or tobacco [5,24,25]. However, this probe does not clearly label the apical domain. Mouse Talin (mTalin) [21,26–28], the second actin binding domain of fimbrin [24], and recently a pollen specific LIM protein have also been used to probe actin in live pollen [25]. These probes have shown utility in other cell types, but in pollen the images are inconsistent and do not detect all of the structures shown in the rapidly frozen fixed cells. Specifically, instead of lily pollen’s cortical actin fringe, the probes reveal a dense mesh throughout the cell’s tip [5,24,25]. In moss, attempts to use the actin binding domain of fimbrin fused to GFP have resulted in growth abnormalities and cytoskeletal anomalies (L. Vidali unpublished observations). Although stable expression of mTalin-GFP inhibits cell growth in moss, recent work using a heat inducible promoter upstream of mTalin-GFP did produce labeling of actin. However, the transient nature of the heat-induction only allows for brief imaging of the actin cytoskeleton [4,29]. In addition, GFP-mTalin has been shown to inhibit tip growth in root hairs [30]. Here we employ Lifeact, a probe first used in animal cells [31], to examine actin in live moss protonemata and pollen tubes. This probe consists of the first 17 amino acids from the budding yeast ABP-140 fused to GFP. In animal cells Lifeact-mEGFP and Lifeact fused to FITC effectively label actin without impairing cell viability [31]. Lifeact peptide fused to FITC has subsequently been used in mouse oocytes to elucidate a novel actin-based mechanism for chromosomal motility [32]. Lifeact-mEGFP allows visualization of actin dynamics in growing moss protonemata and both lily and tobacco pollen tubes. In moss, the probe labels a distinct and consistent apical F- actin network at the growing tip of protonemata with a focal point of F-actin. In lily and tobacco pollen tubes, a highly dynamic apical F-actin network is labeled defining the clear zone. In all three organisms a cortical actin network extends rearward through the cell. In pollen tubes there are also dynamic and distinct interior filaments that appear to be involved in reverse fountain streaming. As a live-cell probe, Lifeact provides a new valuable tool for examining actin organization in tip growing plant cells. Results and Discussion Lifeact-mEGFP labels a clear three dimensional apical F- actin network in moss and pollen We analyzed Lifeact-mEGFP labeling of actin in protonemata of the moss Physcomitrella patens, a model bryophyte, and pollen tubes from Lilium formosanum and Nicotiana tobaccum, representing monocots and dicots respectively. These well characterized cells are notable because of their actin dependent, highly polarized tip growth and the ease with which they are transformed. We constructed a fusion protein consisting of Lifeact [31] fused to mEGFP with a seven amino acid linker (Lifeact-mEGFP). We stably transformed moss with Lifeact-mEGFP under the control of the maize ubiquitin promoter [33]. Several individually trans- formed lines of moss were isolated and characterized. For lily and tobacco pollen, we performed transient transformations with the same fusion construct under the control of the zmC13 and Lat52 promoters, respectively [34,35]. To visualize the actin cytoskeleton throughout the cell volume, we used Laser Scanning Confocal Microscopy (LSCM) and recorded z-stacks of live cells expressing Lifeact-mEGFP. Moss protonemata are composed of two cell types: chloronemal and caulonemal cells. Chloronemata contain many chloroplasts, have perpendicular cell plates, and have a poorly defined clear zone. In contrast, caulonemata have fewer chloroplasts, oblique cell plates, a defined clear zone, and grow about three times faster Lifeact-mEGFP in Plant Cells than chloronemata. In previous studies, fixed caulonemal cells have shown an apical actin fringe, but previous live cell imaging, performed with a heat-shock inducible mTalin-GFP construct, did not corroborate these findings [4,29]. Here, Physcomitrella proto- nemata expressing Lifeact-mEGFP were imaged by collecting serial optical slices in the z axis. In caulonemal cells expressing Lifeact-mEGFP, an apical F-actin network can consistently be visualized near the tip of the growing cell and an actin focal point is also observed (Figure 1A). The brackets define the area rotated and shown in the inset. This highlights the cortical F-actin network consistently seen in caulonemal cells. In chloronemal cells a large amount of F-actin accumulates at the apex, but instead of a small focal point, it appears as a patch (Figure 1B). Filamentous structures are visible within the patch and seem to extend out from it. Towards the back of the cell, the actin microfilaments extend along the cortex. The inset shows the bracketed region of the cell rotated 90u. It demonstrates that although longitudinal fibers exist along the cortex, they are mostly absent from the center of the cell. Collecting z-stacks of live lily pollen proved challenging as the cells grow rapidly enough that the stack blurs unless collected faster than the cell can grow. To partially alleviate this problem, we took rapid, small images of half the cell’s diameter. In lily pollen tubes, a clear fringe, consisting of a palisade of short longitudinally oriented fibers, encircles the cell’s apex (Figure 1C). Forward of this fringe, few filaments are evident. The inset shows the same image rotated 90u along the y axis, clearly demonstrating that the Lifeact-mEGFP signal is largely cortical, with reduced signal in the middle of the tube. Significantly, Lifeact-mEGFP labels both G- and F-actin, so some of the signal in the center of the tube may be G-actin [31]. However, we routinely observed some microfilaments (F-actin) in the center of the tube. These images are consistent with the data from rapidly frozen fixed cells [22]. In tobacco pollen, Lifeact-mEGFP labels longitudinal fibers along the shank of the tube (Figure 1D). An apical F-actin network is also present though it is closer to the tip and more dense in the medial planes than the fringe seen in the lily pollen tube. The inset shows the same image rotated 90u. It highlights the apical F-actin network. Although some F-actin is located in the center of the tube, a great deal is positioned in the cortex. Lifeact-mEGFP confirms the existence of a fringe in lily as shown in the rapidly frozen cells [22], and shows that a similar structure exists in tobacco. Taken together, these data demonstrate the existence of an apical F-actin network in all three cell types. Lifeact-mEGFP and phalloidin label the same structures in MBS-EGS fixed moss cells Our results with Lifeact-mEGFP were not entirely consistent with recent work using fluorescently conjugated phalloidin to label actin microfilaments in moss [11,13]. Though the network labeled by Lifeact-mEGFP is similar in some respects to that seen in the fixed-cell images, there are some differences. We wished to investigate whether the difference between these two F-actin patterns is due to fixation, or whether Lifeact-mEGFP and fluorescently conjugated phalloidin are labeling distinct structures. We began by monitoring Lifeact-mEGFP before and after cross- linking with MBS-EGS (Figure 2A). Clearly some rearrangements in the Lifeact-mEGFP pattern occur; most importantly, the tip localized focal point dissipates and in general the filaments seem thicker as if bundling has occurred. To verify that Lifeact-mEGFP and phalloidin label the same structures, we used MBS-EGS to cross-link cells expressing Lifeact-mEGFP. After cross-linking, we fixed the cells and processed them for labeling with rhodamine-phallodin. We then examined the localization of the two probes (Figure 2B). PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5744