We sincerely acknowledge the members of our new home at LPENS for their warm welcome, and in particular, the ABCDJ team for all their help and support. J.P. acknowledges financial support from the Foundation ARC (Grant PJA 20191209604), and C.S. acknowledges financial support from the Human Frontiers Science Program Organization (Grant RGP0026/2020).

1. Preparation of buffers
NOTE: Use ultrapure H2O for all buffers. It does not have to be sterile. Filter all the solutions described in steps 1.1-1.4 with a 0.2 &#181;m syringe filter, aliquot in portions of 500 &#181;L-2 mL per tube depending on usage, and store at -20 &#176;C.
<ol>
	<li>Prepare 10% BSA by weighing 2 g of BSA into a 50 mL conical tube and filling to the 20 mL mark with H2O. Mix until the BSA is dissolved (about 30 min), and then make up the volume to 20 mL. 
	NOTE: Use high quality BSA (see Table of Materials). The 10% BSA solution is used in both bead preparation and the motility mix.</li>
	<li>For bead preparation, prepare Xb buffer (10 mM HEPES, 0.1 M KCl, 1 mM MgCl2, and 0.1 mM CaCl2, pH 7.5) as a 10x solution, and dilute before use (100 &#181;L of 10x Xb stock solution + 900 &#181;L of H2O). Prepare Xb/1% BSA by mixing 100 &#181;L of 10x Xb stock solution + 100 &#181;L of 10% BSA + 800 &#181;L of H2O.</li>
	<li>Prepare G-buffer (2 mM Tris, 0.2 mM CaCl2, 0.2 mM DTT, 2 mM ATP), which is the buffer used to dilute monomeric actin (G-actin). Adjust to pH 7, not pH 8 as traditionally used (see discussion).</li>
	<li>Prepare the motility buffer MB13 (10 mM HEPES, 1.5 mM ATP, 3 mM DTT, 1.5 mM MgCl2, 1 mM EGTA, 50 mM KCl, 1% BSA, pH 7.5). For some applications, 10x MB13 is useful. However, prepare 10x MB13 without BSA, as it leads to problems during pH adjustment. Add BSA from 10% stock solution (prepared in step 1.1) when reconstituting 1x MB13 from 10x MB13.</li>
</ol>
2. Preparation of protein solutions
NOTE: Use ultrapure H2O for all resuspensions. It does not have to be sterile. Handle all proteins on ice and aliquot into pre-chilled tubes. Manipulate gently so as not to produce bubbles, and never vortex protein solutions. For stocks to be stored at -80&#176;C, flash freezing in liquid nitrogen is not necessary. Adapt aliquot size to avoid more than about five freeze-thaw cycles, as this does not appear to affect the activity of any of the proteins. Working aliquots can be stored at -20 &#176;C for a few weeks.
<ol>
	<li>Prepare G-actin (rabbit skeletal muscle) solution as described below.
	<ol>
		<li>Pulse centrifuge actin powder (see Table of Materials) at 4 &#176;C to gather the solid at the bottom of the tube.</li>
		<li>Add H2O as per manufacturers&#39; instructions (1 mg protein in 100 &#181;L of cold H2O for unlabeled actin, 100 &#181;g protein in 100 &#181;L of cold H2O for ATTO-labeled actin).</li>
		<li>Let it sit on ice for at least 15 min. Mix gently by pipetting up and down, let it sit on ice at least another 15 min, and mix again. Pulse centrifuge at 4 &#176;C to collect the solution at the bottom of the tube, and remix.</li>
		<li>Prepare 10-50 &#181;L aliquots of unlabeled actin depending on use, and 20 &#181;L aliquots of ATTO-labeled actin. Store the aliquots at -80 &#176;C.</li>
		<li>To depolymerize actin oligomers that form during lyophilization and freezing, dilute an aliquot of resuspended actin ~8-fold in G-buffer spiked with additional ATP and DTT (for example, to 20&#181;L of resuspended actin solution from step 2.1.4, add 134 &#181;L of G-buffer, 0.32 &#181;L of 0.2 mM ATP and 0.16 &#181;L of 1 M DTT). For fluorescent labeling, add approximately 10% labeled actin; for example, add 5 &#181;L of ATTO-labeled actin to 40 &#181;L of diluted unlabeled actin. Let it depolymerize on ice with occasional mixing (pipetting) at least a few days to a week before measuring protein concentration by Bradford assay as described in step 3. 
		NOTE: Keep diluted unlabeled and fluorescent actin on ice in a cold room or refrigerator; never freeze or allow to warm. The preparation will continue depolymerizing over time, and when handled properly can be used for at least 6 months.</li>
	</ol>
	</li>
	<li>Resuspend Arp2/3 complex (porcine brain) (see Table of Materials) following the manufacturer&#39;s instructions (20 &#181;g protein in 20 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as described for actin in step 2.1. Combine the protein solutions from resuspending two tubes of powder to have a larger stock for reproducible experiments. Prepare 2 &#181;L aliquots and store at -80 &#176;C.</li>
	<li>Resuspend profilin (human recombinant) (see Table of Materials) at a 4x higher concentration than stipulated in the manufacturer&#39;s instructions (100 &#181;g protein in 25 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as for actin in step 2.1. Combine the protein solutions from resuspending two tubes of powder before determining protein concentration to have a larger stock for reproducible experiments. 
	NOTE: Keep on ice in a cold room or refrigerator; never freeze or allow to warm. When handled properly, resuspended profilin is good for at least 6 months to 1 year.</li>
	<li>Resuspend capping protein (&#945;1&#946;2, human recombinant) (see Table of Materials) following the manufacturer&#39;s instructions (50 &#181;g protein in 50 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing etc. on ice as described for actin in step 2.1. Chill 50 &#181;L of glycerol on ice and add the 50 &#181;L of resuspended capping protein to it; mix gently. Store at -20 &#176;C. 
	NOTE: The solution does not freeze and the activity is robust, so the solution can be kept as a single aliquot for months, or even years, if handled carefully. Mouse recombinant capping protein, the one most commonly used in the past in in vitro experiments13, will soon be commercially available.</li>
	<li>Resuspend gelsolin (human recombinant, His-tagged) (see Table of Materials) following the manufacturer&#39;s instructions (20 &#181;g protein in 20 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as described for actin in step 2.1. About 2 &#181;L of gelsolin is used per day of experiments; therefore, prepare large aliquots (5-10 &#181;L) and store at -80 &#176;C. 
	NOTE: The protocol with gelsolin is provided as an alternative. The use of capping protein in the place of gelsolin is recommended, either purchased or purified as in13.</li>
	<li>Resuspend VCA (human WASP-VCA, GST-tagged) (see Table of Materials) at 2x higher concentration than stipulated in the manufacturer&#39;s instructions (500 &#181;g protein in 250 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as for actin in step 2.1. Make 10 &#181;L aliquots and store at -80 &#176;C. 
	&#8203;NOTE: Once SpVCA (human pVCA, streptavidin and His-tagged) is commercialized or if protein purification14 is possible, the use of SpVCA instead of VCA is recommended. VCA does not reproducibly give comets under the conditions described here.</li>
</ol>
3. Measurement of protein concentrations
<ol>
	<li>Construct a Bradford standard curve made of two overlapping serial dilutions of BSA. 
	NOTE: The standard curve only needs to be constructed every couple of months (or even less frequently) as long as the spectrophotometer does not change.
	<ol>
		<li>In row 1 of a microtube rack, place tubes for BSA dilution series #1: four 2 mL tubes followed by four 1.5 mL tubes. In row 3 of the rack, place tubes for BSA dilution series #2 as for series #1. In row 5 of the rack, place one 2 mL tube for each sample to be measured along with two 1.5 mL tubes. Add a single 1.5 mL tube in row 5 for the blank.</li>
		<li>Measure out Bradford Reagent (see Table of Materials) into the 1.5 mL tubes.
		<ol>
			<li>Fill a 15 mL conical tube to the top with Bradford Reagent for easier pipetting (excess will be returned to the stock bottle in the refrigerator) on ice. Take up 200 &#181;L Bradford Reagent and eject it back into the conical tube to wet the pipette tip. As the solution is viscous, pipette slowly to allow the solution to enter and leave the tip completely without making bubbles.</li>
			<li>With the &#34;pre-wet&#34; tip, slowly pipette 200 &#181;L of Bradford Reagent into each of the 1.5 mL tubes in the rack (four for each BSA dilution, one for the blank and two for each sample to be measured). Do this first to allow the Bradford Reagent to thoroughly warm to room temperature before mixing with protein solutions. Return the remaining contents of the 15 mL conical tube to the bottle.</li>
		</ol>
		</li>
		<li>Measure out H2O into the 2 mL tubes. In row 1 of the rack, add 1,990 &#181;L H2O to the first tube, and 900 &#181;L to the three other tubes. For row 3, add 1,992.5 &#181;L to the first tube and 900 &#181;L to the three other tubes. Add 2,000 &#181;L H2O to each of the sample tubes in row 5. 
		NOTE: For all volumes larger than 1,000 &#181;L, use a 1,000 &#181;L pipette, but administer the full amount by pipetting twice. It is important to get everything ready before starting the subsequent steps, in order to avoid leaving proteins highly diluted in H2O for long periods of time.</li>
		<li>To prepare BSA dilution series #1, mix 10 &#181;L of calibrated 2 mg/mL BSA (see Table of Materials) into the tube with 1,990 &#181;L H2O to make a 10 &#181;g/mL solution. From this, make three serial dilutions (5 &#181;g/mL, 2.5 &#181;g/mL, and 1.25 &#181;g/mL BSA) by transferring 900 &#181;L of each solution into the next tube (containing 900 &#181;L H2O).</li>
		<li>To prepare BSA dilution series #2, mix 7.5 &#181;L of calibrated 2 mg/mL BSA into the tube with 1,992.5 &#181;L H2O to make a 7.5 &#181;g/mL solution. From this, make three serial dilutions (3.75 &#181;g/mL, 1.875 &#181;g/mL and 0.9375 &#181;g/mL BSA) by transferring 900 &#181;L of each solution into the next tube (containing 900 &#181;L H2O).</li>
		<li>Mix and read absorbance to generate the standard curve. Add 800 &#181;L H2O to the Bradford reagent tube for the blank, and start the timer. As efficiently as possible without making bubbles, mix 800 &#181;L of each BSA standard with 200 &#181;L Bradford reagent in the prepared tubes. Once all the standards have been mixed with Bradford reagent (&#60; 5 min), pour each standard into a disposable cuvette and read the absorbance at 600 nm in the spectrophotometer after blanking the machine. 
		NOTE: The same cuvette can be used to read the whole series if the least concentrated standard is read first and the cuvette is well-emptied between reads. Redo the standard curve until the linear fit has an R value of at least 0.99. Only after pipetting and gentle mixing is mastered, proceed to reading samples.</li>
	</ol>
	</li>
	<li>Measure concentrations of actin and actin-binding proteins
	<ol>
		<li>In the 2 mL tubes containing 2,000 &#181;L of H2O (prepared in step 3.1.3), gently mix in the following: 2 &#181;L each of Arp2/3 complex and profilin, 4 &#181;L of labeled G-actin, 5 &#181;L each of gelsolin and VCA and 8 &#181;L of capping protein (human recombinant). Immediately take 800 &#181;L of the solution and mix with the already prepared Bradford reagent, and then repeat to have two readings within 5%-10 % of each other for each sample. A larger difference indicates a problem with resuspension or handling. Read within a few minutes of mixing with Bradford reagent. 
		NOTE: If using a standard curve from another day, only the blank from step 3.1.6 has to be prepared, in addition to the samples.</li>
	</ol>
	</li>
	<li>Calculate the concentrations of actin and actin-binding proteins using the standard curve and the dilution factor. Redo the measurement for each new resuspension. Molecular weights to convert mg/mL readings obtained via Bradford assay to &#181;M are: actin 43 kD, Arp2/3 complex 224 kD, profilin 15 kD, gelsolin 95 kD, capping protein 68 kD (human recombinant) or 63.5 kD (mouse recombinant), VCA 43 kD, and SpVCA 54 kD (monomer molecular weight).</li>
</ol>
4. Coating of beads
<ol>
	<li>Pre-chill the centrifuge to 4 &#176;C and set the agitating dry block (see Table of Materials) to 18 &#176;C.</li>
	<li>Wash the beads: pipette 50 &#181;L of Xb buffer into a 1.5 mL microcentrifuge tube, add 9 &#181;L of 4.5 &#181;m diameter bead suspension or 2 &#181;L of 1 &#181;m diameter bead suspension (2.5 % w/v suspension) (see Table of Materials). Mix thoroughly and centrifuge the samples at 20,000 x g for 10 min at 4 &#176;C. 
	NOTE: Total bead surface area of both bead sizes is 3 cm2: 
	<img alt="Equation 1" src="/files/ftp_upload/64261/64261eq01.jpg" style="margin: auto;" /> 
	derived by calculating the number of beads and then their total surface using classic equations for the volume and surface area of spheres. Other sizes of beads can be used if amounts are adjusted to keep the total surface area constant at 3 cm2.</li>
	<li>Coat the beads: carefully remove the supernatant without disturbing the beads, and resuspend the bead pellet in 40 &#181;L of 2 &#181;M SpVCA (or 7 &#181;M VCA) in Xb buffer by gentle pipetting. Agitate at 18 &#176;C, 1,000 rpm for 20 min.</li>
	<li>Wash coated beads: centrifuge the mix (20,000 x g for 10 min at 4 &#176;C) and carefully remove the supernatant. Resuspend the beads in 50 &#181;L of cold Xb/1% BSA, and centrifuge at 20,000 x g for 10 min at 4 &#176;C. Remove the supernatant and repeat the washing step 1x.</li>
	<li>Resuspend the coated bead pellet from step 4.4 in 120 &#181;L cold Xb/1% BSA for both sizes of beads so that the amount of bead surface area/&#181;L of bead solution is the same. Store on ice in a refrigerator or cold room. Coated beads will continue to function normally for at least several weeks.</li>
</ol>
5. Preparing the motility mix and slides for observation
NOTE: The total volume of the motility mix is 8.4 &#181;L to allow for about 25 &#181;m clearance between the slide and the 18 mm x 18 mm coverslip so beads of all sizes (up to 10 &#181;m diameter) are not squeezed. The basic motility mix is approximately 5 &#181;M G-actin (10% labeled fluorescent actin) with 5 &#181;M profilin, 50 nM Arp2/3 complex, and 25 nM capping protein (or 240 nM gelsolin).
<ol>
	<li>Prepare the motility reaction mixture. Exact amounts of actin (and therefore profilin) depend on the concentration calculated in step 3.3, but a representative reaction is as follows. Mix on ice, in this order: 3.2 &#181;L of MB13, 1.5 &#181;L of profilin at 30 &#181;M diluted in MB13, 1 &#181;L of capping protein at 0.21 &#181;M diluted in MB13 or gelsolin diluted to 2 &#181;M in MB13, 1 &#181;L of Arp2/3 complex at 0.47 &#181;M diluted in MB13, 0.2 &#181;L of bead suspension (vortex just before use), and 1.5 &#181;L of actin at 30 &#181;M in G-buffer. Mix well but quickly and start the timer.</li>
	<li>Spot the entire motility reaction mixture on a slide. Cover with an 18 mm x 18 mm coverslip and seal the coverslip with melted VALAP using a small paintbrush. VALAP is a mix of lanolin, paraffin, and petroleum jelly (see Table of Materials) 1:1:1 by weight, melted and stirred together.</li>
</ol>
6. Microscopy observation
<ol>
	<li>Observe motility reactions immediately using a 100x objective on either an upright or an inverted microscope (see Table of Materials), equipped with phase contrast and/or epifluorescence microscopy (GFP cube, see Table of Materials). Observations are done at room temperature (23-25 &#176;C).
	<ol>
		<li>To obtain average displacement speeds for a whole population of beads, record phase contrast or fluorescent still images over time by scanning the entire slide. Measure comet length by hand and plot versus time. The slope of the linear fit is the average growth speed.</li>
		<li>To evaluate the speed of individual beads, collect time-lapse movies in phase contrast microscopy. Depending on the bead speed and the resolution required, take frames every 1-10 s. Use the tracking tool of any image processing program to obtain bead speeds and trajectories.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Campellone, K. G., Welch, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nucleator+arms+race:+cellular+control+of+actin+assembly.">A nucleator arms race: cellular control of actin assembly.</a> Nature Reviews Molecular Cell Biology. 11 (4), 237-251 (2010).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate+constants+for+the+reactions+of+ATP-+and+ADP-actin+with+the+ends+of+actin+filaments.">Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments.</a> Journal of Cell Biology. 103, 2747-2754 (1986).</li><li>Blanchoin, L., Boujemaa-Paterski, R., Sykes, C., Plastino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics,+architecture+and+mechanics+in+cell+motility.">Actin dynamics, architecture and mechanics in cell motility.</a> Physiological Reviews. 94 (1), 235-263 (2014).</li><li>Tilney, L. G., Tilney, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wily+ways+of+a+parasite:+induction+of+actin+assembly+by+Listeria.">The wily ways of a parasite: induction of actin assembly by Listeria.</a> Trends in Microbiology. 1 (1), 25-31 (1993).</li><li>Moore, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+cables+and+comet+tails+organize+mitochondrial+networks+in+mitosis.">Actin cables and comet tails organize mitochondrial networks in mitosis.</a> Nature. 591 (7851), 659-664 (2021).</li><li>Taunton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-dependent+propulsion+of+endosomes+and+lysosomes+by+recruitment+of+N-WASP.">Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP.</a> Journal of Cell Biology. 148 (3), 519-530 (2000).</li><li>Velarde, N., Gunsalus, K. C., Piano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+roles+of+actin+in+C.+elegans+early+embryogenesis.">Diverse roles of actin in C. elegans early embryogenesis.</a> BMC Developmental Biology. 7, 142 (2007).</li><li>Merrifield, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+vesicles+move+at+the+tips+of+actin+tails+in+cultured+mast+cells.">Endocytic vesicles move at the tips of actin tails in cultured mast cells.</a> Nature Cell Biology. 1 (1), 72-74 (1999).</li><li>Samarin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+VASP+enhances+actin-based+motility.">How VASP enhances actin-based motility.</a> Journal of Cell Biology. 163 (1), 131-142 (2003).</li><li>Bernheim-Groswasser, A., Wiesner, S., Golsteyn, R. M., Carlier, M. -. F., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+actin-based+motility+depend+on+surface+parameters.">The dynamics of actin-based motility depend on surface parameters.</a> Nature. 417 (6886), 308-311 (2002).</li><li>Boujemaa-Paterski, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+heterogeneity+regulates+steering+in+actin-based+motility.">Network heterogeneity regulates steering in actin-based motility.</a> Nature Communications. 8 (1), 655 (2017).</li><li>Akin, O., Mullins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capping+protein+increases+the+rate+of+actin-based+motility+by+promoting+filament+nucleation+by+the+Arp2/3+complex.">Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex.</a> Cell. 133 (5), 841-851 (2008).</li><li>Palmgren, S., Ojala, P. J., Wear, M. A., Cooper, J. A., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+with+PIP2,+ADP-actin+monomers,+and+capping+protein+regulate+the+activity+and+localization+of+yeast+twinfilin.">Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.</a> Journal of Cell Biology. 155 (2), 251-260 (2001).</li><li>Carvalho, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+or+myosin+contraction:+two+ways+to+build+up+cortical+tension+for+symmetry+breaking.">Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking.</a> Philosophical Transactions of the Royal Society B. 368 (1629), 20130005 (2013).</li><li>Marcy, Y., Prost, J., Carlier, M. -. F., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forces+generated+during+actin-based+propulsion:+a+direct+measurement+by+micromanipulation.">Forces generated during actin-based propulsion: a direct measurement by micromanipulation.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 5992-5997 (2004).</li><li>Carlier, M. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin:+protein+structure+and+filament+dynamics.">Actin: protein structure and filament dynamics.</a> Journal of Biological Chemistry. 266 (1), 1-4 (1991).</li><li>Loisel, T. P., Boujemaa, R., Pantaloni, D., Carlier, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+actin-based+motility+of+Listeria+and+Shigella+using+pure+proteins.">Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.</a> Nature. 401 (6753), 613-616 (1999).</li><li>Reymann, A. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+branched+actin+filament+networks+by+stochastic+fragmentation+with+ADF/cofilin.">Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin.</a> Molecular Biology of the Cell. 22 (14), 2541-2550 (2011).</li><li>Wioland, H., Jegou, A., Romet-Lemonne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+variations+with+pH+of+Actin+Depolymerizing+Factor/Cofilin's+multiple+actions+on+actin+filaments.">Quantitative variations with pH of Actin Depolymerizing Factor/Cofilin's multiple actions on actin filaments.</a> Biochemistry. 58 (1), 40-47 (2019).</li><li>Plastino, J., Blanchoin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+stability+of+the+actin+ecosystem.">Dynamic stability of the actin ecosystem.</a> Journal of Cell Science. 132 (4), 219832 (2019).</li><li>Pollard, T. D., Blanchoin, L., Mullins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+controlling+actin+filament+dynamics+in+nonmuscle+cells.">Molecular mechanisms controlling actin filament dynamics in nonmuscle cells.</a> Annual Review of Biophysics and Biomolecular Structure. 29, 545-576 (2000).</li><li>Suarez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+regulates+F-actin+network+homeostasis+by+favoring+formin+over+Arp2/3+complex.">Profilin regulates F-actin network homeostasis by favoring formin over Arp2/3 complex.</a> Developmental Cell. 32 (1), 43-53 (2015).</li><li>Courtemanche, N., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+profilin+with+the+barbed+end+of+actin+filaments.">Interaction of profilin with the barbed end of actin filaments.</a> Biochemistry. 52 (37), 6456-6466 (2013).</li><li>Achard, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&amp;#34;primer&amp;#34;-based+mechanism+underlies+branched+actin+filament+network+formation+and+motility.">A &amp;#34;primer&amp;#34;-based mechanism underlies branched actin filament network formation and motility.</a> Current Biology. 20 (5), 423-428 (2010).</li><li>Sykes, C., Plastino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+filaments+up+against+a+wall.">Actin filaments up against a wall.</a> Nature. 464 (7287), 365-366 (2010).</li><li>vander Gucht, J., Paluch, E., Plastino, J., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+release+drives+symmetry+breaking+for+actin-based+movement.">Stress release drives symmetry breaking for actin-based movement.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (22), 7847-7852 (2005).</li><li>McGough, A. M., Staiger, C. J., Min, J. -. K., Simonetti, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gelsolin+family+of+actin+regulatory+proteins:+modular+structures,+versatile+functions.">The gelsolin family of actin regulatory proteins: modular structures, versatile functions.</a> FEBS Letters. 552 (2-3), 75-81 (2003).</li><li>Abou-Ghali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capping+protein+is+not+necessary+for+polarized+actin+network+growth+and+actin+based+motility.">Capping protein is not necessary for polarized actin network growth and actin based motility.</a> Journal of Biological Chemistry. 295, 15366-15375 (2020).</li><li>Yarar, D., D'Alessio, J. A., Jeng, R. L., Welch, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+determinants+in+WASP+family+proteins.">Motility determinants in WASP family proteins.</a> Molecular Biology of the Cell. 13 (11), 4045-4059 (2002).</li><li>Padrick, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+regulation+of+WASP/WAVE+proteins.">Hierarchical regulation of WASP/WAVE proteins.</a> Molecular Cell. 32 (3), 426-438 (2008).</li><li>Bussonier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+detection+of+a+long-range+actin+network+emanating+from+a+biomimetic+cortex.">Mechanical detection of a long-range actin network emanating from a biomimetic cortex.</a> Biophysical Journal. 107 (4), 854-862 (2014).</li><li>Paluch, E., vander Gucht, J., Joanny, J. -. F., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformations+in+actin+comets+from+rocketing+beads.">Deformations in actin comets from rocketing beads.</a> Biophysical Journal. 91 (8), 3113-3122 (2006).</li></ol>

The authors declare that they have no conflicts of interest with the contents of this article.

The protocol detailed here describes how to obtain actin network growth on bead surfaces, comet formation, and bead motility using commercially available proteins. However, sometimes comets are not reproducibly observed or are inhomogeneous between the slide and coverslip. The following discussion emphasizes some key points in the protocol and suggests some parameters that can be adjusted. One factor to keep in mind is that comet formation and bead speed are affected by temperature, with temperatures much above 25 &#176;C or much below 23 &#176;C negatively impacting comet formation and giving irreproducible data. Use of a temperature-controlled microscope or a microscope in a climate-controlled room is strongly recommended. Although fluorescently labeled actin is often included in the motility mix to observe comets by fluorescence microscopy, once comets are more than a bead diameter in length, they are also visible by phase contrast microscopy as a dark smear next to the bead. Phase contrast visualization is more appropriate for time-lapse imaging as some phototoxicity is associated with fluorescence imaging even via spinning disc. Because beads settle over time, an inverted microscope produces less horizontal bead drift than an upright one and is more appropriate for movies. The use of molten VALAP to seal slides is important as substances such as nail polish interfere with comet formation. Large quantities of VALAP can be made in a beaker, and then scooped out to refill smaller beakers more amenable to rapid melting. VALAP is good for years at room temperature.
Another key technical aspect is meticulous buffer and motility mix preparation. Care should be taken when preparing MB13, in particular at the pH adjustment step. The pH of MB13 should be adjusted rapidly to neutral with NaOH to avoid ATP hydrolysis, but not too quickly as the EGTA solubilizes as the pH approaches neutral. EGTA is a key ingredient because it complexes the calcium bound to actin, giving in the motility mix the more active magnesium form16. MB13 prepared too quickly or too slowly gives suboptimal comet formation or even none at all. An additional key point is to keep careful track of KCl concentration in the motility mix when playing with conditions. For example, when using 1x MB13 in the reaction mix and diluting profilin, capping protein, and the Arp2/3 complex in MB13, the final KCl concentration in the motility reaction is about 40-50 mM due to dilution by G-buffer. This concentration gives the best results in the comet assay, and any more than 60 mM KCl decreases Arp2/3 complex nucleating activity.
On the protein side of things, a critical technical aspect of obtaining actin comets is proper handling of commercial actin-binding proteins, in particular precise pipetting of microliter quantities. The linearity of the Bradford standard curve is a good test of pipetting and the curve can then be used for routine measurements of protein concentrations. Indeed, when using resuspended commercial proteins for the comet procedure, it is important to always verify protein concentrations, as batch variability and user error during resuspension can lead to differences between real and expected concentrations. Sometimes small differences in protein concentrations can lead to the complete absence of comets.
Another important aspect of the method presented here is the use of profilin-complexed G-actin as the fuel for polymerization. Historically, in vitro systems used pre-polymerized filamentous actin (F-actin) as the actin source: depolymerization in the bulk fed polymerization on the surface10,17. This had the advantage of controlling G-actin levels, but added a layer of complexity requiring additional components to catalyze depolymerization. Since turnover of the actin network is not necessary for force production and motility, which are fueled by nucleation and polymerization at the surface of the bead, while actin depolymerization factors such as ADF/cofilin act on the aged networks far from the surface18, most in vitro reconstitution of actin-based motility is now done without turnover for simplicity. However, there are some drawbacks to using G-actin. First, when using commercial actin, which has been lyophilized, oligomers are present. The depolymerization steps described here are very important in obtaining reproducible results. In particular, although G-buffer is traditionally adjusted to pH 8, lower pH (pH 7, for example) appears to work better in the assays described in this article, possibly because low pH enhances depolymerization19. Another disadvantage of using G-actin is that once placed in salt conditions permissive to polymerization, spontaneous nucleation occurs and F-actin forms in the bulk as well as on the bead surface. Complexing G-actin with profilin suppresses spontaneous nucleation in the bulk and pointed end polymerization, thereby focusing both nucleation and barbed end polymerization at the surface20. Profilin-G-actin is physiologically relevant, as much of the actin in the cell is present in this form21. Here, a 1:1 ratio of profilin:actin is used; however, higher ratios (for example 3:1) more thoroughly inhibit polymerization in the bulk, although higher ratios also inhibit the Arp2/3 complex and barbed end elongation to some extent22,23.
Capping activity is also key for comet formation since it ensures insertion of new actin at the surface via cycles of nucleation by surface-activated Arp2/3 complex24,25. Without capping, actin clouds do not break symmetry to form comets because polymerization at the surface does not build up enough tension to break open the cloud26. In the past, we have used home-purified recombinant mouse capping protein13, but tests performed for this article indicate that commercially available recombinant human capping protein is equally effective, as is commercially available gelsolin, although 10x more gelsolin has to be used, and for certain applications, it may not be appropriate as it has actin severing activity as well as capping27.
Finally the robustness of this method resides in the use of a very active Arp2/3 complex activator, streptavidin-pVCA (SpVCA)28. SpVCA includes the profilin-G-actin binding domain of WASP (the p domain) in addition to the Arp2/3 complex binding domain as this is found to be most efficient in profilin-G-actin conditions29. More importantly, the use of the streptavidin tag, originally introduced to allow surface functionalization via the biotin-streptavidin link, has the additional effect of increasing Arp2/3 complex activation, presumably due to the fact that streptavidin is a tetramer and thus clusters the activator, known to increase Arp2/3 complex activity30. Commercially-produced SpVCA is currently in development and will soon be available for purchase. It should further be noted that, although 40 &#181;L of 2 &#181;M SpVCA is routinely used to coat 3 cm2 of bead surface, other coating concentrations (higher and lower) also work, and playing with these conditions gives different comet growth speeds and morphologies. Indeed, when comets do not form or comet size is not homogenous on the slide, different coating conditions should be tested, as well as different KCl and profilin concentrations in the motility mix. The concentrations of actin, Arp2/3 complex, and capping protein in the motility mix can also be altered to optimize comet formation, but in our hands, changing these proportions often gives confusing results.
To conclude, the methods described here produce actin assembly on bead surfaces and motility, but any surface that can be functionalized with SpVCA can be used. In cases where adsorption as described here does not work, the streptavidin moiety can be used to attach SpVCA to the surface of interest after biotinylation. The actin structures thus formed, comets or otherwise, can be used for testing different biochemical and biophysical aspects of actin networks, and are especially appropriate for physical manipulations with micropipettes, optical tweezers, and laser ablations15,26,31,32. In addition to its uses to the research community, the approach described here is appropriate as a teaching tool for undergraduate biophysics students to study active matter concepts such as symmetry breaking and self-organization.

Actin polymerization in cells is spatially and temporally controlled by tight regulation of actin filament nucleation downstream of cell signaling1. Nucleation occurs via the formation of an actin trimer, and then both ends of the nascent filament polymerize spontaneously, although one end is more dynamic (the barbed end) than the other (the pointed end)2. When nucleation and barbed end polymerization are directed toward a surface, they produce enough force (in the pico-to-nano Newton range) to push out the cell membrane for movement and to move micron-sized objects inside the cell with ATP hydrolysis as the energy source3. Some examples include Listeria monocytogenes bacteria that use actin comets to spread from cell to cell, and mitochondria, where actin comet-based movement is important for randomized inheritance during mitosis4,5. Actin comets on endosomes and other intracellular vesicles are implicated in detachment from donor membranes6,7,8.
With the method presented here, the signaling aspects of cellular actin polymerization are bypassed, and actin polymerization is produced on micrometric polystyrene beads by coating them with activators of branched actin nucleation, specifically the active domain of the human WASP protein, VCA (also called WA or WCA)1. The coated beads are then incubated in a mix containing the ingredients necessary for actin polymerization, including the main actin polymerization nucleator in cells, the Arp2/3 complex, which is activated by VCA at the bead surface to form new filaments as branches off the sides of daughter filaments1. Actin initially polymerizes uniformly around the bead, but then spontaneously breaks symmetry to create an actin comet that pushes the bead forward, thereby recreating cell-like protrusive networks and comets in a controlled manner. Similar approaches with beads and other coated surfaces have been used in the past by us and others to study the biochemistry and biophysics of actin polymerization9,10,11,12, but extensive expertise in actin-binding proteins was required for these experiments. The protocol presented here describes how to robustly create actin comets and motility entirely with commercially available (or soon to be available) reagents, making this approach accessible to anyone, including in an educational setting for teaching biophysical concepts. Key features include the importance of gentle and reliable pipetting, the use of profilin-complexed monomer as the actin source, and the essentiality of using a highly active Apr2/3 complex activator as a bead coating reagent.

<table><tbody><tr><td>Actin, rabbit muscle, Alexa Fluor 488 conjugate&amp;nbsp;</td><td>Invitrogen (ThermoFisher Scientific)</td><td>A12373 (recently discontinued)</td><td>This product can be replaced with ATTO-488 actin from Hypermol.</td></tr><tr><td>Actin, rabbit muscle, ATTO-488</td><td>Hypermol</td><td>8153</td><td></td></tr><tr><td>Actin, rabbit skeletal muscle</td><td>Cytoskeleton</td><td>AKL99</td><td></td></tr><tr><td>Arp2/3 complex</td><td>Cytoskeleton</td><td>RP01P</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>BioSpectrometer, basic</td><td>Eppendorf</td><td>035739</td><td></td></tr><tr><td>Bradford Reagent</td><td>Bio-Rad</td><td>500-0006</td><td></td></tr><tr><td>BSA, high quality</td><td>Sigma</td><td>A3059</td><td></td></tr><tr><td>BSA standard 2 mg/mL (Pierce)</td><td>Thermo Scientific</td><td>23209</td><td></td></tr><tr><td>Capping protein (a1b2, mouse recombinant)</td><td>Home-purified (Reference 13)</td><td></td><td>This product will soon be commercially available from Cytoskeleton.</td></tr><tr><td>Capping protein (a1b2, human recombinant)</td><td>Hypermol</td><td>8322</td><td></td></tr><tr><td>Cube, GFP: U-MNIBA3 or U-MWB2</td><td>Olympus</td><td>discontinued</td><td>Any GFP cube, adapted to the microscope being used, can be used.</td></tr><tr><td>Dry block, agitating: ThermoMixer C (refrigerated)</td><td>Eppendorf</td><td>035963</td><td></td></tr><tr><td>** with SmartBlock, 24 microtubes 2 mL</td><td>Eppendorf</td><td>035969</td><td></td></tr><tr><td>Gelsolin (human recombinant, His-tagged)</td><td>Cytoskeleton</td><td>HPG6</td><td></td></tr><tr><td>Lanolin</td><td>Sigma</td><td>49909</td><td></td></tr><tr><td>Microcentrifuge 5427R + rotor</td><td>Eppendorf</td><td>934126</td><td></td></tr><tr><td>Microscope, upright: BX51</td><td>Olympus</td><td>discontinued</td><td>Any epifluorescence upright microscope equipped with phase contrast optics can be used.</td></tr><tr><td>Microscope, inverted: IX70</td><td>Olympus</td><td>discontinued</td><td>Any epifluorescence inverted microscope equipped with phase contrast optics can be used.</td></tr><tr><td>Paraffin</td><td>Sigma</td><td>76244</td><td></td></tr><tr><td>Petroleum jelly: Vaseline</td><td>Sigma</td><td>16415</td><td></td></tr><tr><td>Pipettes Research Plus</td><td>Eppendorf</td><td></td><td>Gilson pipettes don&#039;t work as well for delivery of very small volumes (0.5 &amp;micro;L for example).</td></tr><tr><td>**10 &amp;micro;L</td><td></td><td>933954</td><td></td></tr><tr><td>**2.5 &amp;micro;L</td><td></td><td>933953</td><td>These two sizes are essential, but the use of high-quality pipettes (a full Research Plus set for example) is recommended.</td></tr><tr><td>Polystyrene carboxylate beads</td><td>Polysciences</td><td></td><td></td></tr><tr><td>**approx. 1 &amp;micro;m diameter</td><td></td><td>08226</td><td></td></tr><tr><td>**approx. 4.5 &amp;micro;m diameter</td><td></td><td>17140-5</td><td></td></tr><tr><td>Profilin 1 (human recombinant, untagged)</td><td>Cytoskeleton</td><td>PR02</td><td></td></tr><tr><td>SpVCA (human WASP pVCA domain, N-ter His-tag, C-ter Streptavidin tag)</td><td>Home-purified (Reference 14)</td><td></td><td>This product will soon be commercially available from Cytoskeleton.</td></tr><tr><td>VCA (human WASP VCA domain, GST-tagged)</td><td>Cytoskeleton</td><td>VCG03</td><td></td></tr></tbody></table>

reconstitution of actin-based motility with commercially available proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

One of the key aspects of reproducibly creating actin comets on beads is gentle and precise pipetting of delicate actin-binding proteins. Generating a Bradford standard curve is a good way of evaluating pipetting skills. Figure 1A,B shows the tubes for the standard curve and an example of what the two serial dilutions of BSA look like once mixed with Bradford reagent. Note the graded blue hue (higher protein concentration gives a bluer solution). When read in the spectrophotometer and plotted, these solutions give a standard curve as shown in Figure 1C. To practice careful pipetting, the assay should be repeated until the linear correlation factor is 0.999, as shown.
Once the concentrations of commercial resuspended proteins have been carefully evaluated via the Bradford assay, coated beads and motility mix are prepared and mixed together. Figure 2A shows representative images of the different stages of comet formation: actin clouds form within minutes of mixing SpVCA-coated beads and motility medium; cloud polarization occurs at ~5 min and comet production at 15-20 min. The actin comets, visible with both epifluorescence and phase contrast microscopy (Figure 2A), continue to elongate for many hours, but a consistent speed is not maintained so bead motility is normally evaluated within 1 h. On the other hand, it takes 30 min with VCA-coated beads to obtain bright actin clouds (Figure 2B), and no comets form, although symmetry starts to break at 1-2 h (arrow in Figure 2B) and clouds show polarization after overnight incubation.
Figure 3 shows an example of bead velocity evaluation in the presence of capping protein. Since all beads break symmetry at approximately the same time, &#34;pseudo-time-lapse&#34; recordings are performed where the slide is scanned and pictures of the whole population of comets are taken over time (Figure 3A). Comets do not depolymerize; therefore, the increase in comet lengths measured over time can be used to calculate displacement speed (Figure 3B). Gelsolin can be used in the place of capping protein for comet formation if 10x more gelsolin is added to compensate for its reduced capping activity. Comets formed in the presence of gelsolin are qualitatively the same and move at approximately the same speeds as beads with capping protein (Figure 3C). Capping activity is key for concentrating polymerization at the surface of the bead, and when neither capping protein nor gelsolin are included in the motility mix, actin clouds never polarize to form comets, although bright actin clouds form around the beads (Figure 3D). Comets on beads can be used to measure actin-based force production in different biochemical contexts by altering the motility mix and observing the result on motility using different micromanipulation techniques, for example15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64261/64261fig01.jpg" /> 
Figure 1: Bradford standard curve. (A) Picture of how to set up the tubes for making the Bradford standard curve. Sample tubes are not shown. (B) Picture of the two overlapping BSA serial dilutions once mixed with Bradford reagent. (C) The absorbances at 600 nm of the solutions shown in (B) are measured in the spectrophotometer and are plotted as a function of the protein concentrations of the BSA solutions. The linear fit is used to calculate the sample concentration. The correlation factor, R, of the linear fit is 0.999. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64261/64261fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64261/64261fig02.jpg" /> 
Figure 2: Comet formation on SpVCA-coated beads as opposed to VCA-coated beads. (A) Representative SpVCA-coated beads (different bead in each image) shown over time. Time from the moment of mixing is indicated. Actin clouds form immediately, and cloud polarization gives comets, which continue to elongate for hours. (B) Representative VCA-coated beads (different bead in each image) shown over time. More than 1 h is necessary to see the beginnings of actin cloud polarization (arrow) and even long incubations do not produce comets. All images are of 4.5 &#181;m diameter beads, epifluorescence imaging of fluorescent actin paired with phase contrast visualization for the 15 and 20 min time-points in (A), scale bars = 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64261/64261fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64261/64261fig03.jpg" /> 
Figure 3: Comets and bead velocity analysis. (A) and (C) In the presence of either capping protein (CP) or gelsolin, actin clouds polarize to form comets in the first 20 min of reaction, and comets elongate over time. Time from mixing indicated for each image; each image is a different bead. There is some variability among beads and preparation, but on average beads move at micron/sub-micron per min speeds (0.2-1 &#181;m/min) under the standard conditions described here. (B) Representative graph showing evaluation of comet length (whole population of beads) over time. The slope of the linear correlation corresponds to the average displacement speed, in this case 0.24 &#181;m/min. (D) In the absence of capping activity (no capping protein or gelsolin), actin clouds form around beads, but comets do not form. All images are of 4.5 &#181;m diameter beads, epifluorescence imaging of fluorescent actin, scale bars = 5 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64261/64261fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Reconstitution of Actin-Based Motility with Commercially Available Proteins at JoVE.com

Reconstitution of Actin-Based Motility with Commercially Available Proteins

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

reconstitution-actin-based-motility-with-commercially-available

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.6K Views.  Sorbonne Université, Université Paris Cité. This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Video: Reconstitution of Actin-Based Motility with Commercially Available Proteins

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Biochemistry

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

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

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

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

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

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

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

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol. Contractility gradients generated by the myosin motors drive the flow of the cytosol across the gel pores, which infers that the mechanics of the cytoskeleton and the cytosol are tightly coupled. One main feature of poroelasticity is the diffusive relaxation of stresses in the network, characterized by an effective diffusion constant that depends on the gel elastic modulus, porosity, and cytosol (solvent) viscosity. As cells have many ways to regulate their structure and material properties, our current understanding of how cytoskeleton mechanics and cytosol flow dynamics are coupled remains poorly understood. Here, an in vitro reconstitution approach is employed to characterize the material properties of poroelastic actomyosin gels as a model system for the cell cytoskeleton. Gel contraction is driven by myosin motor contractility, which leads to the emergence of a flow of the penetrating solvent. The paper describes how to prepare these gels and run experiments. We also discuss how to measure and analyze the solvent flow and gel contraction both at the local and global scales. The various scaling relations used for data quantification are given. Finally, the experimental challenges and common pitfalls are discussed, including their relevance to cell cytoskeleton mechanics.

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

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

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This ...

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

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

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

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Biology

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

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Henty-Ridilla, J.L., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64074">Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.</a> J. Vis. Exp. (2022)
This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

Mikroskopia TIRF do wizualizacji dynamiki sprzężenia aktyny i mikrotubul

Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

Inverted Motility Assay: An In Vitro Technique to Visualize Myosin Movement on Immobilized Actin Filaments

Prepare the solutions for myosin 5a inverted motility assay, and keep them on ice.
Wash the chamber with 10 microliters of motility buffer with DTT. Flow in 10 microliters of 1 milligram per milliliter BSA in motility buffer with DTT. Repeat this twice, waiting a minute after the third wash. Use tissue or filter paper to wick the solution through the channel by gently placing the corner of the paper at the flow chamber exit.
Then, wash the chamber with 10 microliters of motility buffer with DTT three times. Flow in 10 microliters of the NeutrAvidin solution in motility buffer with DTT, and wait for one minute. Then, wash with 10 microliters of that solution three times.
Flow in 10 microliters of bRh-actin-containing motility buffer with DTT using a large-bored pipette tip, and wait for one minute. Then, wash with 10 microliters of motility buffer with DTT three times.
Flow in 30 microliters of final buffer with 10 nanomolar myosin 5a, and immediately load onto the total internal reflection fluorescence microscope. Begin recording once the optimum focus for TIRF imaging is found.

Reconstitution of Actin-Based Motility with Commercially Available Proteins

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Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

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

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

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

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

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

Mikroskopia TIRF do wizualizacji dynamiki sprzężenia aktyny i mikrotubul

Test odwróconej ruchliwości: technika in vitro do wizualizacji ruchu miozyny na unieruchomionych włóknach aktyny