Name: measuring protein binding to f-actin by co-sedimentation
Uploaded: 2017-05-18T12:30:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

This work was supported by the National Institutes of Health Grant HL127711 to AVK.

<ol>
	<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=Actin+and+Actin-Binding+Proteins.">Actin and Actin-Binding Proteins.</a> Cold Spring Harb Perspect Biol. 8 (8), (2016).</li><li>Hansen, M. D., Kwiatkowski, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+actin+dynamics+by+allosteric+regulation+of+actin+binding+proteins.">Control of actin dynamics by allosteric regulation of actin binding proteins.</a> Int Rev Cell Mol Biol. 303, 1-25 (2013).</li><li>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=Actin-binding+proteins:+the+long+road+to+understanding+the+dynamic+landscape+of+cellular+actin+networks.">Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.</a> Mol Biol Cell. 27 (16), 2519-2522 (2016).</li><li>Mullins, R. D., Hansen, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+actin+filament+and+network+dynamics.">In vitro studies of actin filament and network dynamics.</a> Curr Opin Cell Biol. 25 (1), 6-13 (2013).</li><li>Miller, P. W., 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=Danio+rerio+alphaE-catenin+is+a+monomeric+F-actin+binding+protein+with+distinct+properties+from+Mus+musculus+alphaE-catenin.">Danio rerio alphaE-catenin is a monomeric F-actin binding protein with distinct properties from Mus musculus alphaE-catenin.</a> J Biol Chem. 288 (31), 22324-22332 (2013).</li><li>Wickline, E. D., 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=alphaT-Catenin+Is+a+Constitutive+Actin-binding+alpha-Catenin+That+Directly+Couples+the+Cadherin.Catenin+Complex+to+Actin+Filaments.">alphaT-Catenin Is a Constitutive Actin-binding alpha-Catenin That Directly Couples the Cadherin.Catenin Complex to Actin Filaments.</a> J Biol Chem. 291 (30), 15687-15699 (2016).</li><li>Simpson, R. J., Adams, P. D., Golemis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic methods in protein purification and analysis : a laboratory manual. , (2009).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Dyballa, N., Metzger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+colloidal+coomassie+G-250+staining+for+proteins+in+polyacrylamide+gels.">Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.</a> J Vis Exp. (30), (2009).</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=A+guide+to+simple+and+informative+binding+assays.">A guide to simple and informative binding assays.</a> Mol Biol Cell. 21 (23), 4061-4067 (2010).</li><li>Hansen, S. D., 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=alphaE-catenin+actin-binding+domain+alters+actin+filament+conformation+and+regulates+binding+of+nucleation+and+disassembly+factors.">alphaE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors.</a> Mol Biol Cell. 24 (23), 3710-3720 (2013).</li><li>Perrin, B. J., Ervasti, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+gene+family:+function+follows+isoform.">The actin gene family: function follows isoform.</a> Cytoskeleton (Hoboken). 67 (10), 630-634 (2010).</li><li>Tondeleir, D., Vandamme, D., Vandekerckhove, J., Ampe, C., Lambrechts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+isoform+expression+patterns+during+mammalian+development+and+in+pathology:+insights+from+mouse+models.">Actin isoform expression patterns during mammalian development and in pathology: insights from mouse models.</a> Cell Motil Cytoskeleton. 66 (10), 798-815 (2009).</li><li>Prochniewicz, E., Janson, N., Thomas, D. D., Dela Cruz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+increases+the+torsional+flexibility+and+dynamics+of+actin+filaments.">Cofilin increases the torsional flexibility and dynamics of actin filaments.</a> J Mol Biol. 353 (5), 990-1000 (2005).</li><li>Kuhn, J. R., 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=Real-time+measurements+of+actin+filament+polymerization+by+total+internal+reflection+fluorescence+microscopy.">Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy.</a> Biophys J. 88 (2), 1387-1402 (2005).</li><li>Hansen, S. D., Zuchero, J. B., 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=Cytoplasmic+actin:+purification+and+single+molecule+assembly+assays.">Cytoplasmic actin: purification and single molecule assembly assays.</a> Methods Mol Biol. 1046, 145-170 (2013).</li></ol>

The authors declare that they have no competing financial interests.

The actin co-sedimentation assay is a straightforward technique that can quickly determine if a protein binds F-actin. With some modifications, the technique can also be used to measure the affinity of the interaction. In addition to points raised in the protocol above, the following issues should be considered when designing, conducting, and interpreting the assay.
Protein of Interest
Freshly prepared or frozen protein can be used in the assay. If frozen protein is used, it is highly recommended that the results be compared with fresh (never frozen) protein to ensure that freezing does not affect F-actin binding.
G-actin Source
Many pelleting experiments use G-actin isolated from muscle because of its relative abundance. There are three main actin isotypes in mammals &#8211; alpha, beta and gamma &#8211; that are remarkably similar (&#62;90% sequence identity). Nonetheless, there are functional differences between the isotypes12,13. If possible, the G-actin isotype used in the binding assay should match the in vivo isotype. For example, if testing a protein expressed in skeletal muscle, alpha-actin is the best choice; if examining a protein expressed in fibroblasts, beta-actin is recommended.
Phalloidin Use
Since phalloidin binds F-actin, it can interfere or even block the binding of some F-actin binding proteins (e.g., phalloidin blocks cofilin from binding to actin filaments)14. Thus, phalloidin should be used with caution and the results compared to non-phalloidin-treated samples when possible.
High Background
It is not uncommon for proteins to sediment in the absence of F-actin (Figure 1A, no F-actin pellet samples). However, high levels of background sedimentation can mask true actin co-sedimentation and make it difficult, if not impossible, to determine if a protein binds F-actin or to measure the affinity of the interaction. Adding polidicanol to the reaction buffer (step 4.1) can significantly reduce the background and is an easy solution. If that does not reduce the background, adjusting the reaction buffer, salt concentration, and/or incubation temperature may help.
Binding Curve
To generate a binding curve, it is necessary to vary the concentration of either the protein of interest or the F-actin over a series of reactions. In practice, it is easier and preferable to maintain F-actin at a fixed concentration and to vary the concentration of the protein of interest. Maintaining F-actin at a fixed concentration (e.g., 2 &#181;M) in the pelleting assay limits non-specific trapping at higher concentrations of F-actin and prevents depolymerization at lower (&#60;0.5 &#181;M) concentrations of F-actin. Depolymerization can be prevented using phalloidin, although this introduces a potential complicating factor into the system (see step 3.3 and above). Maintaining F-actin at a fixed concentration also allows one to compare (and normalize) the F-actin pellet across samples and to identify failed experiments (i.e., where the F-actin pellet is highly variable, preventing analysis across concentrations). Finally, maintaining F-actin at a fixed concentration allows one to determine if the binding to the actin filament is cooperative (Figure 1C).
Saturated Binding
As in all binding experiments, it is critical that the binding to F-actin is saturated and that the concentration of protein plus F-actin plateaus (Figure 1C). Without a plateau, it is not possible to calculate an accurate dissociation equilibrium constant. Thus, it is important to carefully plan the dilution series to be tested and to always include higher concentrations of protein (i.e., at least 5- to 10-fold higher than the expected Kd).
Binding Analysis
In order for the measured dissociation constants to be conclusive, the assay should be performed using an F-actin concentration that allows the concentration of binding sites on F-actin for the protein of interest to be much lower than the affinity. To check whether this criterion was met, estimate the concentration of binding sites from Bmax. For example, if [F-actin] was 2 &#181;M and Bmax = 0.5, then [binding sites] &#8776; 1 &#181;M. The Kd should be at least 5- to 10-fold greater than [binding sites]. If the measured Kd is of the same order of magnitude as [binding sites], then it is possible that the observed binding curve represents a titration of high-affinity binding sites rather than a true binding isotherm. If this is observed, repeat the assay using a 10-fold lower F-actin concentration to measure an accurate affinity. For high-affinity interactions, phalloidin stabilization (step 3.3) may be necessary to achieve an F-actin concentration low enough to accurately measure affinity.
Finally, there are fundamental limitations with the co-sedimentation assay that researchers should be aware of when performing and evaluating the assay. Most importantly, the co-sedimentation assay does not produce a true equilibrium constant. The products of binding (i.e., protein plus F-actin) are separated from the reactants during centrifugation, whereupon the products can then dissociate to create a new equilibrium. As a result, the co-sedimentation assay can miscalculate or fail to detect low-affinity interactions. Since many actin-binding proteins have a low (i.e., micromolar) affinity for F-actin, a negative result (i.e., no detectable binding) in the assay does not necessarily mean that a protein does not bind F-actin. As an alternative, TIRF microscopy-based, single-filament binding assays are more sensitive and more accurate for determining a dissociation constant (for reviews on this technique, see references15,16). Despite these limitations, the pelleting assay is within the means of most researchers and is an effective tool to determine if a protein binds F-actin and to measure the affinity of the interaction.

Actin is an essential cytoskeletal protein that plays a critical role in multiple cellular processes, including motility, contraction, adhesion, and morphology1. Actin exists in two forms: monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin). Within cells, F-actin organization is controlled by a large collection of proteins that regulate the nucleation, growth, cross-linking, and disassembly of actin filaments2,3,4. However, how multiple actin-binding proteins function in concert to regulate actin network organization is still largely unclear.
The measurement of protein-protein interactions is an important approach for understanding how proteins exert their effects on cellular behavior at the biochemical level. Many different assays can be used to detect interactions between purified proteins. Common approaches for soluble proteins include pull-downs, fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Importantly, all of these assays require proteins to be soluble, and are thus difficult to adapt for use with a polymeric, filamentous protein such as F-actin. Here, we describe a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine if a protein or protein domain binds F-actin and to measure the affinity of the interaction.
The actin pelleting assay is a relatively straightforward technique that does not require specialized equipment, aside from an ultracentrifuge. All reagents can be made, assuming knowledge of basic biochemistry, or purchased. Once binding to F-actin is established, the assay can be used to measure the apparent affinity (i.e., the dissociation equilibrium constant)5. Also, once an affinity is established, the pelleting assay is a useful tool to measure how changes to the protein of interest (i.e., post-translational modifications, mutations, or ligand binding) affect its interaction with F-actin6. The technique does have limitations (see the Discussion) that the researcher should be aware of before attempting the assay.

<table border="0" cellpadding="0" cellspacing="0" width="842">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Sorvall MTX 150 Micro-Ultracentrifuge</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">46960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">S100-AT3 rotor</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">45585</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Ultracentrifuge tubes - 0.2 ml</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">45233</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Actin, rabbit skeletal muscle</td>
			<td style="width:224px;">Cytoskeleton</td>
			<td style="width:119px;">AKL99</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Bovine Serum Albumin</td>
			<td style="width:224px;">Sigma</td>
			<td style="width:119px;">A8531</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Polidicanol (Thesit)&#160;</td>
			<td style="width:224px;">Sigma</td>
			<td style="width:119px;">88315</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Phalloidin</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">P3457</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">R0862</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Adenosine triphosphate (ATP)</td>
			<td style="width:224px;">Sigma</td>
			<td style="width:119px;">A2383</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Imidazole</td>
			<td style="width:224px;">Fisher Scientific</td>
			<td style="width:119px;">O3196</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Sodium Chloride (NaCl)</td>
			<td style="width:224px;">Fisher Scientific</td>
			<td style="width:119px;">BP358</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:224px;">Fisher Scientific</td>
			<td style="width:119px;">M33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Potassium Chloride (KCl)</td>
			<td style="width:224px;">Fisher Scientific</td>
			<td style="width:119px;">P217</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:224px;">Sigma</td>
			<td style="width:119px;">3779</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Odyssey CLx Imaging System</td>
			<td style="width:224px;">LI-COR</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Coomassie Brilliant Blue R-250 Dye</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">20278</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colloidal Blue Staining Kit</td>
			<td style="width:224px;">ThermoFisher Scientific</td>
			<td style="width:119px;">LC6025</td>
		</tr>
	</tbody>
</table>

measuring protein binding to f-actin by co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

We examined &#945;E-catenin homodimer binding to F-actin in the co-sedimentation assay. Since past experiments have shown that the affinity of &#945;E-catenin homodimer for F-actin is around 1 &#181;M and the Bmax near 111, we performed the assay with a low concentration of F-actin (0.2 &#181;M rather than 2 &#181;M; see the Discussion). Since 0.2 &#181;M is below the critical concentration, phalloidin was added to stabilize the F-actin polymerized from rabbit skeletal muscle G-actin (step 3.3). Increasing concentrations of &#945;E-catenin homodimer (0.125-12.0 &#181;M) were incubated in the presence or absence of 0.2 &#181;M F-actin. The samples were centrifuged, and the resulting pellets were analyzed (Figure 1A). As expected, the &#945;E-catenin homodimer co-sedimented with F-actin above background (Figure 1A, compare the F-actin pellet samples to the no-F-actin pellet samples). BSA was run as a negative control (Figure 1B). Bound protein was quantified and plotted over free protein to calculate the affinity of the interaction (Figure 1C). The plotted data best fit a Hill equation. The calculated Kd was 2.9 &#181;M, the Bmax was 0.2, and the Hill coefficient (h) was 4.8. Thus, &#945;E-catenin homodimer binds F-actin cooperatively with a low micromolar affinity, consistent with previous work (a Kd of 2.9 &#181;M versus ~1.0 &#181;M)11.
<img alt="Figure 1" src="/files/ftp_upload/55613/55613fig1.jpg" /> 
Figure 1: High-speed F-actin co-sedimentation assay. (A) Increasing concentrations (0.125-12.0 &#181;M) of &#945;E-catenin homodimer were incubated with (left panels) or without (right panels) 0.2 &#181;M F-actin stabilized with phalloidin. They were incubated for 30 min at RT and centrifuged. The total (7.5% of the starting material) and pelleted material (50% of the pelleted material) were separated by SDS-PAGE and stained with Coomassie dye. (B) 4 &#181;M BSA was run as a negative control. Total and pellet samples, with (+) or without (-) F-actin, were separated by SDS-PAGE and stained with Coomassie dye. (C) Bound &#945;E-catenin (&#181;M/&#181;M actin) from A was plotted against free &#945;E-catenin (&#181;M), and the data fit to a Hill equation (red line). The Kd, Bmax, and Hill coefficient (h) are listed. <a href="http://cloudflare.jove.com/files/ftp_upload/55613/55613fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55613/55613fig2.jpg" /> 
Figure 2: Actin pelleting quantification&#160;&#8211; flow chart. This schematic outlines key steps in section 5, with examples of Total and Pellet samples (A, C-E) and the standard curve (B) used for quantification. 5.13 steps: 1) Measure the amount of protein of interest in the Total samples (A). 2) Generate a standard curve by plotting the band intensity versus protein mass (B). 3) Measure the amount of protein of interest that co-sedimented with F-actin (C). 4) Measure the amount of protein of interest that pelleted in the absence of F-actin (D). 5) Subtract D from C to determine the amount of protein bound to F-actin. 6) Measure the amount of F-actin in each pellet (E), calculate the average amount of F-actin per sample, and divide each sample by the average (the numbers below show the ratio). 7) For each sample, divide the amount of bound protein (calculated in step 5) by the F-actin pellet ratio (calculated in step 6) to adjust for differences in the pellet. 8) Use the standard curve (B) to calculate the amount (mass) of normalized bound protein in each sample (step 7). 9) Determine the concentration of free protein and bound protein to create a binding curve. <a href="http://cloudflare.jove.com/files/ftp_upload/55613/55613fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

Measuring Protein Binding to F-actin by Co-sedimentation

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

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