Abstract
In vivo markers for F-actin organization and dynamics are extensively used to investigate cellular functions of the actin cytoskeleton, which are essential for plant development and pathogen defense. The most widely employed markers are GFP variants fused to F-actin binding domains of mouse talin (GFP-mTn), Arabidopsis fimbrin1 (GFP-FABD2) or yeast Abp140 (Lifeact-GFP). Although numerous reports describing applications of one, or occasionally more, of these markers, are available in the literature, a direct quantitative comparison of the performance of all three markers at different expression levels has been missing. Here, we analyze F-actin organization and growth rate displayed by tobacco pollen tubes expressing YFP-mTn, YFP-FABD2 or Lifeact-YFP at different levels. Results obtained establish that: (1) all markers strongly affect F-actin organization and cell expansion at high expression levels, (2) YFP-mTn and Lifeact-YFP non-invasively label the same F-actin structures (longitudinally oriented filaments in the shank, a subapical fringe) at low expression levels, (3) Lifeact-YFP displays a somewhat lower potential to affect F-actin organization and cell expansion than YFP-mTn, and (4) YFP-FABD2 generally fails to label F-actin structures at the pollen tube tip and affects F-actin organization as well as cell expansion already at lowest expression levels. As pointed out in the discussion, these observations (1) are also meaningful for F-actin labeling in other cell types, which generally respond less sensitively to F-actin perturbation than pollen tubes, (2) help selecting suitable markers for future F-actin labeling experiments, and (3) support the assessment of a substantial amount of published data resulting from such experiments.
Highlights
CDNA sequences were constructed coding for Lifeact-yellow fluorescent protein (YFP), YFP-mTn and YFP-FABD2 fusion proteins composed of eYFP (AAX97736; BD Biosciences-Clontech; San Jose, CA, United States) fused via a flexible 5× Gly-Ala linker to different filamentous actin (F-actin) binding domains identical to those reported in the original publications introducing the three F-actin markers: (1) S. cerevisiae Abp1401−17 (AJT97542.1; Lifeact-YFP: Riedl et al, 2008), (2) Mus musculus Talin12345−2541 (NM_011602.5; YFP-mTn: Kost et al, 1998) or (3) A. thaliana Fimbrin1325−687 (NM_001341826/AT4G26700; YFP-FABD2: Sheahan et al, 2004; Voigt et al, 2005)
A direct comparison was performed of effects of high-level expression of either free YFP, or of one of the F-actin markers Lifeact-YFP, YFP-mTn or YFP-FABD2, on the growth of tobacco pollen tubes transiently transformed by particle bombardment
Under the same conditions actin fibers connecting these two structures, which extend from the subapical cell cortex into the shank, are often visualized by each of the two markers (Figure 3). These structures appear to constitute the tobacco pollen tube actin cytoskeleton as it can be non-invasively visualized using in vivo markers tested in this study at normal growth rates at which F-actin organization is unlikely to be substantially affected
Summary
The actin cytoskeleton, which is composed of filamentous actin (F-actin), plays key roles in organelle transport (Wang and Hussey, 2015), membrane trafficking (Henty-Ridilla et al, 2013), cell division (Panteris, 2008), cell expansion (Smith and Oppenheimer, 2005), transport through plasmodesmata (White and Barton, 2011), gravity sensing (Blancaflor, 2013), programmed cell death (Smertenko and Franklin-Tong, 2011) and stomatal movements (Zhao et al, 2016).Performance of In Vivo F-actin MarkersF-actin has essential functions in plant development (Kost et al, 1999; Higaki et al, 2010; Zhu and Geisler, 2015) and pathogen defense (Porter and Day, 2016). Excellent techniques are available to visualize F-actin organization in fixed plant cells either based on electron microscopy, or on fluorescence microscopy after staining with fluorescently labeled actin antibodies or derivatives of phalloidin, a membrane-permeable fungal metabolite that associates with actin filaments (Wulf et al, 1979). These techniques have substantially contributed to our current understanding of the organization and function of the actin cytoskeleton in different plant cell types (Staiger et al, 2000), they do not allow observation of F-actin dynamics. For this purpose, during the past two decades a collection of markers and methods enabling F-actin imaging in living plant cells based on fluorescence microscopy have been developed and were extensively applied (Du and Ren, 2011)
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