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.
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
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.
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
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.
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.
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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