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

Summary

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

Abstract

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.

Introduction

Active biological materials underly mechanical processes in a variety of physiological processes, including intracellular transport, cell migration, cell shape regulation, and biological force generation^1,2,3. 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 processes^4,5,6,7. 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 materials^{8,9,10,11,12,13,14,15}.

Actin is a key biopolymer in cytoskeletal assemblies regulating cell mechanics and dynamics through motor and polymerization-driven forces^3,16. 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 formation^12,17,18,19. 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 coverslip^21,22, coupling of actin to a surface through linker molecules^10,23,24, formation of dense actin bundles attached to beads at a coverslip surface^25,26, and concentration of actin filaments at a coverslip surface with molecular crowding agents^10,27.

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.

Protocol

NOTE: Proteins and labeling: For the purposes of this protocol, it is assumed that the researcher begins with stocks of purified proteins, which are fluorescently labeled when appropriate for fluorescence microscopy investigation. These proteins can be purchased or purified and fluorescently labeled in lab^{31,32,33,34,35,36,37}. In this protocol, stocks of proteins that have been frozen in liquid nitrogen and stored at -80 °C were used. The proteins typically can be labeled through similar methods. The protein can be labeled as a step of purification or from a frozen stock. Thaw frozen protein stocks on ice before proceeding with the labeling. The following is a method for fluorescently labeling skeletal muscle myosin II (myosin) with a maleimide-functionalized fluorophore (adapted from³⁸).

1. Prepare fluorescently-labeled myosin

1. Start with ~2 mL of myosin at a concentration of 5-10 mg/mL.
   NOTE: In this protocol, skeletal muscle myosin II purified from chicken muscle using published methods³⁵ were used. The myosin is stored in Myosin storage buffer (25 mM KPO₄, 0.6 M KCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), pH 6.6), either directly after purification or from frozen stock. It is important to keep the myosin cold at all times to prevent loss of motor activity.
2. Add DTT to the thawed myosin solution to a final concentration of 10 mM. Use a 1 M DTT stock solution to limit the volume.
   NOTE: The DTT reduces the thiol group on cysteines in the myosin protein, preparing them to react with the maleimide for labeling.
3. To prevent interference with the labeling reaction, remove the DTT by dialyzing overnight in 50 mM HEPES, 500 mM KCl, 1 mM EDTA, pH 7.6, at 4 °C.
4. After dialysis, centrifuge the myosin solution at 100,000 x g at 4 °C for 15 min to pellet any aggregates. Collect the supernatant.
5. Determine the concentration of myosin in the supernatant using a spectrophotometer. Estimate the myosin concentration using an extinction coefficient at 280 nm of ~148,000 M^-1cm^-1 for skeletal muscle myosin³⁸. This extinction coefficient assumes a "monomer" to consist of one heavy chain, one essential light chain, and one regulatory light chain.
6. Prepare fluorophore for labeling reaction by resuspending powdered fluorophore in dry dimethyl sulfoxide (DMSO) to a concentration of 5 mM fluorophore.
   NOTE: Any fluorophore with a maleimide reactive group should work. Alexa 647 maleimide was used. DMSO is hygroscopic and accumulates water. "Dry" DMSO refers to DMSO that has been stored in an environment to prevent water accumulation. To ensure the solvent is dry DMSO, a freshly opened ampule of DMSO was used for this step. Do not put DMSO solutions on ice. DMSO has a melting point of 19 °C, so it must be room temperature (or slightly warmer) to pipette³⁹.
   CAUTION: DMSO can penetrate gloves, so it is good practice to wear double nitrile gloves and take care to prevent spills.
7. To prepare the myosin for adding fluorophore, briefly take the myosin solution off the ice and let it warm to room temperature (RT).
   NOTE: Do not leave myosin at RT for longer than necessary. Do this step after the fluorophore is suspended in DMSO.
8. As soon as the myosin solution is near RT, add the fluorophore solution to the myosin and mix rapidly such that there is a 5:1 molar ratio of fluorophore: myosin. Place myosin solution on ice as soon as it is mixed with the fluorophore.
9. Incubate on ice for 1 h, protected from light, then stop the reaction by adding DTT to a concentration of 1 mM. Use a 1 M DTT stock to minimize the volume added.
10. Remove unreacted fluorophores. Use either of the two methods described below:
    1. Option 1:
       1. Polymerize myosin by slowly diluting it into an F-buffer (10 mM Imidazole, 50 mM KCl, 1 mM MgCl₂, 2 mM EGTA, 4 mM ATP, pH 7.5). This is approximately a 10x dilution, depending on the dye volume added in step 1.8.
       2. Incubate on ice for 20 min. Spin at 8,000 x g in a refrigerated centrifuge at 4 °C for 10 min to pellet.
       3. Discard the supernatant containing free dye and resuspend/de-polymerize myosin with Myosin storage buffer (from Step 1). To remove any remaining dye, dialyze into 5 mM 2-[4-(2-sulfoethyl)piperazin-1-yl]ethanesulfonic acid (PIPES), 0.45 M KCl, pH 7.0 (>100 fold volume excess) three times for 15 min each using a 10 kD cutoff dialysis cup.
    2. Option 2:
       1. Use a desalting column for buffer exchange into 5 mM PIPES, 0.45 M KCl, pH 7.0, with the standard protocol from the column manufacturer.
11. Estimate the fluorophore-to-protein ratio by measuring the concentration of myosin and fluorophore with a spectrophotometer, using extinction coefficients for myosin at 280 nm and for the fluorophore as reported by the manufacturer. A ratio of 2-4 is typical.
    NOTE: The labeled myosin is now ready for snap-freezing aliquots in liquid nitrogen. Aliquots are stored at -80 °C for long-term storage.

2. Optional: Procedure to remove inactive myosin

NOTE: Myosin can be used directly from a thawed aliquot in experiments, but some fraction of the myosin will be inactive. The following procedure describes how to remove inactive myosin. This is an optional step, but it can be crucial for reproducibility. Myosin aliquots are used for approximately 3 days after thawing, stored at 4 °C or on ice.

1. Polymerize unlabeled, phalloidin-stabilized actin:
   1. Mix 10 µL of 10x F-buffer, 1 µL of 100 mM ATP, and ddH₂O such that the final volume (including actin in step 2.1.2) will be 100 µL in a microcentrifuge tube.
   2. Add actin monomer to a final concentration of 20 µM and pipette mix.
   3. To stabilize actin filaments, add phalloidin to a final concentration of 6.7 µM using a stock of 100 µM phalloidin in methanol and pipet mix.
      CAUTION: Phalloidin is a toxin⁴⁰. Avoid spills and contamination.
   4. Incubate on ice for 20 min to allow actin to polymerize. After polymerization, store the actin at 4 °C for future spin-downs.
2. Pipet mix 10 µL phalloidin-stabilized actin, 3 µL of 10x F-buffer, 0.3 µL of 100 mM ATP, 6 µL of 2 M KCl, and ddH₂O to a final volume of 30 µL (including volume associated with adding myosin in the next step) in a microcentrifuge tube on ice.
3. Add dimeric labeled myosin from prepared stock to the desired concentration, maintaining a molar ratio of myosin to actin of ≤ 1:6.
4. Centrifuge the resulting solution cold (4 °C) at 100,000 x g for 30 min. Inactive myosin will bind to actin filaments and remain bound. The inactive myosin and actin filaments will form a pellet during centrifugation.
5. Remove the supernatant (containing active myosin) and store it at 4 °C for use in experiments.
6. Estimate the myosin concentration using the same spectrophotometric method to measure myosin concentration after labeling.
   NOTE: At the protein and ATP concentrations that remain in the supernatant, the 280 nm absorbance peak is obscured by the 260 nm ATP absorbance peak, so measuring the concentration through the absorbance associated with the fluorophore or a relative fluorescence measurement is more accurate.

3. Prepare surfactant solution

NOTE: The surfactant solution can also be purchased pre-mixed and ready to use.

1. Start with a clean glass vial. Rinse the vial in water and ethanol to remove potential contaminants in the oil, which is a solvent for the surfactant. Use borosilicate glass vials with a polytetrafluoroethylene (PTFE)-lined cap.
2. Pick up 5-20 mg of fluorosurfactant using a pipet tip and deposit near the bottom of a glass vial. Measure the surfactant amount by weight using a balance with sub-milligram accuracy.
3. Pipet the appropriate volume of fluorinated oil to make a 2 wt% solution and close the vial.
   NOTE: The oil is volatile, so close the vial immediately after measuring.
4. Vortex at low speed to mix.
   NOTE: The solution can now be stored at 4 °C for a few weeks. Surfactant age and quality can influence both crowding and appearance of the actin. Actin that appears speckled, adhered to the surface, or does not crowd to the surface may indicate that a fresh surfactant solution is necessary. Storage under nitrogen gas can improve shelf life.

4. Prepare sample chamber (Three options)

NOTE: This section provides a procedure to construct three standard sample chambers used for preparing samples for microscopy. The following two sections cover preparing the actual sample and passivating the sample chamber for sample loading.

1. Option 1: Flowcell
   NOTE: A simple flow cell is a standard way of making microscope samples in many labs. For this protocol, it is particularly suited to the samples encased in emulsion drops. It can be difficult to achieve large, flat, fields of view with the surfactant surface coating described in this protocol, so for 2D samples, options 2 and 3 are often more reproducible. The channel geometry also may cause uneven mixing of components added at later times, such as myosin.
   1. Place 2 pieces of double-sided tape parallel to each other across the width of the microscope slide to form the boundaries of the flowcell channel. The channel formed between the two pieces of tape should be approximately 2 mm in width.
      NOTE: Smaller channels help prevent emulsions from getting stuck in the beginning part of the channel. To make a good seal, use pieces of tape just longer than the width of the glass slide and trim after constructing the flowcell.
   2. Place the coverslip over the tape channel. Ensure that the coverslip is perpendicular to the microscope slide. Use a 30 mm x 40 mm coverslip for this purpose because it provides a small overhang when placed on a slide.
   3. To create a good seal and eliminate air pockets between the tape and the coverslip, press down on the area of the coverslip that is in contact with double-sided tape.
      NOTE: Be careful not to break the coverslip while pressing around the edges. Rubbing the area with a rounded object, such as the end of a capped marker, works well to press down the tape.
   4. Using a razor blade, trim any excess tape off the microscope slide and coverslip, such that the only visible tape is within the sample channel.
      NOTE: Be careful completing this step, as it is easy to accidentally break the coverslip while trying to remove the tape.
2. Option 2: Cylinder on an untreated coverslip
   NOTE: This option is suited for 2D planar samples. It is convenient for adding components at different times during microscopy experiments. This sample chamber should not be used for emulsions, which cream rather than sediment, rendering imaging difficult.
   1. Rinse the coverslip and glass cloning cylinder with water, ethanol, and water. Air dry.
   2. Using a thin layer of 5-min epoxy, adhere the glass cloning cylinder to the coverslip.
      NOTE: It is helpful to apply force to the cylinder while the epoxy dries by placing a clean object on top of the cylinder. A small Petri dish lid or the coverslip box can work well.
3. Option 3: Cylinder on a silane-coated coverslip
   NOTE: This option reproducibly results in a large, planar region in the center of the chamber and reduces the bulk flow of the sample. To achieve this, a hydrophobic region was created that is surrounded by a hydrophilic region on the coverslip, which results in a final sample that has an actin network on a passivated, confined region where the actin is anchored to the hydrophilic regions at the edge of the chamber. This sample chamber should not be used for emulsions, which cream rather than sediment in buffer, rendering imaging difficult.
   1. Prepare coverslips with a silane coating to render them hydrophobic.
      1. Load clean glass coverslips into a stainless-steel rack. Preclean glass by sonication in detergent or ethanol if desired.
      2. In a glass staining dish, dilute trimethoxy(octyl)silane to a 2% solution in isopropanol.
      3. Immerse coverslips in the solution for 10 min.
      4. To rinse, replace the solution with water. Lift up and re-immerse the coverslip racks six times. Repeat the process four additional times with water exchanges in between.
      5. Cover the racks with aluminum foil to protect coverslips from dust and dry them at 25-30 °C.
   2. Place an octyl-silane-treated coverslip into the bottom of a glass Petri dish or any other open glass container that will fit into the ultraviolet (UV)-ozone cleaner.
   3. Using forceps, place a 2 mm x 2 mm PTFE square (cut from a sheet) onto the coverslip that will act as a mask for the following UV-ozone treatment.
   4. Treat the coverslip with UV-ozone for 10 min. This process makes the glass hydrophilic in the exposed regions that are not covered by the PTFE mask.
   5. Carefully remove the dish without disturbing the coverslip or PTFE square. Adhere a glass cloning cylinder to the coverslip using 5-min epoxy. Ensure the cylinder encircles the PTFE square, which may be removed with forceps after the epoxy dries.
      NOTE: The PTFE square may be reused in future experiments.

5. Assembling the actin network

NOTE: The following is for preparation of a 50 μL sample. For cylinder samples, a larger volume can reduce the effects of the meniscus and increase sample stability.

1. To a microcentrifuge tube, add 5 µL of 10x F buffer, ddH₂O necessary to bring final volume to 50 µL, 1 µL of 25 vol% beta-mercaptoethanol, 1 µL of 225 mg/mL glucose, 1 µL of a mixture of glucose oxidase (135 mg/mL) and catalase (85,000 units/mL), 1 μL of 25 mM ATP, and 10 µL of 2 wt% 15 centipoise methylcellulose. Pipet mix thoroughly.
   NOTE: Beta mercaptoethanol is a toxin. Avoid spills and contamination. The reagents glucose oxidase, catalase, glucose, and beta-mercaptoethanol are in the sample to create an oxygen-scavenging system. The methylcellulose stock solution is prepared by stirring at 4 °C and can be stored at 4 °C for several weeks. Methylcellulose can precipitate out of solution and should be re-prepared if there is visible precipitate or if actin crowding is poor.
2. Optional: Add cross-linking protein (0.1-1 crosslinker protein for every 1 actin monomer). Pipet mix.
   NOTE: Cross-linking protein(s) may also be added after actin polymerization and allowed to bind for 10-20 min before adding myosin. This is preferable for crosslinker concentrations that are high enough to bundle actin filaments-the bundles are more evenly and reproducibly distributed at the surfactant-water interface. The cylinder sample chamber is amenable to adding a crosslinker after the actin polymerizes.
3. Add phalloidin, (1 phalloidin for every 1-3 actin monomers), if desired. Pipet mix.
4. In a separate tube, mix unlabeled and labeled actin such that when the actin mix is all added to the 50 μL sample, the total concentration will be 2.64 µM. Use a ratio of 1 labeled actin monomer for every 9 unlabeled actin monomers. Pipet mix.
   NOTE: The actin monomers will begin to polymerize upon addition to the sample solution. Adding the actin labeled and unlabeled actin separately can result in actin that appears speckled with dark and fluorescent regions along the contour of a single filament. The actin monomer stocks are mixed to ensure uniformly labeled actin filaments. Actin purification and labeling are described in³¹.
5. Optional: To make liquid crystals or other experiments using short actin filaments, add capping protein to the actin mix from step 5.4. The exact amount of capping protein depends on the active fraction of the protein and the length of actin filaments targeted, but typically, around 1-10 mol% capping protein (with respect to the actin) is sufficient to create short filaments for liquid crystal experiments. Capping protein purification is described in³⁶.
6. To begin polymerizing actin, add the actin mix to the F-buffer solution and pipet mix.
7. Leave the actin to polymerize in the microcentrifuge tube at RT, and then add it to the sample cell after 10-30 min.
8. Optional: If preparing a cylinder sample, draw the entire solution into a pipet tip so it is ready to pipet in step 7.4 of loading the cylinder sample chamber.

6. Optional: Prepare emulsions

NOTE: It is sometimes convenient to prepare these samples in emulsions, which provide a confined sample chamber. The emulsions are prepared using similar reagents as Chowdhury et al., which results in an oil continuous phase that has surfactant-mediated aqueous emulsion drops⁴¹. In the presence of a depletion agent, such as methylcellulose, used in this protocol, the actin samples will crowd to the aqueous interface of this emulsion. For samples that this protocol is based on, a difference between polymerizing the actin before or after adding it to the emulsions is not seen; however, it has been reported that the polymerization step is important to consider in some encapsulation experiments⁴².

1. Add 3.5 μL of oil-surfactant to a microcentrifuge tube. Use a transparent, light-colored, or clear microcentrifuge tube for this step.
2. Without mixing, add 5 μL of the actin solution to the top of the oil-surfactant layer. Close the microcentrifuge tube.
3. While holding the top of the microcentrifuge tube, flick the bottom edge of the tube to create a foam with visible emulsions initially. Continue to flick the tube until the foam displays no distinguishable features when backlit, indicating microemulsions.
   NOTE: When preparing a sample in emulsions, a flow cell or other sample chamber should be ready before making the actin sample. The glass cylinder sample chamber does not work well for emulsions as they will cream to the air interface rather than sediment to the coverglass surface.

7. Load the sample chamber (Cylinder chambers)

1. Pipet 5 µL of oil-surfactant solution into the bottom of the glass cylinder chamber
   NOTE: Alternatively, samples can be made by passivating the surface with a supported lipid bilayer^11,29,30.
2. Tilt the coverslip and slowly rotate so the coverslip surface and lower cylinder are coated with the surfactant solution.
3. Remove excess oil-surfactant solution with a pipet to get as thin of an oil layer as possible while not allowing the oil, which is volatile, to evaporate completely.
4. Immediately add the sample solution from step 5.7 to the chamber.
5. Cover the chamber with a small piece of PTFE tape to prevent evaporation and flow.

8. Alternate: Load the sample chamber (flow cell)

1. To prepare for loading the flow cell, mix a small amount of 5 min epoxy. A drop with an area less than 0.5 cm² is typically sufficient.
2. To place the sample in the flow cell, first pipette 1-3 μL of oil-surfactant solution into the sample channel of the flow cell to create a small plug of solution that wets the channel.
3. Immediately pipette a volume of sample solution or emulsion suspension slowly into the entrance of the flow cell to fill the channel. Typically, for a channel that is around 2 mm, the volume is around 8 μL. The entrance is the side to which the oil-surfactant solution was added.
   1. If the sample volume exceeds the flowcell volume, wick the sample through the flowcell by placing a lint-free wipe or filter paper at the other end of the channel.
   2. If the meniscus of the oil-surfactant solution has receded, resulting in an air gap, then first tilt the flowcell to remove the air gap at the entrance prior to adding the sample.
4. If necessary, add additional oil-surfactant solution until air is no longer visible in the channel or to push the sample (particularly for emulsions) further into the channel.
5. Seal each side of the channel with 5-min epoxy (mixed right before loading the flow cell).

9. Image and add myosin

1. Mount the sample on the microscope and start timelapse imaging of actin using either a 20x, 40x, 60x, or 100x objective with oil or water immersion to provide a large enough numerical aperture for high-resolution imaging. If polymerizing in the sample chamber, allow polymerization for ~30 min or until there is no longer visible filament lengthening or motion.
2. Optional: Add crosslinker.
3. Add myosin through options 1 or 2 below.
   1. Add myosin as a dimer pipetted to the top of the sample (no mixing is required because the myosin dimer will diffuse).
   2. Add myosin as pre-polymerized filaments. Pipet mix approximately half of the total volume very slowly to avoid disturbing the crowded actin.
      NOTE: The ideal method for myosin addition to get the desired filament density or size at the coverslip surface may vary for different actin architectures and needs to be determined experimentally.
4. Start time-lapse imaging.

Results

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 ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.6 mL microcentrifuge tube	Fisher Scientific	05-408-123
008-Fluorosurfactant	RAN Biotechnologies	00115081361-6525	Alternate to premixed solution
008-FluoroSurfactant in HFE7500	RAN Biotechnologies	008-FluoroSurfactant-2wtH-50G	Premixed solution
2-mercaptoethanol (β-mercaptoethanol)	Sigma Aldrich	63689	CAS Number: 60-24-2 Stock solution: 25 mM in MilliQ water, stored at 4 °C
4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES)	Sigma Aldrich	H3375	CAS Number: 7365-45-9
5 min Epoxy	Devcon	14250
Actin	Cytoskeleton, Inc.	AKL99-A	>99% pure rabbit skeletal muscle (Alternate to purifying)
Actin (rhodamine labeled)	Cytoskeleton, Inc.	AR05-A	Rabbit skeletal muscle (Alternate to purifying)
Adenosine 5′-triphosphate (ATP)	Sigma Aldrich	A6419	CAS Number: 34369-07-8 Stock solution: 25 mM in MilliQ water, store at -20 °C Keep frozen to avoid hydrolysis
Calcium Chloride (CaCl2)	Sigma Aldrich	C5670	CAS Number: 10043-52-4
Capping Protein (Mouse)			Purified from overexpression in E.coli with a HisTag Stock solution: 20 mM in capping protein buffer at -80 °C
Catalase from bovine liver	Sigma Aldrich	C9322	CAS Number: 9001-05-2 Stock solution: 85 ku/mL, store aliquoted with glucose oxidase at -20°C
Cover Glass	Fisherbrand	12544C	Borosilicate, #1.5, 24 mm x 40 mm Fisherbrand not Fisher Finest
D-(+)-Glucose	Sigma Aldrich	G8270	CAS Number: 50-99-7 Stock solution: 225 mg/mL in MilliQ water, store at -20 °C
Dithiothreitol (DTT)	Sigma Aldrich	3860-OP	CAS Number: 3483-12-3 Stock solution: 1 M in MilliQ water, store at -20 °C
Double Sided Tape	3M	3136
Ethyl alchohol 200 Proof	PHARMCO	111000200	CAS Number: 64-17-5 200 proof
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)	Sigma Aldrich	E3889	CAS Number: 67-42-5
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	E9884	CAS Number: 60-00-4
Glucose Oxidase	Sigma Aldrich	345386	CAS Number: 9001-37-0 Stock solution: 135 mg/mL in MilliQ water, store aliquoted with catalase at -20 °C
Glycerol	Sigma Aldrich	356352M	CAS Number: 56-81-5
Imidazole	Fisher Bioreagents	BP305-50	CAS Number: 288-32-4
Magnesium Chloride (MgCl2)	Fisher Bioreagents	BP214	CAS Number: 7786-30-3, 7791-18-6
Methylcellulose	Sigma Aldrich	M0512	CAS Number: 9004-67-5 15 centipoise
Microscope Slides Plain	Electron Microscopy Sciences/Gold Seal	63710-05	3"x1", 1 mm thick
MilliQ water	Millipore	CUFBI001	Ultrapure water
Novec-7500 Engineered Fluid	3M	7100134816	Alternate to premixed solution
Potassium Chloride (KCl)	Sigma Aldrich	P3911	CAS Number: 7447-40-7
Potassium Phosphate (KPO4)	Sigma Aldrich	P3786	CAS Number: 7758-11-4
Rabbit Skeletal Muscle Acetone Powder	Pel-Freez Biologicals	41995-1	Used when purifying actin (alternate to purchasing) Store around 30 mM in actin Buffer G at -80 °C
Sodium Azide	Sigma Aldrich	71289	CAS Number: 26626-22-8
Tetramethylrhodamine-C6-maleimide	AnaSpec	AS-81445	CAS Number: 174568-68-4 Used when purifying actin (alternate to purchasing) Store around 30 mM in actin Buffer G at -80 °C. Used when labeling actin (alternate to purchasing)
Tris Hydrochloric Acid (Tris HCl)	Sigma Aldrich	648317	CAS Number: 1185-53-1
Ultrasonic Bath	Emerson Branson	CPX2800H