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Lifeact: a versatile marker to visualize F-actin
http://www.nature.com/nature methods
Julia Riedl1,7, Alvaro H Crevenna1,7, Kai Kessenbrock2, Jerry Haochen Yu1, Dorothee Neukirchen3, Michal Bista4, Frank Bradke3, Dieter Jenne2, Tad A Holak4, Zena Werb5, Michael Sixt6 & Roland Wedlich-Soldner1
Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained lamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modied peptide form allowed visualization of actin dynamics in nontransfectable cells.
Reliable visualization of the actin cytoskeleton is essential for various elds of biomedical research. Imaging of actin dynamics has been mostly achieved by injection of uorescently labeled actin (technically demanding) or small amounts of uorescently labeled
phalloidin, an F-actin-binding and stabilizing compound1,2. A
widely used alternative is the expression of actin-GFP fusion proteins. However, all described actin fusions are functionally impaired and rely on nontagged actin3 to buffer the defects. Recently, fusions of GFP to actin-binding domains have been used, notably from moesin in Drosophila melanogaster4, LimE in Dictyostelium discoideum5,ABP120in D. discoideum and mammalian cells6,7, and utrophin in Xenopus laevis8. These probes consist of large domains, compete with their endogenous counterparts and are restricted to cells that can be transfected.
Abp140-GFP is the only probe that has been successfully used to label actin cables, in budding yeast9,10. Using total internal reection
(TIRF) microscopy to monitor localization of Abp140 domains fused to GFP, we found that the rst 17 aa of Abp140 were sufcient to mediate actin localization comparable to the full-length protein (Fig. 1a,b). This short peptide is conserved among close relatives of Saccharomyces cerevisiae (Fig. 1c) but is absent from other organisms. This and its small size make it an attractive actin marker for higher eukaryotes. We therefore introduced the peptide, named Lifeact, as a C-terminal GFP fusion (Lifeact-GFP) into mammalian cell lines to test its suitability as an in vivo marker. We also synthesized Lifeact with an N-terminal FITC uorophore (F-Lifeact) to test its biochemical properties.
8 Nature Publishing Group
a b c
d e
Saccharomyces cerevisiae Saccharomyces mikatae Saccharomyces kudriavzevii
Saccharomyces kluyveri Kluyveromyces...