Name: Reconstituting Myosin-I-Mediated Branched Actin Assembly in a Bead-Based Actin Comet-Tail Motility Assay (Video) | JoVE Journal
Uploaded: 2026-09-03T13:05:00.000+00:00
Duration: 7 min 56 s
Description: Learn to reconstitute myosin-I-mediated branched actin assembly using a bead-based actin comet-tail motility assay. This method enables quantitative analysis of actin network dynamics and force generation at membrane-like interfaces.

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We thank Dr. Daniel Safer and Rick Wike for excellent technical assistance with protein purification. We thank Dr. Roberto Dominguez, especially Dr. Malgorzata Boczkowska and Dr. Grzegorz Rebowski, for generously providing the Arp2/3 complex and CapZ proteins. We also thank Dr. Luther Pollard, Dr. Faviolla A. B&#225;ez-Cruz, and all members of the Ostap and Dominguez laboratories for their valuable input throughout the project. This work was supported by National Institutes of Health grant R37 GM057247 (to E.M.O.). E.M.O. and M.X. were partially supported by the National Science Foundation (CMMI-1548571). ChatGPT was used to assist with language editing during manuscript revision. The authors reviewed and edited all AI-assisted content and take full responsibility for the final manuscript.

<ol>
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The authors declare that they have no competing financial interests or conflicts of interest.

The actin comet-tail bead motility assay is a well-established in vitro reconstitution system for investigating Arp2/3 complex-mediated branched actin assembly and its role in generating protrusive forces14,15,16,17. This system has been broadly applied to dissect the biochemical regulation of branched actin assembly14,15,26,27,31,49,50,51 and force generation20,21,22,23,52, while also providing a framework for quantitative studies of the emergent active matter properties of actin networks19,35,36,53. By incorporating myosin, particularly myosin-I18, into this reconstituted system, the modified assay enables investigation of how myosin&#8211;actin coupling modulates actin assembly and force production at the membrane interface, an area that remains relatively underexplored. Notably, myosin-I in this system can be readily replaced by other myosin motors to examine their respective roles in actin network remodeling and force generation. For example, myosin V (cargo transport)54, myosin VI (endocytic trafficking and membrane dynamics)55,56, myosin VII and XV (stereocilia and microvillar organization)57,58,59, myosin X (filopodia formation)60,61, and myosin XIX (mitochondrial dynamics)62 could be incorporated to investigate how different classes of myosin contribute to actin organization and their associated physiological processes.
The assay is highly reproducible provided that several critical parameters are carefully controlled. First, high-quality monomeric G-actin is essential for robust comet-tail formation. We recommend purifying G-actin from rabbit skeletal muscle acetone powder using established protocols41. After 2&#8211;3 days of dialysis, monomeric actin should be further purified by gel filtration on Sephacryl S-300 in G-buffer (2 mM Tris-HCl, pH 8.0; 0.2 mM ATP; 0.1 mM CaCl2; 1 mM NaN3; and 0.5 mM DTT). Fractions collected immediately following the elution peak should be used. Purified G-actin typically retains strong polymerization activity for 2&#8211;3 weeks when stored on ice at 4&#176;C and may remain usable into the fourth week, although performance generally declines thereafter. Reduced comet-tail robustness, characterized by sparse, brittle actin networks and inconsistent tail growth, often indicates compromised actin quality. Second, NPF activity critically determines comet-tail formation. Commercial GST-tagged WCA domain from human neuronal Wiskott&#8211;Aldrich syndrome protein (N-WASP) (GST-VCA) provides robust and sustained nucleation activity, and GST dimerization further enhances this activity63. We recommend validating any NPF construct using a pyrene-actin polymerization assay before bead coating. The activity of the commercial GST-VCA can serve as a reference standard. Notably, the commercial GST-VCA used here lacks a polyproline region and therefore does not interact with profilin&#8211;actin complexes26. Third, reliable visualization of comet-tail growth requires careful slide and coverslip preparation. We recommend thoroughly cleaning slides and coverslips with water and ethanol, followed by plasma cleaning immediately before use. When available, polyethylene glycol&#8211;silanized coverslips26 are preferred because they minimize nonspecific protein adsorption and improve reproducibility. Including an adequate concentration of BSA in the motility mixture can further reduce nonspecific surface interactions and promote consistent assay performance. Fourth, the volume of sample applied to the chamber is critical for reliable measurements15,16,17. Excessive sample volume results in drifting beads, preventing accurate tracking of comet-tail elongation. In contrast, insufficient sample volume compresses beads between the slide and coverslip, increasing friction and immobilizing the beads. This mechanical constraint can distort comet-tail morphology and alter measured growth kinetics. Therefore, optimizing both chamber height and sample volume is essential for reproducible results. We recommend empirically optimizing the sample volume for each experimental setup. When using a 22 mm &#215; 22 mm coverslip as described here, applying 2.1&#8211;2.3 &#181;L of the motility mixture produces a chamber height of approximately 4.3 &#181;m, yielding stable positioning of 2.0-&#181;m-diameter beads without excessive drift or compression.&#160;When a coverslip of a different size is used, the sample volume should be adjusted accordingly to achieve even chamber filling, stable bead positioning, and minimal sample drift.
If comet tails fail to form, verify actin polymerization competence and NPF activity. If symmetry breaking does not occur, adjust CP concentration to modulate network density. Excessively dense networks resist fracture, whereas overly sparse networks lack structural integrity. If myosin-coated beads show no difference from control beads, confirm myosin surface density and motor activity. For quality control, the acceptance criterion is functional rather than based on an absolute numerical cutoff. A new preparation is considered acceptable if,&#160;it reproduces the comet-tail formation and growth observed with a previously validated preparation ( as shown in&#160;Figure 3). In addition, each new preparation of myosin, Arp2/3 complex, NPF, CapZ, or actin should be evaluated using the appropriate quality-control assays and compared with a previously validated preparation before use to ensure consistency across quantitative experiments (see Protocol section 3.1).
Several limitations of this assay should be considered when interpreting the results. First, fluorescence intensity is used as a relative, uncalibrated proxy for actin network density; therefore, comparisons should be made only across samples imaged under identical acquisition settings and analyzed using the same thresholding workflow. Second, bead motility, comet-tail growth, and growth efficiency provide indirect readouts of force-generating network behavior but do not constitute direct measurements of force. Direct mechanical measurements, such as optical trapping or force-calibrated membrane deformation assays64,65, are required to quantify forces produced by the network. Third, this assay does not directly resolve actin network ultrastructure, e.g. filament orientation, branch density, or local myosin organization.&#160;Complementary approaches such as electron microscopy or super-resolution imaging may be required for nanoscale structure&#160;characterization. Finally, the use of rigid micron-sized beads provides a simplified and experimentally tractable interface but does not capture the deformability, curvature heterogeneity, lipid mobility, or molecular complexity of cellular membranes. Future development of more physiologically relevant interfaces will therefore be important for investigating how cytoskeletal components coordinate at the cell leading edge.
Overall, this reconstitution approach provides precise control over protein composition and concentration, allowing individual components to be dissected mechanistically in ways that are difficult to achieve in cellular systems. It provides a versatile platform for studying how molecular motors remodel branched actin networks and shape force generation at membrane-like interfaces. With appropriate validation, the assay can be extended to examine how different myosin classes, mutant or disease-associated motor variants, and force-sensitive actin-binding proteins regulate mechanical feedback within dynamic actin networks. More broadly, this system helps connect molecular-scale motor activity to mesoscale cytoskeletal organization and mechanical function, with implications for cell motility, endocytosis, and membrane remodeling.

Branched actin polymerization mediated by the actin-related protein 2/3 (Arp2/3) complex generates the primary protrusive forces that drive diverse cellular processes, including cell motility, endocytosis, phagocytosis, and the formation of cell adhesion complexes1,2,3,4,5,6. At the leading edge of migrating cells, nucleation-promoting factors (NPFs) activate the Arp2/3 complex at the plasma membrane, initiating dendritic actin network assembly that pushes the membrane forward. Myosin-I motors frequently colocalize with these branched actin networks at membrane interfaces and have been implicated in regulating&#160;actin organization&#160;and modulating force generation7,8,9,10,11,12,13. Despite accumulating evidence that myosin-I contributes to actin-dependent mechanical processes, the molecular mechanisms by which myosin-I interacts with Arp2/3-mediated branched actin assembly to modulate network architecture and mechanical output remain poorly understood.
To address this gap, we incorporated myosin-I into the well-established actin comet-tail bead motility system14,15,16,17 to reconstitute the interplay among myosin-I, actin, and the Arp2/3 complex on the surface of micron-sized beads. The overall goal of this method is to quantitatively investigate how myosin-actin coupling influences branched actin assembly, network architecture, symmetry breaking, and bead motility at a membrane-like interface. In this assay, myosin-I and NPFs are co-coated onto microscale beads to mimic their colocalization at the plasma membrane18. Upon initiation of polymerization, surface-bound NPFs stimulate Arp2/3-dependent branched actin assembly, leading to actin-shell formation, symmetry breaking, and the development of a polarized comet tail&#160;that drives bead propulsion14,19,20,21,22,23. By systematically varying myosin surface density18 and modulating actin network density through capping protein (CP) concentration15,19,24, this method enables quantitative analysis of actin assembly kinetics, growth efficiency, network architecture, and bead motility, providing indirect readouts related to myosin-I-dependent changes in mechanical output.
The assay also offers distinct advantages over complementary approaches. Compared with bulk polymerization assays, such as pyrene-actin assays, this bead motility system enables direct visualization of branched actin assembly and actin polymerization-driven bead movement. In contrast to cellular studies, where overlapping protein functions and complex cellular environments can obscure individual contributions and complicate interpretation, this assay provides precise control over protein composition, concentration, and surface organization, thereby enabling systematic investigation of how myosin-I interacts with Arp2/3-mediated actin assembly under defined biochemical conditions.
This method is most appropriate for studying how cytoskeletal components coordinate at a membrane-like&#160;interface to regulate actin network architecture and mechanical output. Although the current protocol is optimized for myosin-I-mediated branched actin assembly, its modular design may be adapted in the future to examine other membrane-associated myosin classes (e.g., myosin VI, VII, X, XV, and XIX) and/or actin-binding proteins (e.g., formins, vasodilator-stimulated phosphoprotein (VASP), cortactin, coronin, or tropomyosin). Such adaptations would require validation of protein immobilization, activity, and surface density. In addition, the assay is amenable to pulse-chase labeling strategies. Incorporating photoconvertible or photoactivatable actin, or labeled Arp2/3 subunits, could enable measurement of filament turnover and branch dissociation rates within the growing network25. Overall, this assay provides a versatile platform for examining the molecular interplay among membrane-associated motors, actin filaments, and actin-binding proteins, as well as for probing how motors and actin-binding proteins regulate network remodeling and force generation at membrane-like interfaces14,15,26,27,28,29,30,31,32,33,34. More broadly, it also serves as a minimal, tunable active matter platform for investigating the physical principles underlying motor-driven nonequilibrium dynamics, such as symmetry breaking, self-organization, mechanochemical feedback, and other emergent behaviors in active actomyosin networks18,19,20,21,22,23,35,36, with potential relevance to active matter physics and materials engineering.

<table><tbody><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td><td>Sigma</td><td>A26209</td><td>Used to prepare ATP-containing buffers; aliquot and store at &amp;minus;20&amp;deg;C.</td></tr><tr><td>Arp2/3 complex</td><td>Prepared in-house</td><td>N/A</td><td>Used to nucleate branched actin networks; purified according to Ref. 42.</td></tr><tr><td>Biotin-CF640 fluorescent dye</td><td>Biotium</td><td>80032</td><td>Used to fluorescently label control beads; protect from light and store at &amp;minus;80&amp;deg;C.</td></tr><tr><td>Biotinylated Myo1d</td><td>Prepared in-house</td><td>N/A</td><td>Motor protein used to decorate myosin-coated beads; prepared as described in Refs. 18 and 38&amp;ndash;40.</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A7906</td><td>Used for bead blocking and bead storage buffers; prepare a filtered stock solution.</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C7902</td><td>Buffer component used in X buffer and calcium-disruption experiments.</td></tr><tr><td>Calmodulin</td><td>Prepared in-house</td><td>N/A</td><td>Added to myosin-containing reactions and bead storage buffer; purified according to Ref. 44.</td></tr><tr><td>Carboxylate polystyrene beads (2.0 &amp;micro;m diameter)</td><td>Polysciences</td><td>18327-10</td><td>Used as the solid support for NPF, NeutrAvidin, fluorescent dye, and myosin coating; store at 4&amp;deg;C.</td></tr><tr><td>CCD camera</td><td>Teledyne</td><td>01-RET-R6-R-M-14-C-OC</td><td>Used for fluorescence image acquisition during time-lapse microscopy.</td></tr><tr><td>Coomassie Brilliant Blue stain</td><td>Sigma</td><td>B7920</td><td>Used to stain SDS-PAGE gels before densitometric analysis.</td></tr><tr><td>Coverslips, 22 mm &amp;times; 22 mm, #1.5</td><td>Globe</td><td>1404-15</td><td>Used with microscope slides to assemble squeeze chambers.</td></tr><tr><td>Dithiothreitol (DTT)</td><td>GoldBio</td><td>DTT10</td><td>Reducing agent used in buffers and motility mixtures; store at &amp;minus;20&amp;deg;C.</td></tr><tr><td>Dry scroll vacuum pump</td><td>Agilent Technologies</td><td>IDP3B01</td><td>Used to operate the plasma-cleaning system.</td></tr><tr><td>EGTA</td><td>GoldBio</td><td>E-217-100</td><td>Calcium chelator used in M buffer, motility buffer, and actin pretreatment.</td></tr><tr><td>External light source</td><td>Leica</td><td>EL6000</td><td>Metal-halide light source for fluorescence microscopy.</td></tr><tr><td>Fiji (ImageJ)</td><td>Open source</td><td>N/A</td><td>Used for comet-tail length, fluorescence intensity, and network density analysis (version 2.16.0/1.54p).</td></tr><tr><td>Fluorescence microscope</td><td>Leica</td><td>DMIRB</td><td>Inverted epifluorescence microscope used for imaging actin comet tails.</td></tr><tr><td>Glacial acetic acid</td><td>Fisher Chemical</td><td>BP2401-212</td><td>Component of the SDS-PAGE gel destaining solution; corrosive, handle with appropriate precautions.</td></tr><tr><td>GST-VCA</td><td>Cytoskeleton</td><td>VCG03-A</td><td>Nucleation-promoting factor used for Arp2/3-mediated actin assembly; aliquot and store at &amp;minus;80&amp;deg;C.</td></tr><tr><td>HEPES</td><td>GoldBio</td><td>H-400-1</td><td>Buffering reagent used in X buffer, M buffer, and motility buffer.</td></tr><tr><td>Human CapZ</td><td>Prepared in-house</td><td>N/A</td><td>Capping protein used to regulate actin network density; purified according to Ref. 43.</td></tr><tr><td>Magnesium chloride</td><td>Spectrum Chemical</td><td>M1035</td><td>Buffer component used in reaction buffers and actin pretreatment.</td></tr><tr><td>MetaMorph imaging software</td><td>Molecular Devices</td><td>N/A</td><td>Used for microscope control and image acquisition (version 7.10.4.459).</td></tr><tr><td>Methanol</td><td>Sigma</td><td>MX0485-3</td><td>Component of the SDS-PAGE gel destaining solution; flammable, handle according to institutional safety procedures.</td></tr><tr><td>Methylcellulose</td><td>Sigma</td><td>M0387</td><td>Added to the motility buffer to increase solution viscosity (1500 cP).</td></tr><tr><td>Microscope slides</td><td>Corning</td><td>2947-75X25</td><td>Used to assemble squeeze chambers for microscopy.</td></tr><tr><td>NeutrAvidin protein</td><td>Thermo Fisher Scientific</td><td>31000</td><td>Used for immobilization of biotinylated proteins on bead surfaces; aliquot and store at &amp;minus;80&amp;deg;C.</td></tr><tr><td>PageRuler Plus Prestained Protein Ladder</td><td>Thermo Scientific</td><td>26619</td><td>Molecular weight marker used for SDS-PAGE analysis; store at &amp;minus;20&amp;deg;C.</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td>Used to plasma-clean microscope slides and coverslips before chamber assembly.</td></tr><tr><td>Polyethersulfone syringe filter, 0.22 &amp;micro;m</td><td>Sigma</td><td>SLGPR33RS</td><td>Used to filter the bovine serum albumin stock solution before aliquoting.</td></tr><tr><td>Potassium chloride</td><td>Fisher BioReagents</td><td>BP366-1</td><td>Salt used to maintain ionic strength in assay buffers.</td></tr><tr><td>Rabbit skeletal muscle actin</td><td>Prepared in-house</td><td>N/A</td><td>Purified according to Ref. 41 and used as G-actin for actin comet-tail assembly assays.</td></tr><tr><td>Refrigerated microcentrifuge</td><td>Eppendorf</td><td>EP-5415R</td><td>Used for bead pelleting during bead preparation and washing steps.</td></tr><tr><td>Rotating tube mixer (benchtop rotator)</td><td>Cole-Parmer</td><td>EW-04397-40</td><td>Used during bead-coating incubations to maintain the bead suspension.</td></tr><tr><td>Tris/Glycine/SDS running buffer (10&amp;times;)</td><td>Bio-Rad</td><td>1610732</td><td>Dilute to 1&amp;times; before SDS-PAGE electrophoresis.</td></tr><tr><td>Vacuum grease</td><td>High Vac Depot</td><td>408524</td><td>Used to seal squeeze chambers before fluorescence imaging.</td></tr><tr><td>4%&amp;ndash;20% gradient SDS-polyacrylamide gel</td><td>Thermo Fisher Scientific</td><td>XP04205BOX</td><td>Used for SDS-PAGE analysis of bead-associated proteins; store at 4&amp;deg;C.</td></tr></tbody></table>

reconstituting myosin-i-mediated branched actin assembly in a bead-based actin comet-tail motility assay

Reconstituting Myosin-I-Mediated Branched Actin Assembly in a Bead-Based Actin Comet-Tail Motility Assay

Branched actin polymerization mediated by the actin-related protein 2/3 (Arp2/3) complex provides the primary pushing forces for a variety of cellular processes, including cell migration, endocytosis, and phagocytosis. Myosin-I motors, which frequently colocalize with branched actin networks at the cell leading edge, have also been shown to participate in these processes and are thought to regulate actin network organization and mechanical output. However, the molecular mechanisms by which myosin-I interacts with the Arp2/3 complex to modulate branched actin assembly and force generation remain largely unknown. Here, we describe a highly tunable in vitro actin comet-tail bead motility assay that reconstitutes the interplay among myosin-I, actin, and the Arp2/3 complex at the cell leading edge on the surface of micron-sized beads. This method is adapted from well-established actin comet-tail assays by co-immobilizing myosin-I with nucleation-promoting factors (NPFs) on bead surfaces, thereby creating a membrane-like actin assembly interface. The assay enables visualization and quantitative analysis of actin network assembly, network density, bead motility, and growth efficiency. It also provides indirect, qualitative readouts of myosin-I-mediated force enhancement in branched actin networks. This protocol includes bead functionalization, reaction assembly, fluorescence imaging, quantitative image analysis, and troubleshooting strategies providing a reproducible platform for studying myosin-I-regulated actin assembly at membrane-like interfaces.

The actin comet-tail bead motility assay was used to investigate the effect of myosin-I activity on Arp2/3 complex-mediated branched actin assembly at the cell leading edge, recapitulating scenarios in which membrane-associated myosin-I coordinates with actin polymerization to drive membrane protrusion7,8,9,10,11,12,13. To mimic the colocalization of myosin-I and NPFs at the membrane&#8211;actin interface, both proteins were co-coated onto the surface of microscale beads, thereby reconstituting their spatial proximity in a controlled in vitro system (Figure 1A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/71259/71259fig1.jpg" /> 
Figure 1. Reconstitution of myosin-I-mediated branched actin assembly on microscale beads. (A) Schematic showing interactions among myosin-I, nucleation-promoting factors (NPFs), the actin-related protein 2/3 (Arp2/3) complex, capping protein (CP), and globular actin (G-actin) at the cell leading edge. (B) Schematic of bead functionalization. Control beads and myosin-coated beads were coated with NPF and neutravidin. Neutravidin was conjugated to either biotinylated far-red fluorescent dye for control beads or biotinylated Drosophila Myo1d for myosin-coated beads. (C) Representative time series showing actin comet-tail growth from symmetry breaking to polarized comet-tail formation for a control bead and a myosin-coated bead. The final panel shows both beads in the same field of view. Scale bar, 5 &#181;m. Panels B and C are reproduced/modified from Xu et al., Science Advances, DOI: 10.1126/sciadv.ado5788 (2024), AAAS, with permission. <a href="https://www.jove.com/files/ftp_upload/71259/71259fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To better assess the specific contribution of myosin-I to actin assembly, a corresponding control bead was also constructed. The control beads were identical to the myosin-coated beads except that myosin-I was replaced with a biotinylated far-red fluorescent dye conjugated to neutravidin (Figure 1B). Mixing both bead types within the same reaction chamber and imaging them in the same field of view enabled direct comparison of branched actin assembly in the presence and absence of myosin-I under identical experimental conditions (Figure 1C). Furthermore, by varying the myosin-to-NPF ratio on the bead surface, the effect of myosin-I across different surface densities could be systematically examined (Figure 2A,B). In addition, the actin network density could be tuned by adjusting the concentration of CP, generating networks ranging from densely packed, symmetry-breaking-resistant structures to loosely organized, fracture-prone architectures15,19,24. This approach allowed investigation of how myosin-I influences branched actin assembly across networks of varying density and structural integrity (Figure 2A,B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/71259/71259fig2.jpg" /> 
Figure 2. Actin comet-tail morphology&#160;across myosin surface densities and capping protein concentrations. (A) Representative fluorescence images of 2-&#181;m-diameter beads coated with different myosin surface densities and assembled in reactions containing 30&#8211;200 nM capping protein (CP), 4 &#181;M actin with 5% rhodamine-labeled actin, and 200 nM Arp2/3&#160;complex. For each condition, control beads and myosin-coated beads were imaged in the same field of view. Myosin and nucleation-promoting factor (NPF) surface densities were determined by sodium dodecyl sulfate&#8211;polyacrylamide gel electrophoresis, as described in Step 2.3. Images were acquired 20&#8211;35 min after reaction initiation. Brightness and contrast were adjusted linearly and identically within each control&#8211;myosin bead pair but were scaled independently between conditions for better visualization. Scale bar, 5 &#181;m. (B) Qualitative assay map summarizing comet-tail outcomes across CP concentrations and myosin surface densities. Each data point represents a matched control&#8211;myosin bead pair under the indicated condition. Symbols indicate observed outcomes, including myosin-coated beads promoting symmetry breaking, longer tails, similar tails, shorter tails, no comet-tail formation, or aster-like structures. Data were collected from more than 20 independent experiments, with more than 10 bead pairs analyzed for each condition. This figure is reproduced/modified from Xu et al., Science Advances, DOI: 10.1126/sciadv.ado5788 (2024), AAAS, with permission. <a href="https://www.jove.com/files/ftp_upload/71259/71259fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Upon mixing the assay components, surface-bound NPFs stimulated Arp2/3 complex-mediated branched actin assembly around the bead surface. In a typical successful reaction, actin first appeared as a sparse shell surrounding the bead. As polymerization proceeded, the shell expanded and the network became more densely entangled. Continued actin assembly at the bead surface drove outward network growth, which could lead to symmetry breaking and the formation of a polarized comet tail. Once a comet tail was established, ongoing actin nucleation and polymerization at the bead surface supported sustained bead movement (Figure 1C). These sequential features of actin shell formation, symmetry breaking, comet-tail extension, and bead propulsion served as visual indicators that the assay was functioning properly.
As reported in our original study18 and illustrated in Figure 2A,B, comet-tail morphology was sensitive to both CP concentration and myosin surface density. Under standard assay conditions (200 nM Arp2/3 complex, 50 nM CP, myosin:NPF ratio = 0.43:1), myosin-coated beads typically produced sparser or more diffuse actin comet tails than control beads (Figure 2A). At low CP concentrations (&#60;30 nM), actin assembled into dense, fracture-resistant shells. Under these conditions, control beads often remained encapsulated and failed to undergo symmetry breaking, whereas myosin-coated beads were more likely to break symmetry and escape from the surrounding actin shell (Figure 2A,B). At high CP concentrations (&#62;200 nM), network cohesion was reduced, and the presence of myosin further destabilized the actin network, promoted actin shedding, and prevented the formation of stable comet tails (Figure 2A,B). These condition-dependent outcomes provide useful benchmarks that researchers can use to identify appropriate assay regimes and troubleshoot assay performance.
The effect of myosin-I on actin comet-tail morphology can be assessed by measuring the mean fluorescence intensity of the comet tail. The comet-tail region was defined using an intensity threshold, as described in Step 4.2, and the mean fluorescence intensity within this region was then measured (Figure 3A). Under matched imaging conditions, in which control and myosin-coated beads were imaged in the same field of view, this value served as a relative, uncalibrated proxy for actin network density, with lower mean intensity indicating a sparser network. Under standard assay conditions, myosin-coated beads typically generated comet tails with lower mean fluorescence intensity than control beads, consistent with reduced apparent network density (Figure 3C).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/71259/71259fig3.jpg" /> 
Figure 3. Quantification workflow for actin comet-tail growth kinetics and assembly efficiency. (A) Representative time series of comet-tail growth from a control bead and a 0.43:1 myosin-coated bead in the presence of 50 nM capping protein (CP). Black outlines indicate comet-tail regions of interest (ROIs) identified using Fiji for fluorescence intensity measurements. Reaction conditions: 4 &#181;M actin with 5% rhodamine-labeled actin, 200 nM actin-related protein 2/3 (Arp2/3) complex, and 50 nM CP. Scale bar, 5 &#181;m. (B) Comet-tail length plotted as a function of time for the representative control&#8211;myosin bead pair shown in panel (A). The inset shows the time window used for linear fitting, and tail growth rates were determined from the slopes of the fitted lines. (C) Normalized total comet-tail fluorescence intensity plotted as a function of time for the representative control&#8211;myosin bead pair shown in panel (A). Control and myosin beads were normalized to the average fluorescence intensity of the control beads between 1100 and 1300 s. The inset shows the time window used for linear fitting, and fluorescence assembly rates were calculated from the slopes of the fitted lines. (D) Summary table showing the tail growth rate, fluorescence assembly rate, and growth efficiency for the representative control&#8211;myosin bead pair shown in panel (A). <a href="https://www.jove.com/files/ftp_upload/71259/71259fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The effect of myosin-I on comet-tail growth kinetics can be quantified by measuring both comet-tail elongation and actin fluorescence accumulation over time. In most experiments, comet tails grew at an approximately constant velocity during the first ~10 min following symmetry breaking. As polymerization components became depleted, elongation progressively slowed and typically ceased approximately 30 min after reaction initiation. To quantify growth kinetics, comet-tail length and total fluorescence intensity were plotted as functions of time (Figure 3B,C). The first 7&#8211;10 data points, corresponding to the initial linear phase, were fitted using linear regression (Figure 3B,C, insets). The slopes of these fits represented the initial tail growth rate and actin fluorescence assembly rate, respectively. Growth efficiency was defined as the comet-tail growth rate divided by the actin fluorescence assembly rate, quantifying how effectively actin polymerization was converted into productive bead displacement (Figure 3D). In the representative example shown in Figure 3A&#8211;D, the myosin-coated bead exhibited a tail growth rate of 0.60 &#181;m/min (0.010 &#181;m/s), compared with 0.57 &#181;m/min (0.0095 &#181;m/s) for the control bead (Figure 3B,D). The corresponding growth efficiencies were 5.6 and 4.5 &#181;m/intensity unit, respectively, representing an approximately 1.2-fold higher value for the myosin-coated bead (Figure 3D). Under standard conditions, myosin-coated beads typically exhibited growth rates comparable to those of control beads (myosin: 0.69 &#177; 0.10 &#181;m/min; control: 0.66 &#177; 0.20 &#181;m/min), together with a lower actin fluorescence assembly rate (approximately 0.76-fold of control) and an approximately 1.4-fold higher growth efficiency, as reported previously in our original study18. These values are reported as mean &#177; SD from 11 bead pairs per condition obtained across five independent experiments. Statistical analysis was performed using a two-tailed paired t-test, as described in the original study18.
To assess the contribution of the myosin power stroke and rule out the possibility that the observed effects arose from steric hindrance due to myosin surface occupancy, the bead motility assay can be performed under conditions that disrupt myosin-I power-stroke activity. In this protocol, myosin-I motor activity was inhibited by adding 1.1 mM Ca2+ to the reaction mixture. Elevated Ca2+ promoted dissociation of calmodulin from the myosin-I lever arm, reducing lever-arm rigidity and impairing its ability to generate force46,47,48. Under these conditions, myosin-I remained dynamically associated with actin but did not support actin gliding (Figure 4A,B), providing a useful control for evaluating motor activity-dependent effects18.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/71259/71259fig4.jpg" /> 
Figure 4. Calcium-based control for assessing Myo1d motor activity in the bead motility assay. (A) Time-collapsed images of actin filaments moving on a Myo1d-coated surface in the absence of elevated calcium or forming nonproductive swirling patterns in the presence of 100 &#181;M free calcium. The rainbow color bar indicates time progression over 61 frames acquired at 5 s intervals. Scale bar, 5 &#181;m. (B) Quantification of actin gliding speed in the absence and presence of 100 &#181;M free calcium (N = 2 independent experiments; n = 25 filaments). No productive actin gliding was observed in the presence of 100 &#181;M free calcium under these conditions. Error bars represent SD. (C) Representative control and myosin-coated bead pairs assembled in the absence or presence of 100 &#181;M free calcium. Five representative bead pairs are shown for each condition. Within each pair, the control bead and myosin-coated bead were imaged in the same field of view. Images were acquired approximately 15&#8211;20 min after reaction initiation. Brightness and contrast were adjusted linearly and identically within each control&#8211;myosin bead pair but were scaled independently between conditions. Reaction conditions: 4 &#181;M actin with 5% rhodamine-labeled actin, 200 nM actin-related protein 2/3 (Arp2/3) complex, and 50 nM capping protein (CP). Scale bars, 5 &#181;m. Panels A and B are reproduced/modified from Xu et al., Science Advances, DOI: 10.1126/sciadv.ado5788 (2024), AAAS, with permission. <a href="https://www.jove.com/files/ftp_upload/71259/71259fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
When the assay was performed correctly, myosin-coated beads assayed under elevated Ca2+ conditions generated shorter actin comet tails with network densities comparable to those of control beads (Figure 4C). Five representative control- and myosin-coated bead pairs from two independent experiments are shown. This qualitative outcome was consistently observed across repeated assay preparations and is supported by the quantitative analysis reported in the original study18. This expected outcome indicates that the network differences observed under standard conditions depend on an active myosin power stroke rather than myosin surface occupancy alone. Thus, the elevated Ca2+ condition serves as a key control for assessing whether changes in branched actin network organization arise from myosin motor activity.

This protocol describes a tunable actin comet-tail bead motility assay to quantitatively investigate how myosin motor activity influences branched actin assembly and network architecture at a membrane-like interface.

Branched actin polymerization mediated by the actin-related protein 2/3 (Arp2/3) complex provides the primary pushing forces for a variety of cellular ...

Our research focuses on studying how myosin-I shapes branched actin networks and drives force generation at membrane interfaces. Previous cellular studies make it hard to isolate myosin-I effects. This protocol reconstitutes everything in vitro under well-controlled conditions.To begin, gather the required reagents. After preparing the nucleation-promoting factor, or NPF, and neutravidin coating mixture, gently mix the coating mixture by pipetting up and down 5 to 10 times, avoiding bubble formation. Transfer 5 microliters of bead slurry to a microcentrifuge tube. Add 10 microliters of X buffer to the beads and pipette at least five times. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius. Carefully remove the supernatant by tilting the tube and aspirating from the side opposite the bead pellet. Resuspend the washed beads in 50 microliters of the previously prepared coating mixture. Mix the suspension by gentle pipetting and leave a small air bubble to facilitate mixing during incubation. Incubate the beads on a benchtop rotator at 20 revolutions per minute for two hours at 4 degrees Celsius. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius, and carefully remove the supernatant containing excess neutravidin and GST-VCA. Resuspend the coated beads in 200 to 400 microliters of 10 milligrams per milliliter BSA. Incubate the suspension on ice for 30 minutes to block unoccupied binding sites on the bead surface. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius, and carefully remove the supernatant. Resuspend the beads in 50 microliters of 1 milligram per milliliter BSA prepared in X buffer. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius. Remove the supernatant and repeat the wash twice. Store the coated beads in 1 milligram per milliliter BSA prepared in X buffer on ice and use them within one week.To exchange the storage X buffer with the M buffer before myosin coating, divide 50 microliters of neutravidin-NPF-coated beads equally into two microcentrifuge tubes. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius and carefully remove the supernatant. Wash the beads with 25 microliters of 1 milligram per milliliter BSA prepared in M buffer. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius. Carefully remove the supernatant and repeat the wash twice. Resuspend the control beads in 25 microliters of 1 micromolar Biotin-CF640 fluorescent dye diluted from the stock solution in M buffer. Resuspend the myosin beads in 25 microliters of 500 nanomolar to 1 micromolar biotinylated myosin supplemented with 1 to 5 micromolar calmodulin. After covering the tubes with aluminum foil, incubate the bead suspensions on ice for at least 30 minutes. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius, and carefully remove the supernatant containing excess biotinylated myosin or fluorescent dye. Wash the decorated beads with 25 microliters of 1 milligram per milliliter BSA prepared in M buffer. Pellet the beads by centrifugation at 16,000 x g for two minutes at 4 degrees Celsius. Carefully remove the supernatant and repeat the wash twice. Supplement the myosin-decorated beads with 1 to 5 micromolar calmodulin. Protect all decorated beads from light by covering the tubes with aluminum foil, then store the far-red fluorescent dye-decorated beads or myosin-decorated beads on ice.After bead preparation and decoration, prepare a 50-microliter motility mixture. Initiate the reaction by adding 4 microliters of 50 micromolar G-actin with 5% to 10% of the actin fluorescently labeled. Mix the reaction immediately and designate this time point as time equals 0. Clean the glass slide and coverslip with 70% ethanol, followed by deionized water. Plasma-clean the glass surfaces using ambient air plasma for 10 minutes. Apply 2.3 microliters of the reaction mixture to the glass slide. Place a 22-millimeter by 22-millimeter coverslip over the sample to form a squeeze chamber, with an approximate chamber height of 4.3 micrometers. Seal the chamber with vacuum grease. Using an epifluorescence microscope, acquire fluorescence images.Surface-bound NPFs stimulated branched actin assembly around the bead surface and actin first appeared as a sparse shell surrounding the bead. Continued actin assembly at the bead surface drove outward network growth, which led to symmetry breaking and the formation of a polarized comet tail. Once a comet tail was established, ongoing actin nucleation at the bead surface supported sustained bead movement. Under standard assay conditions, myosin-coated beads typically produce dimmer, sparser, or more diffuse actin comet tails than control beads.This protocol allows researchers to study how membrane-bound myosin motors regulate actin network density, growth kinetics, and polymerization-driven bead movement. The most important consideration is protein quality. Always validate new preparations against a previously validated batch before quantitative experiments. Future studies building on this work can test other membrane-associated myosins, disease-associated motor variants, or actin regulators at the membrane-like interface.

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Learn to reconstitute myosin-I-mediated branched actin assembly using a bead-based actin comet-tail motility assay. This method enables quantitative analysis of actin network dynamics and force generation at membrane-like interfaces.

Reconstituting Myosin-I-Mediated Branched Actin Assembly in a Bead-Based Actin Comet-Tail Motility Assay

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