Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells Luis Vidali., Caleb M. Rounds., Peter K. Hepler, Magdalena Bezanilla* Department of Biology, University of Massachusetts, Amherst, Massachusetts, United States of America Abstract Background: Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings: In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance: Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin’s role in tip growing plant cells. Citation: Vidali L, Rounds CM, Hepler PK, Bezanilla M (2009) Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells. PLoS ONE 4(5): e5744. doi:10.1371/journal.pone.0005744 Editor: Ivan Baxter, Purdue University, United States of America Received April 30, 2009; Accepted May 6, 2009; Published May 29, 2009 Copyright: 2009 Vidali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Science Foundation (http://www.nsf.gov/) (grant nos. MCB-0516852 to P.K.H. and MCB-0516702 and MCB- 0640530 to M.B.) and the David and Lucille Packard foundation (http://www.packard.org/home.aspx) (2007-31750 to M.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bezanilla@bio.umass.edu . These authors contributed equally to this work. Introduction Plants use tip growth to achieve many essential objectives. For instance, in the moss Physcomitrella patens, plant expansion is initially carried out by tip growing protonemata and is thus essential for establishment of the plant [1]. In angiosperms, the pollen grain germinates on the stigma, and then extends a long tip growing tube to deliver the sperm to the ovule. Though the relative importance of various physiological parameters and molecular regulatory pathways involved in tip growth remains controversial, the critical role of actin dynamics in promoting this growth is not. Studies of actin’s role in polarized growth of plant cells have revealed that the dynamic pool of filamentous actin is tightly regulated. Indeed, nanomolar concentrations of the actin depolymerizing drug Latrunculin B disrupt growth without altering cytoplasmic streaming in pollen [2]. In moss as well, Latrunculin B inhibits tip growth [3,4]. Investigations of the role played by different actin binding proteins in moss, root hairs and pollen tubes have shown that alterations in expression level and regulation dramatically alter tip growth [2–13]. These data suggest that the plant cell must maintain a delicate balance between G- and F-actin to promote tip growth. While the actin cytoskeleton’s role in polarized growth has been amply demonstrated, it still remains uncertain at a mechanistic level how actin works to promote tip growth. A robust tool for live cell imaging would reveal the structure of the actin network during tip growth and enable detailed studies of the role of actin in tip growth. Despite many attempts at characterizing actin in live and fixed tip growing plant cells, a consensus has not emerged concerning many features, particularly in the apex. In moss, fixed protone- mata show a tip localized collar or aggregation of filaments along with a cortical mesh-work of actin and a network around chloroplasts [11,13–15]. In pollen tubes, the structures yielded by fixation have varied substantially; some studies revealed a dense apical meshwork [16], whereas in others the apical domain was free of actin [2,17–20]. Other work has pointed to a collar around the apical region, but this was not initially seen as a consistent feature [21]. Recently an optimized procedure has revealed the presence of a consistent apical cortical ‘‘fringe’’ in both rapid- freeze and room temperature fixed pollen tubes [22]. Though imaging actin in fixed cells provides a great deal of information, it ultimately yields a static image and is not as useful as live cell imaging. Imaging live-cells allows for the visualization of cytoskeletal structures as they change in response to growth conditions. This, though, has remained difficult. Injecting rhodamine phalloidin into pollen tubes failed to label actin in the extreme apex [23]; subsequent work has shown that the probe is rapidly sequestered into the vacuole. Attempts to express GFP labeled actin have failed largely because pollen tube growth is inhibited [21]. Several different actin binding proteins have been used in an attempt to image actin in growing pollen tubes [24,25]. Actin depolymerizing factor (ADF) from both lily and tobacco fused to GFP labels actin and does not dramatically alter cell growth in lily PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5744