3 functional assays to investigate actin dynamics

Today, I’d like to give you an overview about methods in actin research with validated R&D products and kits which will allow you to measure binding to actin and effects on the polymerisation dynamics of actin.

Actin functions as one of the major cytoskeleton structures. It is involved in a plethora of processes in cell biology:  stabilizing the cell shape, cell movements (e.g. cell migration)  and intracellular movements and transport mechanisms.

Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group.

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Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)

G-actin polymerizes to form F-actin

Globular-actin (G-actin) readily polymerizes under physiological conditions to form Filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament (Fig. 1).  Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end or barbed-end (+) while the slow growing end is called the minus-end or pointed-end (-).

How to measure binding to actin

Blot

Fig. 2: F-Actin Binding Assay. An F-actin binding assay was performed as described in Section VI Binding Assay (method 1) of the manual (centrifugation at 150 000 x g for 1.5 h). Samples of the supernatant (S) and pellet (P) fractions were collected for each reaction. Samples were separated on a 4-20% SDS-gel and stained with 0.1% Coomassie blue. Reaction 1, F-actin alone. Reaction 2, α-actinin alone. Reaction 3, BSA alone. Reaction 4. α-actinin and F-actin. Reaction 5, BSA and F-actin. Note how in the presence of F-actin filaments greater than 70% of the ABP α-actinin is found in the pellet fraction (4 P). In contrast, greater than 90% of BSA remains in the supernatant fraction when incubated with F-actin filaments (5 S).

The first method I am going to introduce allows you to measure actin binding capabilities of proteins of interest. But it’s not only about the simple fact that a given protein is binding to actin, with the method presented here, you’re also able to get an idea of the functionality of the protein – be it F-actin bundling activity, F-actin severing activity or G-actin binding activity.

Cytoskeleton Inc. have optimized a method to measure actin binding which is based on a simple pull-down experiment. Actin binding occurs when there is an affinity for any site of actin. F-actin binding can be measured by using a spin down assay where centrifugation is used to separate F-actin from G-actin by differential sedimentation. F-actin binding proteins will co-sediment with actin filaments and form a pellet at the bottom of the centrifugation tube (Fig. 2).

Measurement of actin bundling activity

Although simple in terms of experimental set-up, the methods allows you not only to detect actin binding of your protein of interest, but also gives you the opportunity to characterize your protein in terms of effects on the actin cytoskeleton.To measure actin bundling activity of your protein the protocol of the pull-down assay has to be slightly modified by pulling down bundled actin by centrifugation at 14.000 x g (instead of 150.000 x g). A typical result of the bundling activity of α-actinin  is shown in Fig. 3.

Actin Bundling

Fig. 3: Actin bundling assay using Cytoskeleton’s Actin Binding Protein Biochem Kit. F-actin was incubated alone or together with α-actinin or BSA. Bundled F-actin was pelleted by a 14.000 x g centrifugation and pellets (P) and supernatants (S) were run on an SDS-PAGE gel. Only in the presence of the F-actin bundling protein α-actinin is actin pelleted at this centrifugation speed.

Furthermore, the detailed protocol of the kits contains a guide to interpret the results and to distinguish between severing or capping protein, whether the protein of interest enhances actin polymerization,  whether it is G- or F-actin binding and whether it exhibits actin depolymerizing activity.

The Actin Binding Protein Spin-Down Biochem Kits provide either muscle or non-muscle actin plus positive (-actinin) and negative (Bovine Serum Albumin, BSA) binding control proteins.

Measuring actin polymerization

Actin polymerization follows three phases, similar to microtubule assembly; these are lag phase, growth and steady state as depicted in Figure 4.

The method applied in the Actin Polymerization Biochem Kit produced by Cytoskeleton Inc. is based on the enhanced fluorescence of pyrene conjugated actin that occurs during polymerization. The enhanced fluorescence that occurs when pyrene G-actin (monomer) forms pyrene F-actin can be used to follow polymerization over time (Fig. 5). Also, by using preformed pyrene F-actin, it is possible to follow depolymerization. Both cell/tissue extracts and purified proteins can be added to the reaction mixture to identify their effect on actin polymerization.

Actin polymersization - phases

Fig. 4: Polymerization of actin as measured by pyrene actin fluorescence.

The method applied in the kit can be used:

  • to show quantitative / qualitative effects on actin polymerization by the addition of a tissue extract, an actin binding protein, or compound.
  • to show quantitative / qualitative effects on actin polymerization by addition of an F-actin nucleating protein, compound, or extract.
  • to show quantitative / qualitative effects on steady-state F-actin levels by addition of an F-actin severing protein, compound, or tissue extract.
  • to show quantitative / qualitative effects on actin depolymerization by addition of an actin binding protein, compound, or tissue extract.
  • to determine the critical concentration of actin polymerization under various experimental conditions.
Actin polymerisation graph

Fig. 5: Pyrene actin polymerization assay – Actin polymerization was carried out as described in the kit manual. Duplicate samples of pyrene actin and General Actin Buffer alone were assayed for 3 min to establish a baseline fluorescence value. At 3 min 20 μl of 10x Actin Polymerization buffer was added to all the wells and fluorescence was assayed every 30 s for 1 h. NOTE: Actin polymerization resulted in a 7 fold increase in fluorescence compared to monomeric actin levels. Arrowhead indicates the fluorescence signal from pyrene G-actin. Arrow shows the increases fluorescence associated with pyrene F-actin. (provided by Cytoskeleton Inc.)

Graph - G-F actin ratio

Fig. 6: Reorganization of actin in Swiss 3T3 cells after treatment with jasplakinolide. Swiss 3T3 cells were treated with jasplakinolide (Jaspl) or left untreated (Untr) and the G-actin (G) and F-actin (F) content was assayed using kit 027BK037. Treatment with jasplakinolide resulted in a potent accumulation of F-actin.

How to measure G : F actin ratio in cells?

The last method I like to introduce enables you to measure the ratio between monomeric globular actin (G actin) and filamentous actin (F actin) in cells, thus giving a good insight in the status of actin dynamics.

The most reproducible and accurate method of determining the amount of F actin content versus free G actin content in a cell population is to use Western blot quantitation of F actin and G actin cellular fractions. The general approach is to homogenize cells in F-actin stabilization buffer, followed by centrifugation to separate the F-actin from G-actin pool. The fractions are then separated by SDS-PAGE and actin is quantitated by Western blot. The final result gives the most accurate method of determining the ratio of F-actin incorporated into the cytoskeleton versus the G-actin found in the cytosol.

Cytoskeleton Inc. offers this method in an easy-to-handle and ready-to-use G-Actin : F-Actin Assay Kit. Typical results obtained with this kit are shown in Fig. 6. Changes in the amount of G-actin and F-actin were investigated in Swiss 3T3 cells treated with the actin polymerizing drug jasplakinolide, using the G-actin/F-actin in vivo assay kit.  In untreated Swiss 3T3 cells, 80% of actin is soluble G-actin, and is found within the supernatant fraction, 20% of actin is filamentous F-actin and is found in the pellet fraction. In Swiss 3T3 cells treated with jasplakinolide, 80% of actin is reorganized into F-actin and is found in the pellet fraction.

Are you interested in establishing one or more of these assays in your laboratory? Please contact me with the form below for more information.

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Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.