Lifeact-mEGFP in Plant Cells Figure 2. Lifeact-mEGFP and phalloidin label the same structures in MBS-EGS fixed moss. (A) shows a cell before and after fixation. Top is DIC, bottom is GFP signal. An apparent change in actin localization has occurred. (B) Two different cells expressing Lifeact-mEGFP (left) stained with rhodamine phalloidin (middle). Far right panels show the merged signal. Bar is 5 mm. doi:10.1371/journal.pone.0005744.g002 network at the extreme apex, causing a loss of signal at the tip and bundling of filaments towards the rear of the cell. In particular, fixation enhances labeling of the fringe-like structure in the apex of some caulonemal cells. Lifeact-mEGFP circumvents these arti- facts, allowing labeling of actin in living cells. Lifeact-mEGFP reveals actin dynamics during growth To image the remodeling of actin filaments and the apical F- actin network in growing cells, we collected time lapse, medial plane images with LSCM. Lifeact-mEGFP labels dynamic filamentous structures throughout moss caulonemal cells. Signif- icantly, in the medial plane a distinct focal point of actin is seen at the cell apex (Figure 3A). In the full movie from which these stills are taken (Movie S1), one can see actin filaments radiating out from this focal point. The still images, shown at 1 minute intervals, highlight the highly dynamic actin filaments at the cell apex (Figure 3A). Interestingly, although an apical focal point of actin is visible in all growing tip cells, its localization varies. Sometimes it is at the extreme tip, while at other times it is to the side. Images using mTalin-GFP have also noted apical accumulations of actin, although with less distinct filamentous structure [4,29]. In Nicotiana tobaccum pollen tubes, Lifeact-mEGFP labels longitudinal filaments as well as a dense apical F-actin network in the medial plane (Figure 3B). Images are shown at 10 second intervals. Notably, the apical F-actin network is not stationary; it varies in its exact distance from the tip. However, it does maintain its position relative to the clear zone of the pollen tube. In the complete movie it is apparent that short filaments are constantly moving in and out of the center of the apex (see Movie S2). In culture, the larger lily pollen grows much more rapidly than either tobacco or moss. Figure 3C shows medial plane images taken at 6 second intervals from a growing lily pollen tube. The cortical actin fringe is observed along the sides of the clear zone, as seen in fixed cells [22]. In addition, there are some microfilaments in the apical core that are constantly being remodeled and occasionally swept rearward (see Movie S3). Furthermore a funnel- like structure appears to taper backwards from the fringe, also consistent with previous studies [2]. These data demonstrate that Lifeact-mEGFP labels F-actin structures and enables imaging of the rapid remodeling of the actin cytoskeleton in a growing tip cell. Lifeact-mEGFP confirms the presence of a cortical actin fringe in lily in keeping with what has been shown in rapid-freeze fixed pollen tube cells probed with anti-actin antibodies [22]. It also shows a dynamic apical F-actin network and many longitudinal fibers. These observations differ from those generated through the use of GFP-ntADF1 and ntLIM2b-GFP [25], which do not clearly resolve apical filamen- tous structures, in particular the cortical actin fringe in the apex of lily pollen tubes. Rapid actin remodeling occurs at the tip region in moss Our time lapse imaging of Lifeact-mEGFP showed changes in the actin network’s structure that were surprisingly fast. To capture these changes, we imaged Lifeact-mEGFP in moss with a PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5744