Growth-Arrest-Specific Protein 2 Inhibits Cell Division in Xenopus Embryos Tong Zhang1*, Bama Dayanandan2, Isabelle Rouiller2, Elizabeth J. Lawrence2, Craig A. Mandato1,2* 1 Department of Biology, McGill University, Montreal, Quebec, Canada, 2 Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada Abstract Background: Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported. Methodology and Principal Findings: To determine whether the Gas2 protein plays a role in cell division, we over- expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin- binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demon- strated that Gas2 bundled microtubules into higher-order structures. Conclusion and Significance: Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest. Citation: Zhang T, Dayanandan B, Rouiller I, Lawrence EJ, Mandato CA (2011) Growth-Arrest-Specific Protein 2 Inhibits Cell Division in Xenopus Embryos. PLoS ONE 6(9): e24698. doi:10.1371/journal.pone.0024698 Editor: Hui Zou, UT Southwestern Medical Center, United States of America Received April 7, 2011; Accepted August 17, 2011; Published September 9, 2011 Copyright: 2011 Zhang 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 grants from the Canadian Institutes of Health Research (CIHR). C.A.M. is supported by a CIHR grant MOPS68970. 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: tong.zhang1@mail.mcgill.ca (TZ); craig.mandato@mcgill.ca (CAM) Introduction Cytoskeletal dynamics are essential for many fundamental cellular processes, including cell division, wound healing and cell motility [1,2,3]. During cell division, for example, dramatic rearrangements of the actin and microtubule cytoskeletons are required in order for the cell to change morphology, segregate its chromosomes and execute cytokinesis. The ability of the cytoskel- eton to adapt to constant physiological changes is mediated, in part, by actin and microtubule-binding proteins and cross-linking proteins that regulate cytoskeleton dynamics. Many actin-microtu- bule cross-linking proteins have been identified; however, their functions and mechanisms of regulation remain unclear [4]. One such potential cytoskeleton-interacting protein is the growth-arrest- specific (Gas) 2 protein. The Gas2 protein belongs to the growth-arrest-specific protein family and is widely expressed in human tissues [5]. Although Gas2 has a putative N-terminal actin-binding calponin homology (CH) domain [6] and a C-terminal tubulin-binding Gas2 domain, no direct evidence for Gas2-cytoskeleton interactions has been reported. However, immunofluorescence studies demonstrated that the full-length Gas2 co-localizes with filamentous actin (F-actin) at the cell cortex and in stress fibers in growth-arrested NIH 3T3 fibroblasts [6] and the Gas2 domain co-localize with microtubules in COS-7 cells [7]. Although the majority of cells in an organism are quiescent, they are able to re-enter the cell cycle and proliferate after stimulation [8]. Several lines of evidences support a role for Gas2 in cell cycle progression. First, the gas2 gene was originally identified in a genetic screen of murine fibroblasts that were cultured under growth arrest conditions [9]. Second, gas2 is down-regulated upon serum and growth factor stimulation [6]. Furthermore, the Gas2 protein is phosphorylated on a serine residue at the G0 to G1 transition allowing quiescent G0 cells to re-enter the cell cycle [6]. However, whether Gas2 plays a direct role in the mechanism of cell division and whether this function is mediated by its cytoskeletal binding properties are completely unknown. In this study, Xenopus embryos and oocytes were used to study Gas2 functions in cell division. Xenopus embryo undergoes a time-regulated synchronized cell division in the early stages of its development and therefore is a useful in vivo model system for studying cell division. An established Xenopus oocyte wound-induced contractile array assay, which mimics cytokinesis, was used to study Gas2 interactions with the cytoskeleton in vivo [10]. Furthermore, cytoskeleton co-sedimen- PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24698