F-actin visualization using fluorescent markers is an important tool for getting a deeper understanding of the structural cytoskeletal dynamics. For the observation of F-actin-related processes, non-invasive live cell imaging has become the state-of-the-art technique. Depending on the application and the investigated model organism and cell type, there are different F-actin staining techniques available—each of them with its own advantages and disadvantages.
For further details, please read this concise review, which summarizes the actin visualization techniques that are currently available:
M. Melak, M. Plessner, and R. Grosse. Actin visualization at a glance. Journal of Cell Science, 2017, 10.1242/jcs.204487 read abstract
At a Glance: Different Methods of Actin Visualization
This table refers to standard applications in mammalian expression systems.
LifeAct is a short, 17-amino acid peptide that specifically binds to F-actin. It is derived from the budding yeast (Saccharomyces cerevisiae) protein Abp140, which has been successfully used to label actin cables in this model. Conjugated with GPF, LifeAct-GFP can easily be introduced into living and fixed eukaryotic cells to visualize F-actin, while retaining highest actin functionality.
In contrast to other actin labeling agents, such as phalloidin and actin-coupled fluorescent proteins, LifeAct can visualize actin kinetics with the lowest potential interference. It is non-toxic and can thereby be used in both living and fixed cells and tissues.
The biocompatibility of LifeAct has been further proven in a transgenic mouse model in vivo. In this model, either LifeAct-EGFP or LifeAct-mRFPruby—both driven by a chicken actin promoter with a CMV enhancer—were introduced into the murine germline. The resulting LifeAct mice were viable, fertile, and showed highly specific and clear LifeAct staining in nearly all cell types. Importantly, actin was evenly distributed and no changes in the cytoskeletal organization were observed.
B. J. Belin, L. M. Goins, and R. D. Mullins. Comparative analysis of tools for live cell imaging of actin network architecture. Bioarchitecture, 2014, 10.1080/19490992.2014.1047714 read abstract
J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, … R. Wedlich-Soldner. Lifeact: a versatile marker to visualize F-actin. Nature Methods, 2008, 10.1038/nmeth.1220 read abstract
J. Riedl, K. C. Flynn, A. Raducanu, F. Gärtner, G. Beck, M. Bösl, … R. Wedlich-Söldner. Lifeact mice for studying F-actin dynamics. Nature Methods, 2010, 10.1038/nmeth0310-168 read abstract
LifeAct specifically stains F-actin in fibroblasts and co-localizes with phalloidin.
Since 2008, LifeAct is regarded as the gold standard for live cell imaging of F-actin. LifeAct constructs are widely used and published. Compared to other genetically encoded actin markers, such as fluorescent protein-coupled actin monomers, antibodies, and small molecules, LifeAct markers show the least interference with cytoskeleton dynamics and artefacts due to overexpression. This interference generally depends on the transfection system, the expression level, and the cell type. As a precaution, ibidi recommends our customers to perform suitable control experiments. Other potential trouble-causing parameters, such as transfection and transduction toxicity as well as imaging phototoxicity should be considered as well.
Sliogeryte K, et al. (2016) Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration. J Biomech 49(2):310–317. 10.1016/j.jbiomech.2015.12.034. read abstract
Courtemanche N, Pollard TD, Chen Q (2016) Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nat Cell Biol 18(6):676–683. 10.1038/ncb3351. read abstract
Phalloidin is a toxin that originates from the death cap mushroom (Amanita phalloides). It binds to F-actin, thereby preventing its depolymerization—ultimately leading to cell death by the paralysis of the cytoskeleton.
The binding of phalloidin to F-actin is irreversible and highly specific, making it a standardly-applied tool for F-actin visualization in fixed cells. Typically, it is conjugated to a fluorophore such as Rhodamine or FITC. After the staining procedure, the endogenous actin filaments with the bound phalloidin can be imaged by fluorescence microscopy. One major drawback of using phalloidin for F-actin imaging is its high toxicity. As it disturbs actin functionality and even leads to cell death, it is unsuitable for live cell imaging applications and should only be used in fixed cells.
J. Wehland, M. Osborn, and K. Weber. Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth. Proc. Natl. Acad. Sci. USA, 1977, 74, 5613-5617 read abstract
E. Wulf, A. Deboben, F. A. Bautz, H. Faulstich, and T. Wieland. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc. Natl. Acad. Sci. USA, 1979, 76, 4498-4502 read abstract
Scheme of the death cap mushroom (Amanita phalloides), which contains the toxin phalloidin.
Actin-Coupled Fluorescent Proteins (Actin-GFP)
Actin-coupled fluorescent proteins, such as actin-GFP, are widely used for F-actin visualization in living cells. Here, the fusion construct of actin and the fluorescent protein is introduced into the cells of interest (e.g., via plasmid transfection or viral transduction) and can be imaged by fluorescent live cell microscopy afterwards. The application of actin-coupled fluorescent proteins is relatively simple, non-toxic, and proven to be useful for actin visualization in living cells using diverse experimental approaches.
However, one clear disadvantage of this method is the inevitable expression of ectopic actin, which can alter the behavior of the cell. In addition, the relatively large size of the GFP (~27 kDa), can cause unwanted effects such as reduced F-actin functionality. This technique requires the precise establishment and accurate control of the actin-GFP expression for each separate cellular model, in order to prevent artificial effects that might alter the experimental outcome.
K. Sliogeryte, S. D. Thorpe, Z. Wang, C. L. Thompson, N. Gavara, and M. M. Knight. Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration. J. Biomech. 2016, 10.1016/j.jbiomech.2015.12.034 read abstract