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Experimental methods
This protocol is extracted from research article:
Tunable structure and dynamics of active liquid crystals
Sci Adv, Oct 12, 2018; DOI: 10.1126/sciadv.aat7779

Proteins. Monomeric actin was purified from rabbit skeletal muscle acetone powder (Pel-Freez Biologicals, Rogers, AR) (39) and stored at −80°C in G-buffer [2 mM tris-HCl (pH 8.0), 0.2 mM adenosine 5′-triphosphate (ATP), 0.2 mM CaCl2, 0.2 mM dithiothreitol (DTT), and 0.005% NaN3]. Tetramethylrhodamine-6-maleimide (TMR) dye (Life Technologies, Carlsbad, CA) was used to label actin. CP [mouse, with a HisTag, purified from bacteria (21); gift from the D. Kovar laboratory, The University of Chicago, Chicago, IL] was used to regulate actin polymerization and shorten the filament length. Skeletal muscle myosin II was purified from chicken breast (40) and labeled with Alexa-642 maleimide (Life Technologies, Carlsbad, CA) (41).

Experimental assay and microscopy. The actin is polymerized in 1× F-buffer [10 mM imidazole (pH 7.5), 50 mM KCl, 0.2 mM EGTA, 1 mM MgCl2, and 1 mM ATP]. To avoid photobleaching, an oxygen-scavenging system [glucose (4.5 mg/ml), glucose oxidase (2.7 mg/ml; catalog no. 345486, Calbiochem, Billerica, MA), catalase (17,000 U/ml; catalog no. 02071, Sigma, St. Louis, MO), and 0.5 volume % β-mercaptoethanol] was added. Methylcellulose [15 centipoise; 0.3 weight % (wt %)] was used as the crowding agent. Actin from frozen stocks stored in G-buffer was added to a final concentration of 2 μM with a ratio of 1:5 TMR-maleimide labeled/unlabeled actin monomer. Frozen CP stocks were thawed on ice and added at the same time (6.7 and 3.3 nM for 1- and 2-μm long actin filaments). We call this assay “polymerization mixture” from henceforth. Myosin II was mixed with phalloidin-stabilized F-actin at a 1:4 myosin/actin molar ratio in spin-down buffer (20 mM MOPS, 500 mM KCl, 4 mM MgCl2, 0.1 mM EGTA; pH 7.4) and centrifuged for 30 min at 100,000g. The supernatant containing myosin with low affinity to F-actin was used in experiments, whereas the high-affinity myosin was discarded.

The experiment was performed in a glass cylinder (catalog no. 09-552-22, Corning Inc.) glued to a coverslip (36). Coverslips were cleaned by sonicating in water and ethanol. The surface was treated with triethoxy(octyl)silane in isopropanol to produce a hydrophobic surface. To prepare a stable oil-water interface, PFPE-PEG-PFPE surfactant (catalog no. 008, RAN Biotechnologies, Beverly, MA) was dissolved in Novec 7500 Engineered Fluid (3M, St. Paul, MN) to a concentration of 2 wt %. To prevent flows at the surface, a small Teflon mask measuring 2 mm by 2 mm was placed on the treated coverslip before exposing it to ultraviolet-ozone for 10 min. The glass cylinder was thoroughly cleaned with water and ethanol before gluing it to the coverslip using instant epoxy. Then, 3 μl of oil-surfactant solution was added into the chamber and quickly pipetted out to leave a thin coating. The sample was always imaged in the middle of the film over the camera field of view, which was about 200 μm by 250 μm, to make sure that the sample remains in focus over this area, which is far away from the edges. Imaging close to the edges was avoided. The polymerization mixture was immediately added afterward. Thirty to 60 min later, a thin layer of actin LC was formed. Myosin II motors were added to the polymerization mixture at concentrations of 5 to 10 nM.

The sample was imaged using an inverted microscope (Eclipse Ti-E; Nikon, Melville, NY) with a spinning disc confocal head (CSU-X, Yokagawa Electric, Musashino, Tokyo, Japan), equipped with a CMOS camera (Zyla-4.2 USB 3; Andor, Belfast, UK). A 40× 1.15 numerical aperture water-immersion objective (Apo LWD, Nikon) was used for imaging. Images were collected using 568- and 642-nm excitation for actin and myosin, respectively. Image acquisition was controlled by MetaMorph (Molecular Devices, Sunnyvale, CA).

Image and data analysis. The nematic director field was extracted the same way as in (22), which used an algorithm that was described in detail in the methods section of Cetera et al. (24). The optical images were bandpass filtered and unsharp masked in ImageJ software (42) to remove noise and spatial irregularities in brightness. The image algorithm computes 2D fast Fourier transform of a small local square sections (of side ψ) of the image and uses an orthogonal vector to calculate the local actin orientation. The sections were overlapped over a distance ζ to improve statistics. ψ and ζ are varied over 15 to 30 μm and 1 to 3 μm, respectively, for different images to minimize errors in the local director without changing the final director field.

Myosin puncta density was calculated using ImageJ software. Toward the end of the experiment, large clusters of myosin were not counted. Because the number of myosin polymers remains at least 10-fold greater than that of myosin clusters, our results are insensitive to the choice of the cluster cutoff size. We calculated the mean ld, ξθ, and ξv from overlapping 150-s intervals. We explored averaging over shorter time intervals and found that the trend in ld was similar but, as expected, the SD increased (fig. S4). At the fastest rates of decrease, the myosin density does not decrease over this interval but is within the measurement error reported in Fig. 1C. The typical relaxation time of the actin nematic LC is given by τR = γl2/K, where γ, l, and K are the rotational viscosity, the filament length, and the LC elastic modulus, respectively. For γ ~ 0.1 Pa∙s, l = 1 μm, and K = 0.13 pN, we find that τR ~ 1 s. Thus, the LC structure achieves steady state on time scales much faster than the evolution of the myosin density.

The active flows were quantified using particle image velocimetry (available at www.oceanwave.jp/softwares/mpiv/) to extract local velocity field, v. The orientational correlation length, ξθ, was calculated by computing Embedded Image, where g2(r) = 〈 cos[2(θi − θj)]〉, indicating spatial pairs i and j separated by a distance of r. Similarly, Embedded Image.

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