Lifeact-mEGFP in Plant Cells Figure 4. Spinning disc confocal images show possible actin buckling at the tip region in moss. (A) and (B) show a moss cell expressing Lifeact-mEGFP imaged at times indicated. Arrows point to potential buckling event as seen in successive planes. See Movie S4 for complete series. doi:10.1371/journal.pone.0005744.g004 Arabidopsis epidermal cells using variable angle epifluorescence microscopy [36]. Buckling events have been described before in vitro. Actin immobilized by myosin at one end then elongated by formin at the other is forced to buckle, generating significant force [37]. From the Lifeact-mEGFP images it is of course impossible to infer the proteins involved in the event, nor can we conclude that these structures are producing the same forces as seen in the in vitro experiment. However, these images present compelling evidence that buckling events might be playing a role in polarized tip growth in moss. High expression of Lifeact-mEGFP affects moss protonemal growth As many live cell actin probes significantly inhibit growth, we investigated whether Lifeact-mEGFP affects growth in moss protonemata or pollen tubes [5,24]. In moss we characterized Lifeact-mEGFP in two distinct genetic backgrounds: WT and NLS-4. NLS-4, a line important for RNAi based loss-of-function studies, is a stable transgenic line that expresses a GFP-GUS fusion with a nuclear localization signal [11,13,38]. We obtained several independent lines and analyzed the expression and growth in both backgrounds. We isolated protein from seven day old protonemata and used immunoblotting to evaluate the relative amount of Lifeact- mEGFP expressed in each line. In the WT background, line 22 expresses twice as much Lifeact-mEGFP as line 20 and nearly half again as much as line 25 (Figure 5A compare lanes 2, 3 and 4). In the NLS-4 background, line 8 expresses 3 fold more than the lowest expressing line (Figure 5A compare lanes 8 and 9). To determine whether the amount of Lifeact-mEGFP expressed affects plant growth, we examined the growth properties of the seven Lifeact-mEGFP lines. Young moss plants regenerating from protoplasts are composed exclusively of protonemal tissue and one can measure growth by comparing the area of individual plants. We stained plants with the fluorescent dye calcofluor, and used the signal to calculate the area of individual plants. Additionally we used solidity, which is the area divided by the convex hull area as an indication of overall filamentous outgrowth (see Methods). Solidity values approaching one indicate that the plants are solid and lack polarized extensions; lower values indicate the presence of filamentous outgrowths and a higher degree of plant polarization [11]. Line 8 in the NLS-4 background results in smaller plants indicating the slowest growth rate and a concomitant increase in solidity (Figure 5B). This line has the highest level of Lifeact-mEGFP expression. All the other Lifeact- mEGFP lines have similar areas as compared to wildtype or the NLS-4 control. Line 8 appears to produce far fewer caulonemal cells as compared to controls, presumably contributing to the increase in solidity. High levels of GFP do not exhibit these growth defects (data not shown). Interestingly, neither line 8 nor line 22, the two lines expressing the highest levels of Lifeact-mEGFP, exhibited dramatic actin artifacts (Figure 5C for representative images). In contrast, many other actin binding protein probes have been shown to create circles and large bundles when over expressed [24,25]. The Lifeact-mEGFP labeling looks similar to lines with lower levels of expression, although with a higher diffuse cytoplasmic signal (compare to Figure 1). Our data indicate that Lifeact-mEGFP expression levels that allow effective imaging in live cells allow normal cell growth characteristics. Lifeact-mEGFP thus presents several advantages for studying the actin cytoskeleton in moss. It avoids potential artifacts arising from fixation, particularly from the cross-linking; it does not inhibit filamentous or bulk growth; and expression is constant, so that no manipulations are necessary to allow for imaging. Significantly, Lifeact-mEGFP is expressed in a stable line presenting the opportunity for studying other structures and organelles concurrently using different probes. PLoS ONE | www.plosone.org 6 May 2009 | Volume 4 | Issue 5 | e5744