In Vitro Actin Assembly Assays and Purification From Acanthamoeba

Abstract

The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility and endocytosis. Actin polymerization is tightly regulated by various cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. The pyrene actin assembly assay is a powerful tool for determining the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleators or capping factors. In addition, fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how these proteins influence actin filament superstructure.

1 Introduction

Over the past few decades dozens of actin-binding proteins have been discovered, and there is a rapidly growing body of work focused on dissecting how each of these proteins regulates the kinetics and morphology of actin structures (1). Actin dynamics can be divided into three steps, each of which is specifically regulated. Polymerization is a nucleation-condensation reaction (2), which means that the first step (nucleation) is slow, and the second step (elongation) proceeds quickly once a stable nucleus is formed (3). Nucleation refers to the assembly of actin monomers into a stable trimer, which is usually an extremely unfavorable reaction. As the filament elongates, incorporated actin monomers hydrolyze their ATP to ADP, making filaments more susceptible to depolymerization, the third step in the cycle. All steps of this cycle are regulated by actin-binding proteins (1). The rate of nucleation is controlled by three distinct classes of actin nucleators: the Arp2/3 complex (4), formins, and spire (5,6). Growth, maintenance, and structural integrity of filaments are controlled by classes of proteins that cap, bundle, sever, or disassemble filaments.

The primary in vitro assay used to study the effects of actin-binding proteins on actin polymerization is the pyrene actin assembly assay, wherein the environmentally sensitive fluorophore pyrene indicates the polymerization state of actin (7). Pyrene-labeled actin incorporated in a filament fluoresces significantly greater than a labeled monomer in solution (8). Increase in fluorescence over time is directly proportional to the increase in filamentous actin (9).

Actin polymerization assays are conducted using buffer conditions similar to those in vivo. Importantly, actin requires magnesium-ATP in its nucleotide pocket and physiological concentrations of potassium for rapid polymerization at micromolar concentrations. Actin is stored in Buffer A—which contains calcium instead of magnesium and lacks potassium—to prevent polymerization. Immediately before the assay, calcium is exchanged for magnesium by adding ethylene glycol tetraacetic acid (EGTA) and magnesium. Polymerization is triggered by the addition of buffer containing potassium (10).

There are two standard sources of actin for biochemical studies: Acanthamoeba castellani and rabbit skeletal muscle. Actin from ameba is cytoplasmic and is a good model for cytoplasmic actins from other organisms (11). Skeletal actin does not bind to some actin-binding proteins (12). Furthermore, Acanthamoeba is an excellent source of actin-binding proteins, such as the Arp2/3 complex (13,14). Although it is best to use actin and actin-binding proteins from the same organism and tissue, actin is highly conserved, and in most cases ameba actin can be used in conjunction with proteins from other organisms (6,10).

Although the pyrene assay is very powerful for measuring kinetics of actin assembly, it does not reveal the structure of the actin filament networks it produces. There are two commonly used techniques that address this: (1) fixing and staining polymerization reactions with fluorescently labeled phalloidin (6) and (2) electron microscopy (15). Fixing actin with labeled phalloidin, detailed below, is a straightforward method that can be done in parallel with pyrene actin experiments.

2 Materials

2.1 Culture of A. castellani

• 1. Ameba medium: 7.5 g/L proteose peptone, 7.5 g/L yeast extract, 15 g/L glucose, 0.2 mM methionine, 3 mM KH₃PO₄, 10 μM CaCl₂, 1 μM FeCl₃, 0.1 mM MgSO₄, 1 mg/L thiamine, 0.2 mg/L biotin, 0.01 mg/L vitamin B₁₂. Autoclave before use.

• 2. Wash buffer: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl.

2.2 Actin Purification From A. castellani

• 1. Extraction buffer: 10 mM Tris-HCl, pH 8.0, 11.6% (w/v) sucrose, 1 mM EGTA, 1 mM ATP, 5 mM dithiothreitol (DTT) (Roche Applied Science, Mannheim, Germany), 30 mg/L benzamidine (Sigma-Aldrich, St. Louis, MO), 5 mg/L pepstatin A (Sigma-Aldrich), 10 mg/L leupeptin (Sigma-Aldrich), 40 mg/L soybean trypsin inhibitor (Sigma-Aldrich), 1 mM phenylmethylsulfonylfluoride (PMSF; Sigma-Aldrich). The first three ingredients may be combined a day in advance and stored at 4°C. Add the final ingredients fresh, pH to 8.0, and bring up to volume with cold water (see Note 1 ). Add PMSF immediately before use.

• 2. Pepstatin A (1000X): 5 mg/mL pepstatin A (Sigma-Aldrich) in dimethylsulfoxide; store at −20°C.

• 3. Protease inhibitor cocktail (100X): 1 mg/mL leupeptin and 4 mg/mL soybean trypsin inhibitor in water; store at −20°C.

• 4. PMSF (Sigma-Aldrich): 200 mM in ethanol; store at −20°C. Before use, warm to 25°C with rocking to resuspend. The half-life of PMSF in aqueous solution is approx 30 min, and so it is always added to buffers immediately before use.

• 5. 100 mM ATP, pH 7.0, dissolved in water.

• 6. Column buffer: 10 mM Tris-HCl, pH 8.0, 0.2 mM CaCl₂, 30 mg/L benzamidine, 0.5 mM ATP, 0.5 mM DTT. Add last three ingredients fresh and bring buffer up to volume with cold water. Adjust buffer to pH 8.0 while at 4°C, just before use.

• 7. Diethylaminoethyl cellulose (DEAE) (DE52, Whatman, Maidstone, England). After use, cycle with 0.5 M NaOH and HCl to wash, and store in Tris base, pH 8.0, 0.2% (v/v) benzalkonium chloride at 4°C indefinitely (see Note 2 for details on washing DEAE).

• 8. 1 M Tris base (Trizma, Sigma-Aldrich).

• 9. High-salt buffer: 600 mM KCl, 10 mM Tris-HCl, pH 8.0, 0.2 mM CaCl₂, 30 mg/L benzamidine, 0.5 mM ATP, 0.5 mM DTT. Prepared as extraction buffer (step 1).

• 10. 200 mM MgCl₂.

• 11. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Invitrogen, Carlsbad, CA): prepare 0.5 M stock in cold water and pH to 7.0 with KOH.

• 12. Buffer A (1X): 2 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.5 mM TCEP, 0.1 mM CaCl₂. Bring pH to 8.0 with KOH, and bring up to volume with cold water (see Note 3 ).

• 13. KMEI buffer (10X): 500 mM KCl, 10 mM MgCl₂, 10 mM EGTA, 100 mM imidazole HCl, pH 7.0 (see Note 4 ).

• 14. Gel filtration column: flex column (25 mm × 100 cm, Fisher Scientific, Pittsburgh, PA).

• 15. Sephacryl S-200 HR resin (GE Healthcare, Little Chalfont, UK). Resin comes stored in 20% (v/v) ethanol and will swell when fully hydrated. To avoid cracking the resin, equilibrate in buffer before decanting into gel filtration column. Pack the column by flowing one to two column volumes of buffer at a faster rate than will be used in gel filtration.

2.3 Pyrene Labeling of Actin

• 1. Actin: If actin is from Acanthamoeba, it must be gel filtered before labeling. Skeletal actin does not have this requirement.

• 2. KMEI buffer (10X): see Subheading 2.2. , item 13.

• 3. Dialysis buffer: 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM imidazole HCl, pH 7.0, 0.2 mM ATP.

• 4. Pyrenyl iodoacetamide (Invitrogen): 10 mM stock solution in DMF, stored at −20°C in the dark.

• 5. Buffer A (1X): see Subheading 2.2 , step 12.

• 6. Dithiothreitol (DTT): 1 M stock in water.

2.4 Pyrene Actin Assembly Assay

• 1. Buffer A (1X): see Subheading 2.2 , step 12.

• 2. KMEI buffer (10X): see Subheading 2.2 , step 13.

• 3. ME buffer (10X): 0.5 mM MgCl₂, 2 mM EGTA.

• 4. Ethanol, high-performance liquid chromatography grade, for washing cuvets.

• 5. Vakuwash cuvet washer (Fisher Scientific).

2.5 Microscopy of Labeled Actin Filaments

• 1. Reagents for pyrene actin assembly assay ( Subheading 2.4. ).

• 2. Alexa-fluor 488 Phalloidin (Invitrogen): make stocks in methanol, to be in molar excess to actin in reaction. For example, 5 μL of a 6.6 μM stock of phalloidin was used to stabilize an equal volume of 4 μM actin (6). Store stocks at −20°C for several months. TRITC phalloidin (Sigma) also works well.

• 3. Wide-bore pipet tips: cut pipet tips with a sterile razor, and be consistent.

• 4. Poly-L-lysine: prepare a 1 mg/mL stock in water.

• 5. Poly-L-lysine-coated cover slips: clean slides by sonication first in 1 M KOH, then in ethanol. Rinse in water and air-dry. Spot 1 mg/mL poly-L-lysine on parafilm and float cover slips on drops (500 μL) for a few minutes; rinse with water, and use within a few days.

• 6. Clear nail polish.

3 Methods

3.1 Culture of A. castellani

• 1. Passage ameba in 10-mL culture tubes with moderate bubbling of humidified air at 25°C (16,17).

• 2. Fill beveled Fernbach flasks (3 L) with 1 L ameba medium and inoculate with cultures. Grow with moderate shaking at 25°C until growth plateaus (see Note 5 ). Use Fernbachs to inoculate 14-L carboys outfitted with bubblers. Practice sterile technique to avoid contaminating the cultures, which grow without any antibiotic.

• 3. When cultures plateau, harvest by centrifugation at 4500g (4000 rpm in a Sorvall RC-3B) for 5 min. Refill tubes and centrifuge again until all ameba have been pelleted.

• 4. Wash ameba with wash buffer. Resuspend cell pellets in the smallest volume of buffer possible. Pool these and centrifuge as above. In this way all ameba are transferred into one or two tubes. During the pairing-down of tubes, weigh ameba to get approx 500 g (enough for actin purification) into a single tube.

3.2 Purification of Actin From A. castellani

• 1. (Example volumes given are for purification from 500 g ameba. Buffer and resin volumes can be scaled linearly as needed.) Resuspend pelleted ameba in 1 L cold extraction buffer. If ameba are frozen, thaw in cold extraction buffer (see Note 6 ). After resuspension, conduct all subsequent steps at 4°C (see Note 7 ).

• 2. Lyse cells by nitrogen decompression in a Parr Bomb (Parr Instrument Company, Moline, IL). Rinse the Parr Bomb with water and cool on ice. Transfer cells into the Parr Bomb and pressurize with nitrogen gas at 400 psi for 5 min. To lyse, release cells from the Parr Bomb slowly, while maintaining constant pressure.

• 3. To remove cellular debris, centrifuge lysate at more than 5000g (7000 rpm in a GS-3 rotor) for 10 min at 4°C. Centrifuge supernatant from this spin at more than 100,000g (38,000 rpm in a Beckman Ti45 rotor) for 2 h at 4°C.

• 4. Equilibrate 500 g DEAE resin in 2 L of column buffer. To equilibrate large volumes of resin, mix resin and buffer in beaker, then pour over a Büchner funnel fitted with a Whatman filter 541 (ashless) and pull vacuum until buffer has passed through but the resin is still damp. It is essential that the pH of the resin is 8.0 before use.

• 5. After the second spin, siphon off the lipid layer at the very top of each tube. Decant supernatants into a rinsed glass beaker, on ice. Mix supernatant with approx 450 mL equilibrated DEAE resin, and stir for 30–60 min at 4°C to batch bind.

• 6. In the cold room, pour remaining approx 50 mL equilibrated DEAE resin into a glass column (diameter 5 cm; height 60 cm) and let resin settle and excess liquid drip through. Gently pour DEAE slurry above this, and collect flow-through (see Note 8 ). Wash resin with 1 L column buffer, pumped at 6 mL per min.

• 7. To elute actin, use a gradient maker to run a linear gradient from 0 to 600 mM KCl using 1 L column buffer (low-salt buffer) and 1 L high-salt buffer, pumped at 3 mL per min. Actin normally elutes as a broad (∼200 mL) peak between 300 and 400 mM KCl. Keep actin fractions in the cold room overnight, covered. (See Note 2 regarding recycling of DEAE resin.)

• 8. The next day, move fractions to 25°C to speed up polymerization. Actin will polymerize in a few hours. Detect polymerized actin fractions by tipping fraction tubes sideways; if there is sufficient polymerized actin, the fraction will be a gel.

• 9. Pool actin fractions into a beaker. Add MgCl₂ to 2 mM and ATP to 1 mM. Stir actin gently for 30 min at 25°C to complete polymerization.

• 10. Centrifuge actin at 1000g (3000 rpm in a Sorvall GSA rotor) for 5 min at 4°C. Pour off supernatant, and wash with 1X KMEI buffer. Centrifuge at more than 100,000g (38,000 rpm in a Ti45 rotor) for 2 h at 4°C. Discard supernatant and rinse the pellet (polymerized actin) quickly with Buffer A. Scrape actin into a Dounce homogenizer. Homogenize 5–10 times with a “loose” pestle in Buffer A to 40–80 mL final volume without creating air bubbles.

• 11. Dialyze into Buffer A for 2–3 d at 4°C to depolymerize actin. Change dialysis buffer daily. Actin can be stored in dialysis at 4°C for several weeks.

• 12. Equilibrate an S-200 column with Buffer A. Centrifuge 15–20 mL depolymerized actin at more than 100,000g (38,000 rpm in a Ti45 rotor) for 2 h at 4°C. Load actin onto S-200 column and gel filter in Buffer A, collecting approx 3-mL fractions.

• 13. Determine the concentration of unlabeled actin by measuring absorbance at 290 nm, using an early fraction from the column to blank the spectrophotometer (see Note 9 ). The concentration of actin is 38.5 μM per O.D. 290, assuming a 10-mm pathlength.

• 14. Gel-filtered actin can now be pyrene labeled or used unlabeled in polymerization assays ( Fig. 1 A). For pyrene labeling, use the entire second (major) peak. Dark actin for fluorimetry is taken from the back end (later fractions) of this peak. The front end of the peak contains actin dimers, which will interfere with kinetic measurements (10).

• 15. Assay the purity of actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining. Actin is 42 kDA and should appear as a single band with little to no breakdown.

Fig. 1.

Gel filtration of actin from Acanthamoeba castellani. (A) Depolymerized actin was gel filtered over S-200 resin in Buffer A. Absorbance at 290 nm was measured and concentration of actin plotted for each fraction. The first peak contains contaminants, including α-actinin. The second (major) peak is actin. It is important to use only the back end of the peak for fluorimetry (20). (B) Pyrene-labeled actin was gel purified over S-200 resin in Buffer A. Absorbances at 290 and 344 nm were measured, and concentrations of total actin (black line) and pyrene actin (gray line) were plotted. Percent labeled equals the ratio of pyrene actin to total actin. As above, use the back end of the peak for fluorimetry.

3.3 Pyrene Labeling of Actin

• 1. Pool 30–80 mg of actin and measure concentration. Dilute actin to 1.1 mg/mL (26.4 μM; because of the limited solubility of pyrene iodoacetamide, the final concentration of actin should be 1 mg/mL) in Buffer A.

• 2. Polymerize actin by adding one-tenth the final volume of 10X KMEI buffer and let stand at 25°C. Actin will be completely polymerized within a few minutes; look for bubbles in the actin solution that do not move.

• 3. To remove reducing reagent for labeling, carefully transfer polymerized actin into dialysis tubing, and dialyze into dialysis buffer for a few hours at 4°C.

• 4. Transfer actin to a beaker and measure the volume. Add 4–7 mol pyrenyl iodoacetamide per mol actin while stirring. Cover the beaker to keep dark, and gently stir overnight at 4°C. Quench reaction by adding DTT to 10 mM.

• 5. Centrifuge labeled actin at 2000g (5000 rpm in a Beckman JA-20) rotor for 5 min at 4°C to pellet precipitated dye.

• 6. Centrifuge supernatant at more than 100,000g (38,000 rpm in a Ti45 rotor) for 2 h at 4°C to pellet actin filaments.

• 7. Homogenize the pellet with a Dounce homogenizer, as above, in Buffer A, into a final concentration of about 2–6 mg/mL (48–144 μM) (see Note 10 ).

• 8. Dialyze into Buffer A at 4°C using at least two changes of buffer over at least 2 d to depolymerize actin.

• 9. Centrifuge depolymerized pyrene actin at more than 100,000g (38,000 rpm in a Ti45 rotor) for 2 h at 4°C to remove remaining actin filaments.

• 10. Gel filter supernatant over S-200 resin equilibrated with Buffer A, as before.

• 11. Determine the concentration and percent labeling by measuring absorbance at 290 and 344 nm. To correct for pyrene absorption at 290 nm: The concentration of total actin is thus 38.5 μM per corrected OD₂₉₀. The concentration of pyrene actin is 45.0 μM per OD 344. To calculate percent labeling:

• 12. Store pyrene actin fractions on ice, in the dark, in the cold room. Pyrene actin is more stable than unlabeled actin and can be used for several months.

3.4 Pyrene Actin Assembly Assay

• 1. Mix unlabeled actin, pyrene actin, and Buffer A to achieve a working stock that is 5–10% labeled and 5–20 times the concentration to be used (see Note 11 ). Store on ice, in the dark.

• 2. Set up the fluorimeter. Set excitation wavelength to 365 nm and emission wavelength to 407 nm.

• 3. Assemble reagents by the fluorimeter. (All volumes listed assume 100-μL reaction.) Prealiquot buffers into labeled Eppendorf tubes to ensure that concentrations of each reagent are exactly the same for each experimental condition: (1) 10-μL aliquots of 10X KMEI buffer and (2) aliquots of 10X ME buffer (one-tenth the volume of actin added to the reaction). Make a dilution series of the protein to be tested in Buffer A and store on ice (see Note 12 ). Clean cuvets extensively with water, ethanol, and water, and wipe dry with lens paper.

• 4. Set a stopwatch to 3 min and start time. Immediately, add actin to prealiquotted 10X ME buffer to exchange Ca²⁺ for Mg²⁺. Exchange for 2 min exactly.

• 5. During this 2-min incubation, assemble the rest of the reagents: Add proteins to be tested to 10 μL prealiquotted 10X KMEI buffer (see Note 13 ). Add Buffer A to bring the total reaction volume (including actin and ME buffer) to 100 μL. Set a pipetteman to 110 μL, and pipet up the KMEI-protein mixture.

• 6. After 2-min exchange, pipet KMEI-protein mixture into the actin. Triturate the reaction one to three times (be consistent) to mix thoroughly, then pipet into the cuvet, being careful to avoid bubbles. Load the cuvet into the fluorimeter and activate data collection. Record dead time: the time from addition of the KMEI-protein mixture to actin until the second the first data point is collected. Always collect complete data sets until pyrene fluorescence plateaus. Try the same condition at least three times to ensure reproducibility. (See Note 14 for tips on improving data.)

• 7. Every time the fluorimeter is turned on, or a different concentration of actin is used, optimize settings to maximize signal while eliminating photobleaching (see Note 15 ). Polymerize actin as described, and monitor the plateau over the course of 1 h, maximizing the signal that causes no appreciable drop in plateau fluorescence over time. This is usually achieved by adjusting the diaphragm between the light source and the cuvet (see Note 16 ).

• 8. Figure 2 illustrates polymerization data and how they are analyzed. Plot fluorescence (arbitrary units) as a function of time (in seconds). Add back dead time to x-axis data (time). Subtract baseline fluorescence from all y-axis data. Baseline fluorescence is fluorescence at time zero, assuming the reaction is slow enough to catch. If the conditions used do not alter the critical concentration of actin (i.e., protein being tested does not cap actin filaments or sequester monomers), normalize data by dividing all y-axis data by the fluorescence at plateau. See ref. 10 for methods to determine critical concentration change.

• 9. Reaction half-times are convenient metrics to measure ( Fig. 2 C, inset). Replot the linear region of the curve surrounding half-maximal fluorescence, and fit a linear equation to these points. Use this equation to solve for half time (the value of x where y = 0.5, if data are normalized).

Fig. 2.

Pyrene actin assembly assay. (A) Actin was polymerized as described; curves show spontaneous nucleation of 2 μM (black), 4 μM (dark gray), and 8 μM (light gray) actin. These data have been zeroed by subtracting baseline fluorescence from all fluorescence values. (B) Data from above were normalized by dividing all fluorescence values by average fluorescence at plateau. (C) 2 μM actin was polymerized in the presence of 10 nM Arp2/3 complex and 5 nM Listeria ActA. Inset: To calculate time to half-maximal fluorescence (t _1/2), the linear region of the curve in (C) spanning 0.5 was replotted and a linear trendline was fit to the data. Solving for time (x) where y = 0.5 yields t _1/2.

3.5 Microscopy of Labeled Actin Filaments

• 1. Polymerize unlabeled actin using conditions described above in an Eppendorf tube. Allow reaction to come to plateau.

• 2. In the meantime, desiccate Alexa-Fluor 488 phalloidin in a speed vacuum to remove all methanol. Labeled phalloidin should be superstoichiometric to actin.

• 3. Pipet 5 μL of polymerized actin—using a wide-bore pipet tip—into the tube containing desiccated phalloidin to stabilize actin filaments.

• 4. Add 45 μL of 1X KMEI buffer to dilute the reaction. Dilute to low nanomolar concentrations to resolve individual filaments.

• 5. Pipet 10 μL of the dilutions onto slides and cover with poly-L-lysine cover slips. Seal slides with clear nail polish.

• 6. Once nail polish dries, image filaments with an epifluorescence microscope.

4 Notes

• 1. All solutions should be prepared in double-distilled water with resistivity of 18.2Ω. Cold water refers to double-distilled water at 4°C.

• 2. More complete protocol: dry DEAE resin (vacuum resin until just damp; not white). Transfer to beaker and add water to 4 L. Add solid NaOH to 0.5 M and stir 5 min at 25°C. Dry and wash with 4 L water. Repeat NaOH wash; this time stir for 30 min. Dry and wash with 8 L water. Resuspend in 2870 mL water and add 130 mL concentrated HCl. Stir 30 min at 25°C. Dry and wash with 8 L water. Resuspend in 3–4 L water and add unbuffered 1 M Tris base until pH is 8.0. Add 0.2% (v/v) benzalkonium chloride and store at 4°C.

• 3. Make Buffer A as a 100X stock without CaCl₂—calcium-ATP precipitates at this concentration—and freeze at −20°C. Add CaCl₂ during dilution to 1X, and aliquot in 15-mL conical tubes and freeze at −20°C. Once thawed, one aliquot may be used for up to 1 wk.

• 4. Imidazole is best stored in the dark, at 4°C, as a 1–2 M stock solution. Store 10X KMEI buffer for fluorimetry in the dark at 4°C. Discard when autofluorescence of the buffer becomes noticeable during fluorimetry.

• 5. Assay growth by absorbance at 600 nm. A typical OD 600 of ameba for purifying actin is 6. OD 600 values greater than 1.0 are out of the linear range in most cases, so dilute the ameba before measuring. Alternatively, ameba can be grown in a fermentor.

• 6. We have noticed inconsistencies with actin purified from ameba that have been frozen for longer than 6 mo.

• 7. The first day of the actin purification protocol is significantly long and is aided by making buffers and gathering equipment and reagents the day before. Once started, the entire purification takes 5–7 d, with 2–3 d waiting for the actin to depolymerize in the middle.

• 8. To eliminate bubbles when decanting resin slurry into the column, pour it slowly down the outside of a Pasteur pipet or glass stir rod onto the inside wall of the column. Do not let the resin drip directly into the column.

• 9. Actin requires ATP, which absorbs significantly less at 290 nm than at 280 nm. To limit the time that actin fractions are out of the cold room, it is practical to reuse pipets and not wash the cuvet between each reading. To avoid contaminating monomeric actin with actin dimers, start at the highest fraction and take readings backwards.

• 10. The critical concentration of calcium-actin is 150 μM (10). Actin will not depolymerize above this concentration.

• 11. Concentrations of both dark actin and pyrene actin stocks must be measured. Because some proteins do not bind as tightly to pyrene actin as to dark actin, the actin used in fluorimetry is normally only 5–10% pyrene actin (10). To calculate how much pyrene actin and dark actin to combine to yield a given percent labeling and a given concentration, use the following equations:

  • Let L _i = starting percent labeling of pyrene actin (100% labeled = 100)

  • Let C _p = starting concentration of pyrene actin (μM)

  • Let C _d = starting concentration of dark actin (μM)

  • Let L _f = final percent labeled (i.e., 5–10)

  • Let C _f = final concentration of actin (μM)

  • Let V _f = final volume of actin (μL)

  Bring up to volume (V _f) with Buffer A.

  Because ATP absorbs significantly at 280 nm, proteins other than actin should not be in Buffer A for quantification by spectrophotometry.

  It may be desirable to premix proteins before starting actin exchange to allow them to come to equilibrium. Although not recommended for detailed kinetic analysis, it is possible to add Escherichia coli lysate containing expressed protein to pyrene actin assembly assays as a first-pass functional test. Uninduced (control) lysate should have no effect on actin polymerization.

  There are many factors to consider for collecting meaningful and reproducible fluorimetry data. To list key points:

  • a. Always do an actin-alone (control) reaction to test the actin first.

  • b. The assay is very sensitive to the concentration of potassium, so keep that constant in all reactions. In the event that a protein component requires potassium, adjust the molarity of KCl in 10X KMEI buffer to keep the final concentration of potassium in the reaction at 50 mM.

  • c. Exchange in 10X ME buffer for exactly 2 min and be consistent.

  • d. Eliminate photobleaching.

  • e. Always collect complete data sets—until pyrene fluorescence plateaus.

  • f. Wash cuvets extensively between each reaction, because polymerized actin remaining in the cuvet will nucleate new filaments in the next run.

  • g. Do your best to avoid bubbles, which both scatter light and cause oxidative damage to proteins.

  • h. Store proteins on ice, but ensure that all components are at 25°C at the start of the reaction. Preincubating tubes in a water bath works well.

  • i. When expressing glutathione-S-transferase (GST)-tagged recombinant proteins, it is critical to remove the GST, because it will mediate the dimerization of the fusion protein. For example, Arp2/3 activators display faster kinetics when the GST remains uncleaved, which is thought to be an artifact of this dimerization (18,19).

• 15. Photobleaching is a complex phenomenon that cannot be corrected for mathematically (10). If the overall signal is too low after eliminating photobleaching, try widening emission slits, increasing the voltage of the photomultiplier, or increasing pyrene-doping of actin, and again measuring photobleaching after the change.

• 16. We polymerize the concentration of actin that will be used in the experiments in the presence of Arp2/3 and an activator (like Listeria ActA), which comes to plateau very quickly. It is also possible to polymerize a high concentration of actin, and then dilute down to working concentration and measure in the fluorimeter.

• 17. First, to determine when a given condition will plateau, perform pyrene actin assembly assay as above. For microscopy, reaction volumes can be scaled down to less than 100 μL.