Name: Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops (Video) | JoVE Journal
Uploaded: 2025-07-11T16:50:00.000+00:00
Duration: 6 min 48 s
Description: Learn to prepare quasi-two-dimensional actin assemblies for studying soft biological materials and contractility mechanisms. This protocol improves reproducibility in protein assembly experiments.

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5 minutes

We thank Todd Thorenson, Mike Murrell, Jennifer Ross, Patrick McCall, and other members of the Gardel and Kovar labs (University of Chicago) for useful discussions while developing the methods. M.A.C. and S.R. were partially supported by Clemson University&#39;s College of Engineering Computing and Applied Sciences Undergraduate Research Opportunity Grants, and V. J. A. was supported by the Clemson Biophysics REU under NSF Award #2349368 with funding from the DBI and EPSCoR programs. This work was also supported in part by the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740, in part by and in part by the Clemson Creative Inquiry + Undergraduate Research program.

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The authors have no conflicts to disclose.

There are many considerations to producing reproducible actin assemblies. One of the most critical to producing reproducible, analyzable data is the coverglass surface of the sample chamber. The proteins in in vitro actin samples, particularly myosin, are extremely sticky and will adhere to untreated or poorly treated glass surfaces, rendering a sample that is unusable (Figure 5A). We have discussed a standard way of prepping the surface to yield reproducible samples through passiva...

<a href="https://app.jove.com/t/6643">This corrects</a> the article <a href="https://dx.doi.org/10.3791/68127">10.3791/68127</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/68127">Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops</a>. The Affiliations&#160;section was&#160;updated.

Erratum: Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops

Active biological materials underly mechanical processes in a variety of physiological processes, including intracellular transport, cell migration, cell shape regulation, and biological force generation1,2,3. In vitro assemblies of cytoskeletal systems, constructed from purified and engineered protein components self-assembled in buffer, are an established tool for characterizing fundamental biophysical and biochemical processes4,5,6,7. These simplified model systems allow proteins to be studied without the complexity of cellular environments, allowing the roles of individual components to be identified. In addition to supporting fundamental biological studies, in vitro cytoskeletal assemblies are also widely developed as experimental systems to investigate liquid crystalline and active or out-of-equilibrium materials8,9,10,11,12,13,14,15.
Actin is a key biopolymer in cytoskeletal assemblies regulating cell mechanics and dynamics through motor and polymerization-driven forces3,16. Fluorescence microscopy experiments allow visualization of actin network structural changes during processes such as motor-driven contraction and actin bundling by cross-linking proteins. Imaging three-dimensional actin networks as they are remodeled by embedded motor proteins has illuminated the role of actin crosslinkers for network contraction and pattern formation12,17,18,19. However, quasi-two-dimensional actin networks overcome challenges in imaging with sufficient spatiotemporal resolution to capture network structural changes. Additionally, high-density, quasi-two-dimensional actin structures are a closer mimic of cellular assemblies such as the dense actin cortex of a cell20. Strategies to produce quasi-two-dimensional actin structures have included attachment of actin nucleating proteins to a coverslip21,22, coupling of actin to a surface through linker molecules10,23,24, formation of dense actin bundles attached to beads at a coverslip surface25,26, and concentration of actin filaments at a coverslip surface with molecular crowding agents10,27.
Here, we detail a method for creating reproducible model actin-based assemblies and how to modify it to prepare variations from active networks to quasi-2D liquid crystals and nematic droplets. We have previously used similar methods to study the formation of phase-separated liquid crystalline droplets of short actin filaments with crosslinker addition11, changes in myosin II-generated actin network deformation modes (e.g., filament buckling or relative sliding) with filament cross-linking and connectivity28, differences in myosin II-generated forces on actin bundles with varying spacing and compliance29, and myosin II transport30. The use of cross-linking proteins and actin capping proteins allows for systematic variation of actin filament length and assembly architecture.

<table><tbody><tr><td>0.6 mL microcentrifuge tube</td><td>Fisher Scientific</td><td>05-408-123</td><td></td></tr><tr><td>008-Fluorosurfactant</td><td>RAN Biotechnologies</td><td>00115081361-6525</td><td>Alternate to premixed solution</td></tr><tr><td>008-FluoroSurfactant in HFE7500</td><td>RAN Biotechnologies</td><td>008-FluoroSurfactant-2wtH-50G</td><td>Premixed solution</td></tr><tr><td>2-mercaptoethanol (&amp;beta;-mercaptoethanol)</td><td>Sigma Aldrich</td><td>63689</td><td>CAS Number: 60-24-2&lt;br/&gt; Stock solution: 25 mM in MilliQ water, stored at 4 &amp;deg;C</td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES)</td><td>Sigma Aldrich</td><td>H3375</td><td>CAS Number: 7365-45-9</td></tr><tr><td>5 min Epoxy</td><td>Devcon</td><td>14250</td><td></td></tr><tr><td>Actin</td><td>Cytoskeleton, Inc.</td><td>AKL99-A</td><td>&gt;99% pure rabbit skeletal muscle&lt;br/&gt; (Alternate to purifying)</td></tr><tr><td>Actin (rhodamine labeled)</td><td>Cytoskeleton, Inc.</td><td>AR05-A</td><td>Rabbit skeletal muscle&lt;br/&gt; (Alternate to purifying)</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate (ATP)</td><td>Sigma Aldrich</td><td>A6419</td><td>CAS Number: 34369-07-8&lt;br/&gt; Stock solution: 25 mM in MilliQ water, store at -20 &amp;deg;C&lt;br/&gt; Keep frozen to avoid hydrolysis</td></tr><tr><td>Calcium Chloride (CaCl2)</td><td>Sigma Aldrich</td><td>C5670</td><td>CAS Number: 10043-52-4</td></tr><tr><td>Capping Protein (Mouse)</td><td></td><td></td><td>Purified from overexpression in &lt;em&gt;E.coli &lt;/em&gt;with a HisTag&lt;br/&gt; Stock solution: 20 mM in capping protein buffer at -80 &amp;deg;C</td></tr><tr><td>Catalase from bovine liver</td><td>Sigma Aldrich</td><td>C9322</td><td>CAS Number: 9001-05-2&lt;br/&gt; Stock solution: 85 ku/mL, store aliquoted with glucose oxidase at -20&amp;deg;C</td></tr><tr><td>Cover Glass</td><td>Fisherbrand</td><td>12544C</td><td>Borosilicate, #1.5, 24 mm x 40 mm&lt;br/&gt; Fisherbrand not Fisher Finest</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G8270</td><td>CAS Number: 50-99-7&lt;br/&gt; Stock solution: 225 mg/mL in MilliQ water, store at -20 &amp;deg;C</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma Aldrich</td><td>3860-OP</td><td>CAS Number: 3483-12-3&lt;br/&gt; Stock solution: 1 M in MilliQ water, store at -20 &amp;deg;C</td></tr><tr><td>Double Sided Tape</td><td>3M</td><td>3136</td><td></td></tr><tr><td>Ethyl alchohol 200 Proof</td><td>PHARMCO</td><td>111000200</td><td>CAS Number: 64-17-5&lt;br/&gt; 200 proof</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Sigma Aldrich</td><td>E3889</td><td>CAS Number: 67-42-5</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>E9884</td><td>CAS Number: 60-00-4</td></tr><tr><td>Glucose Oxidase</td><td>Sigma Aldrich</td><td>345386</td><td>CAS Number: 9001-37-0&lt;br/&gt; Stock solution: 135 mg/mL in MilliQ water, store aliquoted with catalase at -20 &amp;deg;C</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>356352M</td><td>CAS Number: 56-81-5</td></tr><tr><td>Imidazole</td><td>Fisher Bioreagents&amp;nbsp;</td><td>BP305-50</td><td>CAS Number: 288-32-4</td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Bioreagents&amp;nbsp;</td><td>BP214</td><td>CAS Number: 7786-30-3, 7791-18-6</td></tr><tr><td>Methylcellulose</td><td>Sigma Aldrich</td><td>M0512</td><td>CAS Number: 9004-67-5&lt;br/&gt; 15 centipoise</td></tr><tr><td>Microscope Slides Plain</td><td>Electron Microscopy Sciences/Gold Seal</td><td>63710-05</td><td>&amp;nbsp;3&quot;x1&quot;, 1 mm thick</td></tr><tr><td>MilliQ water</td><td>Millipore</td><td>CUFBI001</td><td>Ultrapure water</td></tr><tr><td>Novec-7500 Engineered Fluid</td><td>3M</td><td>7100134816</td><td>Alternate to premixed solution</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma Aldrich</td><td>P3911</td><td>CAS Number: 7447-40-7</td></tr><tr><td>Potassium Phosphate (KPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>P3786</td><td>CAS Number: 7758-11-4</td></tr><tr><td>Rabbit Skeletal Muscle Acetone Powder</td><td>Pel-Freez Biologicals</td><td>41995-1</td><td>Used when purifying actin (alternate to purchasing)&lt;br/&gt; Store around 30 mM in actin Buffer G at -80 &amp;deg;C</td></tr><tr><td>Sodium Azide</td><td>Sigma Aldrich</td><td>71289</td><td>CAS Number: 26626-22-8</td></tr><tr><td>Tetramethylrhodamine-C6-maleimide</td><td>AnaSpec</td><td>AS-81445</td><td>CAS Number: 174568-68-4 Used when purifying actin (alternate to purchasing)&lt;br/&gt; Store around 30 mM in actin Buffer G at -80 &amp;deg;C. Used when labeling actin (alternate to purchasing)</td></tr><tr><td>Tris Hydrochloric Acid (Tris HCl)</td><td>Sigma Aldrich</td><td>648317</td><td>CAS Number: 1185-53-1</td></tr><tr><td>Ultrasonic Bath</td><td>Emerson Branson&amp;nbsp;</td><td>CPX2800H</td><td></td></tr></tbody></table>

tuning the contractility and deformation modes of active actin-based assemblies in vitro: from two-dimensional active networks to liquid crystal drops

Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops

Actin cytoskeleton-based materials are widely investigated as model cellular materials to elucidate physical mechanisms of cell mechanics, such as shape regulation and force production, as well as intriguing soft polymeric materials. In this method, we detail creating actin-based assemblies in vitro using purified protein for fluorescence microscopy studies. We polymerize long actin filaments in a sample chamber and use a polymer depletant to crowd filaments into a two-dimensional (2D)-entangled network against a surface passivated with a surfactant layer. Adding skeletal muscle myosin II filaments in the presence of adenosine triphosphate (ATP) induces contraction of the actin network. By bundling actin filaments with a crosslinker, we tune the contractility of the assembly, transitioning from a material that buckles to a material that slides at the microscale. By reducing the length of the actin filaments through co-polymerizing actin in the presence of capping protein, we tune the material from being a 2D network to a liquid crystal. Cross-linking of dispersed short actin filaments results in three-dimensional (3D) liquid crystal droplet formation.

A broad range of actin assemblies can be formed in model systems using purified proteins by following the general strategy described in these methods, where actin is polymerized into filaments in the presence of accessory proteins that modify the assembly architecture (Figure 1). When actin is polymerized into filaments without crosslinkers or capping protein, it forms entangled filament networks (Figure 1A). Adding crosslinker to the entangled networks results ...

Here, we present methods for preparing quasi-two-dimensional (2D) entangled, cross-linked, and liquid crystal actin assemblies from purified proteins.

Actin cytoskeleton-based materials are widely investigated as model cellular materials to elucidate physical mechanisms of cell mechanics, such as shape ...

tuning-contractility-deformation-modes-active-actin-based-assemblies

Research

JoVE Journal

Biology

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

Here, we fabricate 3D polymeric micro/nano structures in which both the shape and the molecular alignment can be engineered with nanometer scale accuracy by the use of direct laser writing. Light induced deformation of several types of liquid crystalline elastomer microstructures can be controlled in the microscopic scale.

Free-form Light Actuators &#8212; Fabrication and Control of Actuation in Microscopic Scale

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

The goal of the protocol is to create liquid crystalline polymer films that can mechanically oscillate under continuous light irradiation. We describe in great detail the conception of free-standing films, from the liquid crystal alignment method to the photo-actuation. The experimental protocol applied to prepare this material is broadly applicable.

Preparation of Liquid Crystal Networks for Macroscopic Oscillatory Motion Induced by Light

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites 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.

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

Presented here are methods for preparing active nematics from microtubules and kinesin motors, including protein preparation and construction and the use of wells for active nematic confinement.

Forming, Confining, and Observing Microtubule-Based Active Nematics

In this work, an in vitro reconstitution approach is employed to study the poroelasticity of actomyosin gels under controlled conditions. The dynamics of the actomyosin gel and the embedded solvent are quantified, through which the network poroelasticity is demonstrated. We also discuss the experimental challenges, common pitfalls, and relevance to cell cytoskeleton mechanics.

The Mechanics of (Poro-)Elastic Contractile Actomyosin Networks As a Model System of the Cell Cytoskeleton

Learn to prepare quasi-two-dimensional actin assemblies for studying soft biological materials and contractility mechanisms. This protocol improves reproducibility in protein assembly experiments.

Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops (Video) | JoVE Journal

Tuning the Contractility and Deformation Modes of Active Actin-Based Assemblies In Vitro: From Two-Dimensional Active Networks to Liquid Crystal Drops

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