Visualization of the actin cytoskeleton: Different F‐actin‐binding probes tell different stories

Abstract

The actin cytoskeleton is necessary for cell viability and plays crucial roles in cell motility, endocytosis, growth, and cytokinesis. Hence visualization of dynamic changes in F-actin distribution in vivo is of central importance in cell biology. This has been accomplished by the development of fluorescent protein fusions to actin itself or to various actin-binding proteins, actin cross-linking proteins, and their respective actin-binding domains (ABDs). Although these protein fusions have been shown to bind to F-actin in vivo, we show that the fluorescent protein used for visualization changes the subset of F-actin labeled by an F-actin ABD probe. Further, different amino acid linkers between the fluorescent protein and ABD induced a similar change in localization. Although different linkers and fluorescent proteins can alter the subset of actin bound by a particular ABD, in most cases, the fusion protein did not label all of a cell's F-actin all of the time. Even LimEΔcoil and GFP-actin, which have been used extensively for cytoskeletal visualization, were highly variable in the subsets of actin that they labeled. Lifeact, conversely, clearly labeled cortical F-actin as well as F-actin in the anterior pseudopods of motile cells and in macropinocytotic cups. We conclude that Lifeact most accurately labels F-actin and is the best currently available probe for visualization of dynamic changes in F-actin networks.

Actin visualization at a glance

“…Using the example of LifeAct, it has been proposed to be the most suitable actin probe to visualize the dynamic rearrangement of the actin cytoskeleton in Dictyostelium, as it labels a more complete subset of actin structures than other actin-binding probes (Lemieux et al, 2014). The application of LifeAct was also favored over actin fused to fluorophore in the context of visualizing actin rearrangements during cellular mechanotransduction events (Sliogeryte et al, 2016;Deibler et al, 2011).…”

“…For instance, a recent publication has demonstrated that the subset of F-actin that is recognized by diverse ABDs can be altered by the fluorescent protein or the linker sequence between the ABD and the fluorescent protein (Lemieux et al, 2014). New applications for fluorescent labeling of proteins have become available recently, such as the use of Y-FAST, a small monomeric protein tag enabling reversible binding and activation of a cell-permeant and nontoxic fluorogenic molecule (Plamont et al, 2016), or the flavoprotein improved LOV (iLOV), a fluorescent protein based on the light, oxygen or voltage domain (LOV) from a variety of sources (Buckley et al, 2015).…”

“…Recently, several publications have discussed in great detail the benefits and disadvantages of different fluorescent actin reporters (Belin et al, 2014;Spracklen et al, 2014;Lemieux et al, 2014;Du et al, 2015). In this short article, and the accompanying poster, we present an at-a-glance view of the current approaches to visualize actin filaments, emphasizing the advantages and pitfalls of the available tools to investigate F-actin not only in the cytoplasm, but also in the somatic cell nucleus.…”

Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody

“…The expression of fluorescent actin has not proved useful in plants because most of it stays in monomeric form and diffuse in the cytoplasm resulting in a strong fluorescent background [23]. Phalloidin, a toxin extracted from death cup Amanita phalloides , binds and stabilizes F-actin and when conjugated to the fluorescent dye rhodamine selectively stains actin filaments in permeabilised and fixed plant cells.…”

Chemotaxis of a model organism: progress with Dictyostelium

“…A detailed comparison of F-actin reporters showed that they can recognize different portions of the cytoskeleton, such as the cortex, and that LifeAct is the most representative reporter of F-actin in general[23].…”