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.

In previous posts,  I invited you to look at versatile methods to

• Visualize actin: Focus on Actin staining and visualization
• Measure actin binding: Focus on Actin – Detection of actin binding and actin binding proteins
• Measure actin polymerization: Focus on Actin – Measuring actin polymerization

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

Today, let’s focus on a method which enables researchers 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.

Actin can be stained in living and fixed cells to determine and follow the structure and function of the cytoskeleton. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed by either cold methanol or paraformaldehyde prior to probing or staining for actin structures.

Actin staining in fixed cells

Fluorescent phalloidin binding to F-Actin, Source: Cytoskeleton Inc.

In fixed cells, actin structures can be visualized by actin antibodies, fluorescent phalloidins, or even electron microscopy.
Antibodies recognize both monomer and polymer (filamentous or F-actin) actin and hence tend to have a high background compared to probes that bind only F-actin. Well designed fluorescent phalloidins only bind to the native quaternary structure of F-actin and therefore have a low background. To create the correct fixation conditions for phalloidin binding, paraformaldehyde must be used as the fixative because it retains the quanternary protein structure which is necessary for high affinity. Methanol destroys the native conformation and hence is not suitable for actin staining with phalloidin. 

Cytoskeletal live cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics in living cells.