Fluorescent protein‐based reporters of the actin cytoskeleton in living plant cells: Fluorophore variant, actin binding domain, and promoter considerations

Abstract

Genetically encoded filamentous actin (F-actin) reporters designed based on fluorescent protein fusions to F-actin binding domains of actin regulatory proteins have emerged as powerful tools to decipher the role of the actin cytoskeleton in plant growth and development. However, these probes could interfere with the function of endogenous actin binding proteins and in turn impact actin organization and plant growth. We therefore surveyed F-actin labeling and compared organ growth in Arabidopsis thaliana lines expressing a variety of F-actin markers. Here we show that the variant of fluorescent protein, type of actin binding domain, and the promoter that drives reporter expression can influence the quality of F-actin labeling particularly in stable plant lines. For example, older red fluorescent protein (RFP)-based probes such as DsRed2 and mOrange induced more aberrant labeling compared to the newer RFP-based, mCherry, GFP, and GFP-derived fluorophores such as YFP and CFP. Moreover, qualitative and quantitative analyses revealed differences in F-actin organization in seedlings expressing Talin- and Lifeact-based reporters in some cell types compared to the fimbrin actin binding domain 2 (ABD2)-based reporters. Finally, the use of the ubiquitin10 (UBQ10) promoter to drive expression of the GFP-ABD2-GFP probe minimized loss of fluorescence and growth defects observed in the 35S-driven version. Taken together, this study shows that care must be taken in the interpretation of data derived from stable expression of certain F-actin reporters and that using alternative promoters such as UBQ10 can overcome some of the pitfalls that accompany the use of in vivo F-actin probes in plants. © 2014 Wiley Periodicals, Inc.

Differential Growth in Periclinal and Anticlinal Walls during Lobe Formation in Arabidopsis Cotyledon Pavement Cells

“…This difference in actin localization may be a result of the different GFP-tagged actin binding proteins used to highlight actin filaments within pavement cells. Actin filaments tagged with GFP-fABD2, used in this study, tend to be less bundled and exhibit faster dynamics than those tagged with GFP-mTalin, another actin-bundling protein (Sheahan et al, 2004;Holweg, 2007;Dyachok et al, 2014). Here, GFP-fABD2-tagged filamentous actin arrays changed in their distribution over 7 h, but in a previous study using wild-type pavement cells transiently expressing GFP-mTalin, the distribution of actin filaments showed little change over a 10-h period (Fu et al, 2002).…”

Nuclear Function of Subclass I Actin-Depolymerizing Factor Contributes to Susceptibility in Arabidopsis to an Adapted Powdery Mildew Fungus

“…Although those lines had been surveyed previously and compared for effects on plant development and AF organization (Dyachok et al, 2014) and the GFP-ABD2 line is generally considered an optimal line that reflects an intact AF organization (Higaki et al, 2010a;Dyachok et al, 2014), the investigation was limited to seedlings. We found a loss of GFP-ABD2 fluorescence in most mature leaf epidermal cells.…”

“…Thus, it is possible that an increased level of AF bundling in GFP-hTalin (Higaki et al, 2010a;Dyachok et al, 2014;Supplemental Fig. S8) had masked the changes in the AF organization in adf4.…”

RopGEF1 Plays a Critical Role in Polar Auxin Transport in Early Development

“…To quantify percent occupancy (density) of F-actin, we used images of epidermal cells in root maturation zone from at least nine serial optical sections (1-mm-step) following the methods of Higaki et al (2010) and Dyachok et al (2014). The original confocal images were converted into binary images and skeletonized images for the measurement of F-actin pixels.…”

“…Filament occupancy was calculated with the total pixel numbers of all marked filaments. Density was defined as the ratio of the filament occupancy and cell area as described by Dyachok et al (2014). The above measurements were performed by using the software Image J (version 1.4.3.67).…”