Focus on Actin – Measuring actin polymerization

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

Actin dynamics – i.e. polymerization of G-actin to form filamentous F-actin or de-polymerization of F-actin – is a fundamental process in cell biology, as it is the basis of cell movement (e.g. cell migration)  and intracellular movements and transport mechanisms. 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 (-).

In this post, I’d like to concentrate on a method to measure polymerization of actin in biochemical assays. It’s the third post in a series of actin related publications which started with Focus on Actin staining and visualization and  Focus on Actin – Detection of actin binding and actin binding proteins. In an upcoming blog I’ll be focussing on G-F actin ratio detection in cells.

Measuring actin polymerization

Actin polymersization - phases

Fig. 2: Polymerization of actin as measured by pyrene actin fluorescence.

Actin polymerization follows three phases, similar to microtubule assembly; these are lag phase, growth and steady state as depicted in Figure 2.
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. 3). 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 polymerisation graph

Fig. 3: 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.)

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

If you are interested in this method, leave your comment or questions below!

 

 

 

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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.