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 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.
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.
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 µm syringe filter, aliquot in portions of 500 µL-2 mL per tube depending on usage, and store at -20 °C.
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°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 °C for a few weeks.
3. Measurement of protein concentrations
4. Coating of beads
5. Preparing the motility mix and slides for observation
NOTE: The total volume of the motility mix is 8.4 µL to allow for about 25 µm clearance between the slide and the 18 mm x 18 mm coverslip so beads of all sizes (up to 10 µm diameter) are not squeezed. The basic motility mix is approximately 5 µM G-actin (10% labeled fluorescent actin) with 5 µM profilin, 50 nM Arp2/3 complex, and 25 nM capping protein (or 240 nM gelsolin).
6. Microscopy observation
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, "pseudo-time-lapse" 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.
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. Please click here to view a larger version of this figure.
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 µm 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 µm. Please click here to view a larger version of this figure.
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 µm/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 µm/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 µm diameter beads, epifluorescence imaging of fluorescent actin, scale bars = 5 µm. Please click here to view a larger version of this figure.
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 °C or much below 23 °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 µL of 2 µM 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.
The authors have nothing to disclose.
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).
Actin, rabbit muscle, Alexa Fluor 488 conjugate | Invitrogen (ThermoFisher Scientific) | A12373 (recently discontinued) | This product can be replaced with ATTO-488 actin from Hypermol. |
Actin, rabbit muscle, ATTO-488 | Hypermol | 8153 | |
Actin, rabbit skeletal muscle | Cytoskeleton | AKL99 | |
Arp2/3 complex | Cytoskeleton | RP01P | |
ATP | Sigma | A7699 | |
BioSpectrometer, basic | Eppendorf | 035739 | |
Bradford Reagent | Bio-Rad | 500-0006 | |
BSA, high quality | Sigma | A3059 | |
BSA standard 2 mg/mL (Pierce) | Thermo Scientific | 23209 | |
Capping protein (a1b2, mouse recombinant) | Home-purified (Reference 13) | This product will soon be commercially available from Cytoskeleton. | |
Capping protein (a1b2, human recombinant) | Hypermol | 8322 | |
Cube, GFP: U-MNIBA3 or U-MWB2 | Olympus | discontinued | Any GFP cube, adapted to the microscope being used, can be used. |
Dry block, agitating: ThermoMixer C (refrigerated) | Eppendorf | 035963 | |
** with SmartBlock, 24 microtubes 2 mL | Eppendorf | 035969 | |
Gelsolin (human recombinant, His-tagged) | Cytoskeleton | HPG6 | |
Lanolin | Sigma | 49909 | |
Microcentrifuge 5427R + rotor | Eppendorf | 934126 | |
Microscope, upright: BX51 | Olympus | discontinued | Any epifluorescence upright microscope equipped with phase contrast optics can be used. |
Microscope, inverted: IX70 | Olympus | discontinued | Any epifluorescence inverted microscope equipped with phase contrast optics can be used. |
Paraffin | Sigma | 76244 | |
Petroleum jelly: Vaseline | Sigma | 16415 | |
Pipettes Research Plus | Eppendorf | Gilson pipettes don't work as well for delivery of very small volumes (0.5 µL for example). | |
**10 µL | 933954 | ||
**2.5 µL | 933953 | These two sizes are essential, but the use of high-quality pipettes (a full Research Plus set for example) is recommended. | |
Polystyrene carboxylate beads | Polysciences | ||
**approx. 1 µm diameter | 08226 | ||
**approx. 4.5 µm diameter | 17140-5 | ||
Profilin 1 (human recombinant, untagged) | Cytoskeleton | PR02 | |
SpVCA (human WASP pVCA domain, N-ter His-tag, C-ter Streptavidin tag) | Home-purified (Reference 14) | This product will soon be commercially available from Cytoskeleton. | |
VCA (human WASP VCA domain, GST-tagged) | Cytoskeleton | VCG03 |