Lifeact: a versatile marker to visualize F-actin

Abstract

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 filamentous 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 modified peptide form allowed visualization of actin dynamics in nontransfectable cells.Reliable visualization of the actin cytoskeleton is essential for various fields of biomedical research. Imaging of actin dynamics has been mostly achieved by injection of fluorescently labeled actin (technically demanding) or small amounts of fluorescently labeled phalloidin, an F-actin-binding and stabilizing compound 1,2 . A widely used alternative is the expression of actin-GFP fusion proteins. However, all described actin fusions are functionally impaired and rely on nontagged actin 3 to buffer the defects. Recently, fusions of GFP to actin-binding domains have been used, notably from moesin in Drosophila melanogaster 4 , LimE in Dictyostelium discoideum 5 , ABP120 in D. discoideum and mammalian cells 6,7 , and utrophin in Xenopus laevis 8 . 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 yeast 9,10 . Using total internal reflection (TIRF) microscopy to monitor localization of Abp140 domains fused to GFP, we found that the first 17 aa of Abp140 were sufficient to mediate actin Correspondence should be addressed to M.S.

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…To visualize potential changes in cytoskeletal structures, we used a B16.F10 melanoma cell line stably transfected with Lifeact-GFP, a fluorescent probe for F-actin. 55 The pDNA molecules were covalently labeled with a red fluorescent dye, tetramethylrhodamine, to track their locations. Comparing with non-treated control cells, in which actin filaments were evenly distributed in the cytosol, exposure of cells to electric pulses in the presence of DNA led to formation of various structures of F-actin, including punctate-like aggregates, membrane ruffles and filopodia-like protrusions (see Figures 1A and S1A-S1C).…”

Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation

“…Lifeact is a 17aa peptide from a yeast actin binding protein, Abp140, sufficient to bind F-actin and, to a much less extent, G-actin [Riedl et al, 2008]. The actin cloud recognized by Lifeact is absent from the same region in formin-2 null oocytes, indicating that this F-actin population is nucleated by formin-2.…”

The actin-associating protein Tm5NM1 blocks mesenchymal motility without transition to amoeboid motility

“…We next determined the composition of the short pericellular protrusions, observed in the B35.Tm5NM1 cells (Figure 1c), by transfecting the cells with GFPtagged monomeric LifeACT to detect filamentous actin (Riedl et al, 2008). Confocal microscopy revealed that the short spikes emanating from the Tm5NM1-overexpressing cells are actin-rich structures, visible both in the main population of elliptical cells and the 25% of the elongated cells (Figures 3c and d).…”