Year
Publication title Cited by 1	2022
Publication title Cited by 1	2022

Article	Year
Direct Observation of Liquid Crystal Droplet Configurational Transitions using Optical Tweezers. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 31990557	2020
Molecular investigations into the unfoldase action of severing enzymes on microtubules. Cytoskeleton (Hoboken, N.J.)\| PubMed ID: 32170815	2020
Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics. Science advances\| PubMed ID: 33547082	2021
Crowder and surface effects on self-organization of microtubules. Physical review. E\| PubMed ID: 34271669	2021
Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics. Frontiers in physics\| PubMed ID: 34368112	2020
Active cytoskeletal composites display emergent tunable contractility and restructuring. Soft matter\| PubMed ID: 34792082	2021
Controlling Liquid Crystal Configuration and Phase Using Multiple Molecular Triggers. Molecules (Basel, Switzerland)\| PubMed ID: 35164141	2022
Motor-Driven Restructuring of Cytoskeleton Composites Leads to Tunable Time-Varying Elasticity. ACS macro letters\| PubMed ID: 35549081	2021

Jennifer L. Ross

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