Lifeact-mEGFP and GFP alone have equivalent affects on pollen tube growth Previous studies with actin binding probes have shown that the amount of DNA used to transform pollen affects both expression level and growth rates [5,24,25]. To determine if high expression levels of Lifeact-mEGFP correlated with mortality, we compared growth in pollen transformed with either 3 mg of plasmid encoding Lifeact-mEGFP or GFP alone under the control of the zmC13 pollen specific promoter. We imaged transformed pollen tubes after allowing them to grow for three hours. From these data, we measured the length and the average fluorescence per pixel in each pollen tube (Figure 6A). Though the highest expressing tubes tend to be shorter, this is true for both GFP and Lifeact-mEGFP. This shows that both GFP and Lifeact-mEGFP at high expression levels reduce growth, suggesting that high levels of Lifeact-mEGFP are no more toxic than high levels of GFP. We wished to compare growth of transformed pollen tubes to untransformed pollen tubes. To this end we transformed pollen with Lifeact-mEGFP and collected high-resolution images of trans- formed and untransformed cells. We then tracked the growth of 10 individual cells of both types for comparison (Figure 6B). Once again, Lifeact-mEGFP transformed cells exhibit a growth rate that is not significantly different from untransformed pollen tubes. To address whether Lifeact-mEGFP negatively affects growth, we transformed pollen with increasing amounts of Lifeact-mEGFP and GFP plasmid DNA from 0.75 mg to 3.0 mg. To ensure that the length of pollen tubes did not change over the course of the experiment, we fixed the pollen tubes in 3.7% formaldehyde for 30 minutes prior to imaging [2]. The pollen tube lengths were then measured. At all amounts of DNA, GFP and Lifeact-mEGFP showed very similar distributions of pollen tube length (Figure 6C). These findings further validate that Lifeact-mEGFP is no more toxic to the pollen tube than GFP. To investigate whether Lifeact- mEGFP was altering actin dynamics, we collected high resolution z-stack images of pollen that exhibited cytoplasmic streaming but lacked growth. At no time did we observe the dramatic actin artifacts seen with other GFP labeled actin binding probes (Figure 6D) [24,25]. The major difference between these pollen tubes and the pollen that was growing well, is the lack of a cortical actin fringe. As the length of Lifeact-mEGFP transformed pollen tubes was equivalent to pollen tubes expressing GFP alone, it seems unlikely that alteration in actin dynamics caused these pollen tubes to grow poorly. These data indicate that Lifeact-mEGFP can be used to reliably label actin in growing cells. Alterations to growth due to of high levels of Lifeact-mEGFP appear to be dependent on the species, with moss more sensitive than lily pollen. Nevertheless, Lifeact- mEGFP’s robust labeling suggests that it will provide a tool for visualizing actin while imaging structures, organelles, or ions. Similarly, it will serve as an excellent tool for studying the changes in actin structure as growth oscillates and as manipulations to the cell’s environment alter growth. Latrunculin B induced actin depolymerization and disrupted filamentous localization of Lifeact-mEGFP signal The depolymerizing drug Latrunculin B has been widely used to investigate actin’s role in various cell processes. For example, nanomolar concentrations abolish growth in pollen tubes but do not alter cytoplasmic streaming [2,39]. Experiments in moss have also demonstrated sensitivity to Latrunculin B, though at micromolar concentrations in multi-day experiments [3,4]. As Lifeact-mEGFP appears to be labeling actin faithfully, we sought Lifeact-mEGFP in Plant Cells to investigate whether its localization would be altered by Latrunculin B treatment. Specifically, we predicted that concen- trations of Latrunculin B that inhibit growth would dissipate the apical F-actin network. As a first step we examined the effect of Latrunculin B on moss protonemata by comparing the growth of WT plants to the three Lifeact-mEGFP lines in the WT background. Lifeact-mEGFP and WT plants were regenerated from single protoplasts. After four days, the protoplasts were transferred to media containing increasing concentrations of Latrunculin B. Two days after incubation in Latrunculin B, whole plants were imaged to determine plant area and solidity (see Methods). Increasing concentrations of Latrunculin B inhibits tip growth, which is represented by an increase in plant solidity. All assayed lines, including lines 22 and 25, which contain 2- and 1.5-fold more Lifeact-mEGFP respectively (Figure 5A), had similar IC50 for Latrunculin B (Figure 7A). This is particularly significant because it suggests that actin is not stabilized by increasing amounts of the Lifeact-mEGFP probe, consistent with previous in vitro results [31]. To examine the effects of Latrunculin B on actin localization as visualized by Lifeact-mEGFP, we transferred Lifeact-mEGFP expressing plants to agar pads containing DMSO, 0.5 mM or 1.25 mM Latrunculin B. Cells were then imaged after 10 minutes. Cells in DMSO showed no alteration in either growth or Lifeact- mEGFP signal (Figure 7B and data not shown). Cells imaged from the 0.5 mM treatment had stopped growing. They also showed fewer actin filaments, and manifested punctate fluorescence suggesting F- actin depolymerization. The cells incubated in 1.25 mM Latrunculin B also stopped growing and lost any clear actin localization; instead the fluorescence was diffuse throughout the cytoplasm. To analyze Latrunculin B treatment on pollen tubes, we transformed lily pollen with Lifeact-mEGFP and allowed it to grow for two hours in standard media. After imaging selected cells for several minutes, we replaced the growth media with media supplemented with 2 nM Latrunculin B. This concentration has been used in the past to reversibly terminate growth [2,39]. We then collected a time lapse image series of the growing pollen tube as it reacted to the drug. Before treatment, the clear zone is apparent and the fringe appears as cortical brightness in a medial plane view (Figure 7C, representative image). Figure 7D shows the same tube after growth has stopped. In line with previous results, the clear zone has collapsed and the tip has swollen. Cytoplasmic streaming continues, but it is no longer organized into the typical reverse fountain [2] (Movies S5 and S6). The fringe has dissipated. Some actin filaments remain, but they are disorganized and largely cortical (Movie S6). Imaging multiple planes in the z-axis reveals filamentous staining in the cortex, but the microfilaments appear to be randomly oriented (Figure S1). These data indicate that the apical F-actin network is important for growth. Our results indicate that Lifeact-mEGFP expressing moss protonemata and pollen tubes both respond to Latrunculin B treatment. In moss, protonemal cells cease growing and filamen- tous Lifeact-mEGFP fluorescence is lost. In lily pollen tubes, Lifeact-mEGFP fluorescence is reduced, the tubes stop growing and the tip swells. The tremendous difference in sensitivity to the drug likely results from the wide variance in growth rates; pollen tubes endocytose at a fast pace [40] and therefore will take up a great deal of the drug quickly, whereas the slowly growing moss may take it up more slowly. Lifeact-mEGFP robustly labels actin in moss subapical protonemal and gametophore cells As the moss is stably transformed, we were able to monitor actin labeling in a variety of different cell types using line 25. We took PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5744