CHAPTER

Reconstituting cytoskeletal contraction events with biomimetic actinemyosin active gels

6

Jose´ Alvarado*, x, Gijsje H. Koenderink*, 1 x

*FOM Institute AMOLF, Amsterdam, The Netherlands Massachusetts Institute of Technology, Cambridge, MA, USA 1

Corresponding author: E-mail: [email protected]

CHAPTER OUTLINE Introduction .............................................................................................................. 84 1. Building Contraction Chambers ............................................................................. 87 1.1 Cleaning Glass ...................................................................................... 87 1.2 Constructing Contraction Chambers ........................................................ 88 1.3 Passivating Surfaces.............................................................................. 89 2. Preparing Contractile Active Gels.......................................................................... 90 2.1 Purifying Proteins.................................................................................. 90 2.2 Preparing Protein Stock Solutions........................................................... 90 2.3 Buffers and Components........................................................................ 91 2.4 Loading Gels into Chambers................................................................... 92 2.5 Microscopy ........................................................................................... 93 3. Manipulating Contractile Active Gels..................................................................... 93 3.1 Sticky Surfaces Decrease Contraction Length Scale ................................. 94 3.2 Excess Monovalent Salt and Nucleotides Weaken Motors .......................... 95 3.3 Lower Actin Concentration Decreases Contraction Length Scale ................ 97 3.4 Gelsolin Shortens Actin Filaments and Prevents Contraction ..................... 99 Outlook .................................................................................................................... 99 Acknowledgments ................................................................................................... 100 References ............................................................................................................. 101

Abstract The actinemyosin cytoskeleton allows cells to move, change shape, and exert forces. These fascinating functions involve active contraction of cross-linked networks of actin filaments by myosin II motor proteins. Unlike muscle cells, where actin and myosin form Methods in Cell Biology, Volume 128, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.02.001 © 2015 Elsevier Inc. All rights reserved.

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ordered bundles that contract homogeneously, nonmuscle cells have a variety of more disordered types of actinemyosin meshworks. Active gels reconstituted from purified actin and myosin proteins offer a useful in vitro model system to systematically and quantitatively investigate the mechanisms of contraction and the role of physical parameters like motor activity and network connectivity. In order to quantify the effect of these physical parameters on contraction, time-lapse microscopy combined with quantitative image analysis is required. Here we describe an assay that we developed specifically to record contraction events of entire biomimetic active gels in contraction chambers, which enables one to systematically quantify the dependence of contraction time and length scales on experimental parameters such as protein concentrations, adenosine triphosphate concentration, ionic strength, and surface adhesion.

INTRODUCTION Animal cells constantly perform a variety of mechanical tasks: they change shape, spatially arrange organelles, exert forces on neighboring cells or extracellular matrix, and migrate to targeted locations. An important driver of these mechanical tasks is the actinemyosin cytoskeleton. Actin and myosin proteins are best known as the essential components which drive contraction in mammalian muscle tissue (Rayment et al., 1993). Yet these two proteins also appear outside of muscle cells across eukaryotes to perform a diverse set of mechanical functions. For example, animal cells divide by a contractile actinemyosin ring which pinches the mother cell into two daughter cells (Guertin, Trautmann, & McCollum, 2002). Starfish oocytes localize chromosomes to the nucleus by a space-filling, contractile actinemyosin meshwork (Le´na´rt et al., 2005). Crawling cells like fish keratocytes move across substrates by a balance between pushing forces from actin polymerization and pulling forces from myosin-mediated contraction (Rafelski & Theriot, 2004). Actinemyosin contraction relies on the interaction between actin filaments and myosin II bipolar filaments. Globular, monomeric actin proteins (G-actin) polymerize to form filaments (F-actin), which undergo sliding due to the enzymatic ATPase activity of myosin motors organized in bipolar filaments of 10e20 molecules (Verkhovsky, 1993). But how does actin filament sliding translate to contraction? In muscle tissue, a host of accessory proteins arrange actin filaments and myosin motors into quasicrystalline sarcomeres. This arrangement converts motor-induced sliding to uniform shortening of sarcomeres and thus muscular contractions. In nonmuscle cells, actin filaments are typically disordered, forming a thin contractile cortex underneath the plasma membrane in single cells and epithelial cell monolayers (Salbreux, Charras, & Paluch, 2012), 3D meshworks in oocytes (Le´na´rt et al., 2005), and bundles known as stress fibers in adherent cells (Naumanen, Lappalainen, & Hotulainen, 2008). Despite the lack of sarcomeric order in these nonmuscle cells, the actinemyosin cytoskeleton is still capable of contraction events (Figure 1(A) and (B)).

Introduction

How can myosin-induced sliding result in contraction events in the absence of sarcomeric order? This question has been difficult to address using in vivo model systems because these are inherently complex and can only be manipulated to a limited extent. Meanwhile, in vitro systems of purified actin and myosin proteins offer a biomimetic model system which exhibits contraction that is very similar to the contraction events found in vivo (Figure 1(C) and (D)). These model systems are well suited to study the physical mechanisms underlying contraction because they comprise a minimal number of components whose concentrations can be systematically varied (Murrell & Gardel, 2012; Soares e Silva et al., 2011). This simplicity allows for quantitative comparison to theoretical models which address the physical mechanisms of contraction (Banerjee, Liverpool, & Marchetti, 2011; Lenz, Gardel, & Dinner, 2012; Lenz, Thoresen, Gardel, & Dinner, 2012; Wang & Wolynes, 2012a, 2012b). Additionally, biomimetic contractile systems are a fascinating example of active gels: materials in which macroscopic behavior emerges from molecular-scale nonequilibrium driving that is coordinated over long length scales (Marchetti et al., 2013). An important advantage of in vitro systems is that they allow one to identify the minimal requirements needed for contraction to occur. In a disorganized network, one would expect motors to both push and pull actin filaments due to symmetry. In order for net contraction to occur, this symmetry must be somehow broken. Quantitative comparison between experiment and theory has established that the asymmetric mechanical response of actin filaments originating from their semiflexible nature favors net contraction: actin filaments strongly resist extension but readily buckle under compression. However, what remains less understood is how contractile stresses propagate across a network of actin. In particular, many studies had suggested over the years that an interplay between network connectivity and motor activity governs the propagation of motor stresses over long length scales (Alvarado, Sheinman, Sharma, MacKintosh, & Koenderink, 2013; Bendix et al., 2008; Janson, Kolega, & Taylor, 1991; Ko¨hler, Schaller, & Bausch, 2011; Murrell & Gardel, 2012; Smith et al., 2007; Soares e Silva et al., 2011). However, the length scales of contraction remained poorly characterized. These length scales were difficult to quantify, partially because most studies so far had only visualized portions of active gels using microscopy techniques. This is unavoidable when using high-power objectives, which are necessary to investigate the behavior of individual actin filaments and myosin motors. But the question of how contractile stresses propagate over longer length scales cannot be addressed using this zoomedin approach. Active gels contract unpredictably over long length scales. This unpredictability either compels microscope users to manually follow contraction events as they occur or miss events outside the field of view. In order to address this experimental shortcoming, we developed a new assay to record time-lapse movies of contracting active gels confined in contraction chambers with a square geometry. This confinement facilitates the visualization of entire contraction events using microscopy techniques. Visualizing entire contraction events allowed us to quantify contraction length scales and relate them to physical parameters such as network connectivity and motor activity (Alvarado et al., 2013).

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(A)

(C)

(B)

(D)

FIGURE 1 Actin filaments and myosin motors are responsible for contraction events, both in vivo and in vitro. (A) Schematic of a starfish oocyte. Depicted are actin filaments (lines), myosin motor filaments (barbells), and chromosomes (blue circles). Cross-link proteins, which control the network connectivity by connecting actin filaments at junctions, are not shown. (B) Chromosome congression in a starfish oocyte 30 min after nuclear envelope breakdown. Chromosomes are fluorescently labeled and transported by a contractile meshwork of actin filaments and myosin motors (unlabeled). Color corresponds to time. A time span of 23 min elapses between the beginning of contraction (black-blue-purple pixels) and the end of contraction (yellow-white pixels). Scale bar approximately 20 mm. (C) Schematic of a reconstituted active gel of actin filaments (unlabeled) and myosin proteins (labeled). (d) Close-up of a contraction event in an active gel. Color corresponds to time. A time span of 118 min elapses between the beginning of contraction (black-blue-purple pixels) and the end of contraction (yellow-white pixels). Scale bar 200 mm. Panel (B) is assembled from selected frames of Video S2 of Le´na´rt et al. (2005). (See color plate)

1. Building contraction chambers

Here we furthermore show how this assay can be conveniently used to screen for the influence of various experimental parameters on network contractility. We expect future experimental research on contractile active gels to further investigate the physical mechanisms which propagate stresses from myosin motors over long distances. In this chapter, we describe the assembly process of contraction chambers and the protocol for reconstituting active gels of actin and myosin. Finally, we demonstrate the effect of various experimental parameters on the length scale of contraction.

1. BUILDING CONTRACTION CHAMBERS In order to visualize entire contraction events, we designed contraction chambers which have a flat square shape similar to a pizza box, with dimensions of w2.5 mm in length and width and 0.08 mm in height. These dimensions allow the contraction chamber to fit in the field of view of a 4 microscope objective. The 0.08 mm height allows the entire chamber to remain in focus. A typical assembly yields eight contraction chambers (Figure 2). In this section, we describe the process of building these contraction chambers.

1.1 CLEANING GLASS To avoid network adhesion to the chambers walls, the contraction chambers require clean surfaces that are free of impurities. We use a base piranha solution which is safer than the standard acid piranha solution common in many laboratories. Whereas acid piranha instantly reacts upon mixing, the base piranha reaction only activates at temperatures of 60  C or higher. 1. Load glass coverslips (24  60 mm as well as 22  40 mm, thickness #1 ¼ 0.15 mm, Menzel Gla¨ser) into a Teflon carousel.

FIGURE 2 Schematic of contraction chamber assembly with top view (center), front view (bottom), and side view (right).

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2. Rinse coverslips by placing into a clean beaker filled with MilliQ water. Sonicate using a bath sonicator for 5 min at room temperature. Drain and blow-dry with a stream of clean nitrogen gas. 3. Fill a beaker with five parts MilliQ water. Heat on a hot plate to 80  C while continuously stirring with a magnetic bar. Keep the beaker in a fume hood. Add one part ammonium hydroxide (30% stock) and one part hydrogen peroxide (30% stock). Upon mixing, the solution will spontaneously form bubbles, indicating an active reaction. The amount of base piranha solution should suffice to completely submerge coverslips and carousel. 4. Place carousel into beaker. Incubate coverslips in solution for 30 min, maintaining a temperature of 80  C. Cover the beaker with aluminum foil to prevent bubbles from splashing base piranha solution to surroundings, but do not cover so tightly as to prevent fumes and water vapor from escaping. 5. Rinse and dry coverslips according to Step 2. Store in isopropanol. a. Tip: The hydrogen peroxide should be pure and not stabilized with additives. Some additives may prevent the base piranha reaction from proceeding. Hydrogen peroxide stock should also be fresh, as it gradually breaks down to water and oxygen. To keep stocks fresh, ensure that bottles remain closed and refrigerated when not in use.

1.2 CONSTRUCTING CONTRACTION CHAMBERS Here we describe the process for constructing contraction chambers, which consist of a thin glass substrate, Parafilm wax strips, and a thin strip of glass: 1. Using a ruler and diamond-tip glass cutter, cut a clean microscope coverslip (22  40 mm) along its long edge to recover glass strips that are w2.5 mm wide and 40 mm in length. The width of the glass strips determines the length of the contraction chambers. Always clean rulers and workbench surfaces with ethanol or isopropanol to avoid contaminating the glass strips. 2. Cut a 22  58 mm rectangle of clean Parafilm. Place it centered on the glass coverslip and press firmly enough to adhere the film to the glass but gently enough to not leave waxy residues on the glass when peeled away. One way of achieving this is by coating with wax paper (obtained from the lining of the Parafilm) and gently rolling with gloved fingertips. 3. Use a scalpel to cut w2.5 mm strips of the Parafilm layer. Cut the strips parallel to the short edge of the coverslip. When cutting, always use a fresh, sharp razor or scalpel blade and do not apply too much pressure while cutting. Otherwise, wax will deposit on the glass surface and/or the coverslip may fracture and crack. Remove strips gently using forceps. The width of the removed Parafilm strips sets the width of the contraction chambers. 4. Place a clean glass strip on top of and perpendicular to the Parafilm strips, centered with respect to the coverslip. The thickness of the Parafilm strips determines the height of the contraction chambers.

1. Building contraction chambers

5. Place the assembly on a hot plate set to 120  C. The Parafilm strips will melt, which is evident by a change in appearance from milky white to transparent. Once melted, the Parafilm strips bond to the glass surfaces. While the Parafilm is melted, apply gentle pressure on the glass strip with forceps to maximize the contact area between wax and glass. Do not apply so much pressure that the Parafilm flattens and bulges outward into the chamber. Remove assembly from hot plate and leave to cool at room temperature. a. Tip: Often, bubbles form as the Parafilm melts in Step 5. This occurs because before melting, Parafilm and the glass surfaces are not in full contact and are separated by an air layer. Once the Parafilm melts, the air layer is trapped under molten Parafilm. In order to minimize the formation of bubbles, the contact surface between the Parafilm and glass surfaces should be increased before placing onto the hot plate. This can be accomplished at the end of Step 3 by applying more pressure to the remaining Parafilm strips using wax paper and the curved end of a glass rod or ballpoint pen; and at the end of Step 4 by applying more pressure to the glass strip at the locations where it contacts the Parafilm using forceps. The contact area between the glass and the Parafilm can be seen with the naked eye as dark regions when viewing the glasseParafilm interface up close and at an angle. This protocol yields one coverslip with up to eight contraction chambers. The chambers are bounded on the two broad sides by glass, two walls with Parafilm, and two sides are open which allow fluid to flow (Figure 2).

1.3 PASSIVATING SURFACES Clean glass surfaces are essential for contraction assays. But in order for proteins to contract freely, glass surfaces must furthermore be passivated to prevent nonspecific adhesive interactions. We use the block copolymer poly(L-lysine)-graft-poly (ethylene glycol) (PLL-PEG) to passivate glass surfaces and employ a protocol which is derived from TIRF microscopy assays (Preciado Lo´pez et al., 2014): 1. Rinse chambers by flowing through several chamber volumes of MilliQ water. The chamber volume described here is 0.5 mL, therefore pipetting 10 mL of MilliQ water suffices. Blow-dry with clean nitrogen gas. 2. Flow in 2 mL of 1 M potassium hydroxide per chamber and incubate in a humid environment for 10 min. The humid environment minimizes evaporation of the potassium hydroxide droplets and can be made, for instance, by placing the chamber assembly in a petri dish together with wet tissues. After incubation, rinse and dry chambers as in Step 1. Potassium hydroxide activates hydroxyl groups on the glass surface, giving the surface a net negative charge to which the positively charged lysine block of PLL-PEG attaches. 3. Flow in 2 mL of 0.2 mg/mL PLL-PEG. Incubate in a humid environment for 45 min. After incubation, rinse and dry chambers as in Step 1. Use immediately for experiments.

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2. PREPARING CONTRACTILE ACTIVE GELS Active gels serve as a useful model system to study contraction in a well-controlled laboratory setting. Actin filaments, myosin motors, and cross-links are the essential proteins needed for contraction to take place. In this section, we briefly discuss how to prepare these proteins. We also discuss additional components which are required to mimic the intracellular environment and minimize damage due to fluorescence imaging. Finally, we describe how to load active gels into contraction chambers.

2.1 PURIFYING PROTEINS In order to perform experiments with purified proteins, first several biochemical preparations must be performed in order to purify protein from different sources. We purified monomeric (G-) actin and myosin II from rabbit psoas skeletal muscle (Soares e Silva et al., 2011). We further purified G-actin with a Superdex 200 size-exclusion column to remove oligomers (GE Healthcare, Waukesha, WI, USA). We snap-freeze G-actin aliquots and store at 80  C in G-buffer (2 mM tris-hydrochloride pH 8.0, 0.2 mM disodium adenosine triphosphate, 0.2 mM calcium chloride, 0.2 mM dithiothreitol). Actin is maintained in monomeric form due to the low ionic strength of G-buffer. We prepare myosin II in a high-salt buffer with glycerol for long-term storage at 20  C (25 mM monopotassium phosphate pH 6.5, 600 mM potassium chloride, 10 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 50% w/w glycerol). In addition, we prepared fluorescent protein stocks by labeling myosin II with Alexa Fluor 488 NHS ester (Invitrogen, Paisley, UK) and G-actin with Alexa Fluor 594 carboxylic acid, succinimidyl ester (Soares e Silva et al., 2011). We purified mouse fascin by transforming T7 Escherichia coli bacteria with a glutathione S-transferase (GST)efascin pGEX vector (Gentry et al., 2012). The protein is expressed together with a GST tag and purified by cleaving the tag while bound to a glutathione sepharose column (GE Healthcare). We snap-freeze aliquots of fascin protein and store at 80  C in 20 mM imidazole pH 7.4, 150 mM potassium chloride, 1 mM dithiothreitol, and 10% v/v glycerol.

2.2 PREPARING PROTEIN STOCK SOLUTIONS Before experiments, fresh protein stocks should be prepared from the storage stocks, which should be used within 4 days. Frozen aliquots of actin and fascin proteins are first clarified of aggregated proteins upon thawing by centrifuging at 120,000 g and keeping the pellet. Myosin proteins are dialyzed overnight at 4  C to transfer them to a storage buffer without glycerol and with a lower monovalent salt concentration (20 mM imidazole pH 7.4, 300 mM potassium chloride, 4 mM magnesium chloride, 1 mM dithiothreitol). Next, we determine all proteins’ concentrations by measuring the solution absorbance at 280 nm with a NanoDrop 2000 (ThermoScientific, Wilmington,

2. Preparing contractile active gels

DE, USA) and using extinction coefficients, in M1 cm, of 26,600 (actin (Pardee & Spudich, 1982)), 249,000 (myosin (Margossian & Lowey, 1982)), and 66,280 (fascin, computed from amino acid sequence (Artimo et al., 2012)). Finally, we mixed fluorescently labeled proteins with unlabeled proteins to yield on average a 10% molar ratio of dye to protein.

2.3 BUFFERS AND COMPONENTS Contractile active gels were mixed with the following components to yield these final concentrations: • • • • • • • • • • •

20 mM imidazole pH 7.4 50 mM potassium chloride (KCl) 2 mM magnesium chloride (MgCl2) 0.1 mM adenosine triphosphate (ATP) 10 mM creatine phosphate (CP) 0.1 mg/mL creatine kinase (CK) 1 mM dithiothreitol (DTT) 1 mM trolox 2 mM protocatechuic acid (PCA) 0.1 mM protocatechuase 3,4-dioxygenase (PCD) Note: When determining the mixing ratio of these solutions, take into consideration that the G-actin stock contains 0.2 mM ATP, the fascin stock has 50 mM KCl, and the myosin protein stock contains 150 mM KCl and 4 mM MgCl2.

Imidazole is commonly used as a buffer because it maintains a constant physiological pH over a broad range of temperatures. We adjust the pH of imidazole stocks using potassium hydroxide, to match the choice of potassium chloride as a monovalent salt. A pH of 7.4 mimics intracellular pH. If the pH is below 6, the actinemyosin bond is too strong to allow cross-linked active gels to contract (Ko¨hler, Schmoller, Crevenna, & Bausch, 2012). The monovalent potassium cations from KCl mimic intracellular ionic conditions and prevent nonspecific electrostatic interactions between proteins. The divalent magnesium cations from MgCl2 interact with the b and g phosphate groups of ATP and are needed for actin polymerization (Estes, Selden, Kinosian, & Gershman, 1992) and myosin ATPase activity (Watterson, Kohler, & Schaub, 1979). The low ATP concentration of 0.1 mM was found to be optimal for sliding actin filaments in myosin motility assays (Cooke & Bialek, 1979) and for contraction in active gels (Mizuno, Tardin, Schmidt, & MacKintosh, 2007). A gel with 0.1 mM myosin motors would exhaust all available ATP after only 1000 steps on average. But significantly increasing ATP concentration past 0.1 mM interferes with contraction (see below). In order to provide myosin motors with a continuing supply of free energy while maintaining a low ATP concentration, we use CP and CK, a substrateeenzyme pair derived from muscle. CK transfers a phosphate group from CP to ADP, which replenishes the pool of available ATP and maintains it at a constant value close to 0.1 mM, provided that the solution contains enough CP. DTT

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is a strong reducing agent which can even reduce disulfide bonds. It can thus prevent the formation of permanent disulfide cross-links between actin filaments, which would otherwise build up over time as the solution is exposed to oxygen (Tang, Janmey, Stossel, & Ito, 1999). Trolox is a water-soluble derivative of vitamin E which similarly maintains a reducing environment. It is commonly used in combination with fluorescent molecules to quench triplet states and prevent photobleaching due to reactions of the triplet states with oxygen free radicals (Cordes, Vogelsang, & Tinnefeld, 2009). PCA and PCD are a substrateeenzyme pair derived from plants which scavenge oxygen free radicals and therefore also aid in preventing photobleaching (Shi, Lim, & Ha, 2010). The main advantage of PCA/PCD over the more common oxygen scavenger glucose/glucose-oxidase is that PCA/PCD does not acidify the gel over time.

2.4 LOADING GELS INTO CHAMBERS We prepare contractile active gel samples with a final volume of 10 mL. Although 10 mL is much larger than the volume of 0.5 mL obtained from the dimensions of contraction chambers, preparing gels of a smaller volume is not advisable because pipettes are more susceptible to error as volume decreases. Mixing and loading the contractile gel should be done with care to ensure that the sample is homogeneous before it is loaded and that time-lapse imaging can be started immediately upon loading, according to the following protocol: 1. In one Eppendorf tube, place 8 mL of buffer, salts, and all proteins except actin. In a second tube, place 2 mL of actin at 2.5 mg/mL. 2. Place the contraction chambers on the microscope stage. Focus the microscope objective on an empty chamber. Begin image acquisition and proceed immediately to the next step. 3. Using a 2e20 mL pipette, aspirate the contents of the first tube and mix them into the second tube. Further mix the contents by aspirating up and down 2e3 times. The contents of the two tubes should be thoroughly mixed. This can be visually checked by holding the tube against a light source and verifying that Schlieren lines completely diminish. (Schlieren lines are optical streaks in transparent media that are visible to the naked eye. They form when spatial gradients in index of refraction are present, for instance, when diluting protein-rich solutions.) Take care not to introduce air bubbles while mixing. 4. Immediately load the mixed solution into the contraction chamber. Load only enough gel to fill the chamber. Do not overfill, or else the gel may contract to regions outside of the chamber and hence outside of the objective’s field of view. 5. Seal the two open ends of the contraction chamber with Baysilone silicone grease. a. Tip: When mixing components in Step 3, use the long pipette tips which are designed for use with 20 and 200 mL Eppendorf pipettes in combination with

3. Manipulating contractile active gels

b.

c.

d. e.

0.5 mL Eppendorf tubes. These tips are longer than the depth of the Eppendorf tubes, which allow free movement and greatly aids in homogeneously mixing the contents of the two tubes. The shorter tips meant for 2 mL Eppendorf pipettes cannot move freely inside the tubes, which impedes mixing and results in samples with inhomogeneous protein density and unpredictable contractile behavior. Tip: We dilute actin down to an intermediate concentration of 2.5 mg/mL with fresh G-buffer for two reasons. First, it is easier to design recipes and account for the extra ATP added by actin stocks when the concentration of actin remains constant. Second, the mixing process in Step 3 is more difficult when the actin concentration is too high. Dense actin solutions have a very high viscosity and are difficult to mix. They also polymerize very quickly and can thus further hinder homogeneous mixing. Tip: Once actin monomers are mixed with salts, they will polymerize to form filaments. Myosin motors will also activate and slide filaments. Therefore, it is important to execute the above protocol quickly. Starting image acquisition immediately before Step 3 provides a convenient way to measure sample age. Tip: A convenient way of applying Baysilone grease in Step 5 is to build a dispenser: cut a pipette tip and glue it to the outlet of a plastic syringe. Tip: We found Baysilone grease to be best suited for sealing contraction chambers because it flows along chamber edges to automatically create an airtight seal at corners. Vacuum grease does not tend to flow, often leaving corners unsealed. Silicone oil does seal corners but tends to flow too thinly and readily, and thus often creeps into nearby unused chambers, rendering them unusable for experiments. VALAP, a combination of vaseline petroleum jelly, lanolin, and paraffin, is not recommended since it must first be heated to temperatures exceeding 80  C, which may introduce undesirable temperature gradients in the gel.

2.5 MICROSCOPY We acquired images using a Nikon A1 R-MP confocal point scanner on an EclipseTi inverted microscope with a PlanFluor 4 objective (NA 0.13) and an A1 photomultiplier tube detector. Fluorescent molecules were excited with 488 and 561 nm laser light (Coherent).

3. MANIPULATING CONTRACTILE ACTIVE GELS So far we have described the procedure for preparing contractile active gels of actin filaments and myosin proteins, as well as chambers which accommodate these gels. The chambers are designed to visualize contraction events across an entire gel. This kind of visualization allows us to investigate how physical parameters affect

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contraction events over length scales of w30 mm and larger, which is longer than the lengths of individual actin filaments (w10 mm) and myosin motor filaments (w1 mm). In this section, we investigate the role of surface adhesion, monovalent salt concentration, nucleotide concentration, actin filament concentration, and actin filament length on the length scale of contraction.

3.1 STICKY SURFACES DECREASE CONTRACTION LENGTH SCALE Clean and passivated surfaces are essential in allowing active gels to freely contract. Networks in passivated chambers are free to contract macroscopically (Figure 3(A), top). After the experiment, confocal stacks reveal that proteins are distributed homogeneously in the z-direction (Figure 3(A), bottom). However, in the presence of sticky, nonpassivated surfaces, networks do not contract macroscopically (Figure 3(B), top). Furthermore, surfaces appear to be uniformly coated with actin and myosin (Figure 3(B), bottom). Similar behavior has been observed in twodimensional active gels, where fimbrin molecules were attached to a substrate-supported lipid-bilayer (Murrell & Gardel, 2012). Sticky surfaces attenuate contraction length scales, most likely because they allow myosin-generated stresses to build up and rupture the network into small clusters.

(A)

(B)

FIGURE 3 Sticky surfaces decrease the cluster size of contractile networks. (A) A macroscopically contracting network with passivated glass surfaces. (B) A macroscopically contracting network with nonpassivated, sticky glass surfaces. Top row: Time-overlay images. Color corresponds to time (calibration bar, left). Scale bars 1 mm. Times (tstart, tend) of color overlays, given as time after initiating actin polymerization: (A): (1 min, 38 min), (B): (1 min, 30 min). Bottom row: Average-y-projections of confocal stacks corresponding to dashed cyan boxes of top row. Vertical direction: z-direction, horizontal direction: x-direction. In sticky chambers, myosin is clearly localized on the bottom and top surfaces. (See color plate)

3. Manipulating contractile active gels

3.2 EXCESS MONOVALENT SALT AND NUCLEOTIDES WEAKEN MOTORS In our previous study (Alvarado et al., 2013), we modulated motor activity in crosslinked actinefascin gels by varying the concentration of motors. Here we demonstrate that motor activity can alternatively be modulated at constant motor density by varying the monovalent potassium chloride (KCl) concentration. Increasing [KCl] is expected to weaken motor forces, since the binding affinity of myosin II for actin is reduced with increasing ionic strength (Brenner, Schoenberg, Chalovich, Greene, & Eisenberg, 1982; Takiguchi, Hayashi, Kurimoto, & Higashi-Fujime, 1990), thus reducing motor processivity. The KCl concentration has also been reported to influence the size of myosin filaments (Davis, 1988; Kaminer & Bell, 1966; Koretz, 1979; Pinset-Ha¨rstro¨m & Truffy, 1979; Pollard, 1982; Reisler, Smith, & Seegan, 1980). Myosin is generally stored in nonfilamentous form in high-salt buffers (at least 300 mM KCl) and assembly is triggered by lowering the KCl concentration to below 150 mM. The myosin filament size depends on [KCl] as well as other buffer components, temperature, and the method of preparation (rapid dilution, gradual dilution, or dialysis). Here, we prepared myosin filaments by rapid dilution into a standard imidazole-based buffer, in which the filaments have an average length of 0.85 mm when formed at 50 mM KCl, and 0.63 mm when formed at 150 mM KCl (Soares e Silva et al., 2011). Figure 4 shows time-projections of movies of three different contractile networks, which all have the same initial network connectivity ([actin] ¼ 12 mM, [fascin] ¼ 260 nM) and motor concentration ([myosin] ¼ 340 nM). In the standard buffer with 50 mM KCl, the network contracts into many small clusters (Figure 4(A)). In our previous study (Alvarado et al., 2013), we attributed this rupture to the high myosin concentration used in the assay, which causes crosslink unbinding and thus a progressive reduction of the network connectivity. When we increase [KCl] to 75 mM, we again observe rupture into multiple clusters (Figure 4(B)), but the clusters are larger than at 50 mM KCl. Strikingly, a further increase of [KCl] to 100 mM results in the entire network contracting to one large cluster (Figure 4(C)). These experiments show that increasing the KCl concentration results in contraction events with a longer length scale. In our previous study, we found that decreasing myosin concentration similarly increased the contraction length scale because fewer motor-induced rupture events occurred (Alvarado et al., 2013). These results together suggest that adding excess monovalent salts weakens total motor activity (by reducing myosin’s binding affinity to actin as well as myosin filament length, both of which reduce motor processivity) and hence increases contraction length scale. To independently test this interpretation, we performed an additional contraction assay in which we kept [KCl] ¼ 50 mM as in the standard buffer, but increased the ATP concentration from 0.1 to 1 mM. According to single molecule force measurements (Debold, Patlak, & Warshaw, 2005; Finer, Simmons, & Spudich, 1994), the duty ratio of skeletal muscle myosin II is about 4% at millimolar levels of ATP,

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(A)

(B)

(C)

(D)

FIGURE 4 Lowering the motor activity increases the length scale of contraction. All images are timeoverlay images where color corresponds to time (see calibration bar, left). Assays are performed at fixed initial network connectivity ([actin] ¼ 12 mM and [fascin] ¼ 260 nM) and motor density ([myosin] ¼ 340 nM). (A) Under reference conditions ([KCl] ¼ 50 mM, [ATP] ¼ 0.1 mM), networks contract into many clusters with a scale-free size distribution. (B) Increasing [KCl] to 75 mM promotes larger-scale contraction. Moreover, we observe a second wave of contraction (reddish color). (C) Increasing [KCl] further to 100 mM promotes macroscopic contraction. (D) Increasing [ATP] to 1 mM increases the scale of contraction. For all panels, scale bars are 1 mm. Times (tstart, tend) of color overlays, given as time after initiating actin polymerization: (A): (30 s, 6 min), (B): (40 s, 4 min), (C): (11 min, 14 min), (D) (40 s, 6 min). (See color plate)

3. Manipulating contractile active gels

but increases by a factor of four when the ATP levels are reduced to 0.1 mM. When we increase [ATP] to 1 mM, we again observe a marked increase in the sizes of contracting clusters (Figure 4(D)) compared to the standard buffer conditions. This result is consistent with the interpretation that weaker myosin motor forces result in larger contraction events. To further investigate the effect of weakening motor activity, we also varied the KCl concentration in networks that contract macroscopically in the standard buffer (Figure 5(A)). This condition is attained by increasing the fascin concentration (from 260 nM in Figure 4 to 1.2 mM in Figure 5). When we increase [KCl] to 75 mM, we observe macroscopic network contraction, resembling the contraction at 50 mM KCl (Figure 5(B), top). However, increasing [KCl] further to 100 mM prevents contraction: the network remains static on macroscopic length scales (Figure 5(C), top). Apparently, the motors are not sufficiently processive to cause network contraction. To investigate how the network microstructure is influenced by variations in KCl concentration, we acquire confocal snapshots of the actinemyosin networks 2 h after initiating actin polymerization by a high-NA 100 microscopy objective (middle and bottom row). For the standard KCl concentration of 50 mM, we find a heterogeneous network of clearly distinguishable actin bundles in the contracted network (Figure 5(A)). This network is decorated with myosin foci of variable size, which appear alongside actin bundles but do not appear to be integrated in the network. Increasing [KCl] to 75 mM results in a finer meshwork of actin without pronounced actin bundles (Figure 5(B)). Myosin foci appear to be integrated in the actin network, often surrounded by shell of locally enhanced actin fluorescence intensity indicative of local network condensation. The foci have similar sizes as at [KCl] ¼ 50 mM, but they exhibit more irregular and nonconvex shapes. Further increasing [KCl] to 100 mM, where the network does not contract, results in a homogeneous network of actin bundles (Figure 5(C)). Large myosin foci seldom occur in these networks, although small puncta of myosin fluorescence (potentially single myosin filaments) are present all across the network (Figure 5(C), bottom, white arrows).

3.3 LOWER ACTIN CONCENTRATION DECREASES CONTRACTION LENGTH SCALE In our previous study (Alvarado et al., 2013), we modulated the network connectivity solely by varying the concentration of fascin cross-links. However, network connectivity also depends on the concentration of actin filaments as well as on their length. In semidilute solutions of long actin filaments, above the overlap concentration, steric entanglements can effectively act as cross-links on timescales of minutes to hours, depending on actin filament length (MacKintosh, Ka¨s, & Janmey, 1995; Schmidt, Hinner, & Sackmann, 2000). To test how actin concentration influences connectivity, we perform contraction assays at three different actin concentrations, keeping the myosin concentration and fascin concentration constant

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(A)

(B)

(C)

FIGURE 5 Degree of actin bundling around myosin foci depends on monovalent salt concentration: (A) [KCl] ¼ 50 mM, (B) [KCl] ¼ 75 mM, (C) [KCl] ¼ 100 mM. Top row: Time-overlay images of network contractions based on actin fluorescence. Color corresponds to time (calibration bar, left) and scale bars are 1 mm. Times (tstart, tend) of color overlays, given as time after initiating actin polymerization: (A): (45 s, 6 min), (B): (30 s, 3 min), (C): (1 min, 13 min). Middle row: Close-up of network structures corresponding to the areas indicated by the black squares in the top row. Actin is shown in red, myosin in cyan. Snapshots were acquired 2 h after initiating actin polymerization. Scale bars 30 mm. Bottom row: Further close-up of network structures corresponding to the areas indicated by the white squares in the middle row. Note in panel (C) the presence of small myosin puncta, likely corresponding to individual myosin filaments (white arrows). Scale bars 5 mm. For all panels, [actin] ¼ 12 mM, [myosin] ¼ 120 nM, [fascin] ¼ 100 nM. (See color plate)

([myosin] ¼ 120 nM, [fascin] ¼ 500 nM). At the largest actin concentration (12 mM), we observe macroscopic contraction, consistent with the high cross-link concentration and low motor density used (Figure 6(A)). When we decrease the actin concentration to 6 mM, the network is instead ruptured into multiple large clusters, indicative of lower network connectivity (Figure 6(B)). Further decreasing actin

Outlook

(A)

(B)

(C)

FIGURE 6 Reducing the network connectivity by decreasing the actin concentration causes network contraction into smaller clusters. (A) Time-overlay image of a macroscopically contracting network ([actin] ¼ 12 mM). Color corresponds to time (calibration bar, left). (B) Decreasing [actin] to 6 mM. (C) Decreasing [actin] further to 3 mM. For all panels, [myosin] ¼ 120 nM, [fascin] ¼ 500 nM. Times (tstart, tend) of color overlays, given as time after initiating actin polymerization: (A): (40 s, 20 min), (B): (1 min, 14 min), (C): (1 min, 1.5 h). (See color plate)

concentration to 3 mM results in the formation of many more, much smaller clusters (Figure 6(C)), indicative of a further reduction of network connectivity.

3.4 GELSOLIN SHORTENS ACTIN FILAMENTS AND PREVENTS CONTRACTION To test the influence of filament length, which also influences connectivity, on network contraction, we polymerize actin in the presence of the protein gelsolin, which caps actin filaments at the barbed end. We use 120 nM gelsolin, which is expected to result in an average filament length of w300 nm (Janmey, Peetermans, Zaner, Stossel, & Tanaka, 1986). This drastic reduction in filament length abolishes network connectivity. Instead of a connected network, we observe isolated foci of myosin motors surrounded by a halo of actin filaments/bundles (Figure 7(A)). The foci freely diffusive and coalesce to form larger foci when they collide, as demonstrated in the kymograph in Figure 7(B), where the arrow points to the coalescence event. The coalescence events are irreversible (Figure 7(C)). This coalescence behavior is in marked contrast to the coalescence of actinemyosin foci in the absence of gelsolin, where the actin filaments are sufficiently long to form a connected network (Figure 7(D)). In connected networks, coalescing foci do not diffuse, but move in a directed manner toward one another at typical velocities of w2 mm/ min (Figure 7(E)), consistent with prior reports (Soares e Silva et al., 2011). In this case, coalescence is also irreversible (Figure 7(F)).

OUTLOOK In this chapter, we have described how to mimic contraction events in a wellcontrolled model system of reconstituted proteins. In particular, the contraction

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(A)

(B)

(D)

(E)

(C)

(F)

FIGURE 7 Shortening the actin filaments by copolymerizing actin with gelsolin results in disconnected actinemyosin foci, which freely diffuse and coalesce when they collide. (A) In the presence of 120 nM gelsolin, foci of actin and myosin are freely diffusing in solution. [actin] ¼ 12 mM, [myosin] ¼ 120 nM, [fascin] ¼ 260 nM. Scale bar 10 mm. (B) Kymograph constructed along dashed line in (A). Scale bar 10 mm 20 s. (C) Snapshot of actinemyosin foci after coalescence. (D) In dense networks of long filaments ([actin] ¼ 12 mM, no gelsolin, [myosin] ¼ 120 nM, [fascin] ¼ 120 nM), foci of actin and myosin are connected by a background meshwork of cross-linked actin filaments. Actin is shown in red (gray in print versions), myosin in cyan (light gray in print versions). Scale bar 10 mm. (E) Kymograph showing time along the horizontal direction and fluorescence intensity along the dashed yellow (white in print versions) line from panel (D) along the vertical direction. Scale bar 10 mm  20 s. (F) Maximum-z-projection over 17 mm of actinemyosin foci after coalescence.

chambers we developed allow for the visualization of an entire contractile gel, which is necessary to quantify the effect of network connectivity and motor activity on contraction length scales which can be much larger than the size of the individual protein constituents. It is at these length scales where contraction events remain most poorly understood. The physical mechanisms of long-length-scale contractile force propagation should provide fertile ground for future experimental and theoretical research.

ACKNOWLEDGMENTS We thank M. Kuit-Vinkenoog (AMOLF) for assistance with protein purification, M. PreciadoLo´pez (AMOLF) for assistance with surface passivation, and S. Hansen and R.D. Mullins (UC, San Francisco) for the fascin plasmid. This research was supported by a VIDI grant, which was financed by the Netherlands Organisation for Scientific Research (NWO). This work was furthermore part of the research programme of the Foundation for Fundamental Research on Matter (FOM), which is part of NWO.

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