Gas2 Inhibits Cell Division Figure 2. Exogenous Gas2 inhibits cell division in Xenopus embryos. (A) The experimental flowchart and schematically represented results. The darker side of the egg represents the animal pole and the lighter bottom side represents the vegetal pole. The 90 degree angle rotation views show cell numbers in BSA-injected control vs. Gas2-injected embryos. (B–D) BSA-injected Xenopus embryos continue through normal cell divisions (Video S1). (E–G) Gas2-injected embryo cells divide once, then arrest in subsequent cell divisions. The non-injected control cell of the Gas2-injected embryo divides normally, and the cell number increases 2-fold every 30 minutes at room temperature (Video S2). The red circles in B and E indicate the needle injection sites. Time was set to 0 at 8-cell stage for demonstration purposes. (G’) The rotation view of Fig. 2G embryo shows one of the large arrested cells on the left. Bars, 0.3 mm. (H) Stereo microscopy examination of a Gas2-injected embryo at the 32-cell stage. The normal dividing cells are on the left and the two large arrested cells are on the right. Bar, 0.3 mm. (I and J) Confocal microscopy analysis of post-injected embryo cells with tubulin antibody staining. The white arrow in J indicates that the two large, arrested cells remain connected with microtubules. Bars, 20 mm. (K) Statistical analysis of cell division rate in BSA- and Gas2-injected embryos. Only 7.264.2% of BSA-injected embryos arrest in cell division; however, 79.265.5% of Gas2-injected embryos arrest in cell division (n = 300 embryos and from 5 experiments, p,0.001). (L) Dosage dependent analysis of cell division rate in Gas2-injected embryos. The calculated LD50 from the dosage dependent analysis graph is 5.5 ng or 31 mM for one cell of the 2-cell stage Xenopus embryo. The x-axis of the graph is in logarithmic scale. doi:10.1371/journal.pone.0024698.g002 shown that Gas2 bundles purified microtubules into higher-order structures in vitro, indicating a direct Gas2-microtubule interaction (Fig. 6D). The full-length Gas2 microtubule-bundling ability is preserved when F-actin is present in the same sample, but no F- actin-microtubule cross-linking has been observed in vitro (Fig. 6E). We hypothesize that Gas2-microtubule bundling ability inhibits microtubule dynamics and thereby causes cell division arrest and cytokinesis failure in Xenopus embryos. To investigate the mechanism of Gas2-induced cell division arrest and cytokinesis failure, we used a wound-induced contractile array assay in Xenopus oocytes, which is also a model of cytokinesis [10]. This assay permitted us to study Gas2-cytoskeletal interac- tions during microtubule-dependent actomyosin array formation and contraction in vivo [10]. Oocyte wound healing and cytokinesis are highly dynamic processes that require coordination between the actin and microtubule cytoskeletons [13]. We have shown that Gas2 possesses both actin- and tubulin-binding properties in vitro; therefore, we propose that Gas2 functions as a structural cross- linking protein between these two cytoskeleton systems. When oocytes are treated with taxol to stabilize microtubules prior to wounding, an abnormal double ring of actin forms around the wound border [2]. Staining with phalloidin for F-actin showed that the internal ring is composed of contractile F-actin, while staining for actin monomers showed that the outer ring is formed by de novo actin polymerization [2,13]. Interestingly, we found that when full-length Gas2 or its Gas2 domain was over-expressed, oocytes developed the abnormal double actin ring phenotype upon wounding. Thus, Gas2 mimics the taxol-treatment phenotype, suggesting that Gas2 has microtubule-stabilizing activity. The Gas2-induced double rings can be rescued by de-polymerizing microtubules with nocodazole (Fig. 4L–O) perhaps because the Gas2 protein binds dynamically to microtubules. Full-length Gas2 co-localizes with the actin rings, presumably by binding actin via its CH domain (Fig. 4H–K). It is interesting to note that the Gas2 domain alone also forms a ring structure and localizes between two actin rings during the wound healing (Fig. 4T–W), but it does not overlap with either actin ring since it has no actin-binding domain. In summary, we have demonstrated that Gas2 can inhibit cell division in Xenopus embryos. We have also provided evidences that Gas2 has the ability to change microtubule dynamics and bundle microtubules in vitro. We believe that the microtubule-bundling ability of Gas2 causes Xenopus embryo cell division arrest and the formation of the abnormal double actin rings in oocyte wound healing. Drosophila contains a spectraplakin family protein, Short stop (Shot), that modulates microtubule dynamics via its Gas2 domains [14]. Ro¨ per and Brown showed that Shot has a Gas2 domain at its C-terminus and is required for organizing microtubules in a branched membrane structure called the fusome in meiotic cysts. They demonstrated that, in the absence of Shot, microtubules did not assemble, and oocytes failed to become specified. The authors postulated that Shot elicits this effect via its Gas2 domain influencing microtubule dynamics. Similarly, Pines et al. showed that a Gas2-like protein, Pickled eggs (Pigs) co-localizes with both F-actin and microtubules in the giant epithelial cells in Drosophila [15]. Pigs is postulated to facilitate cytoskeleton rearrangements by binding and stabilizing F-actin and microtubules. Consistent with our data, it is likely that Gas2 has conserved cytoskeleton-binding properties in different species. Although a recent paper by Stroud et al., showed that the GAR domain (the other name of Gas2 domain) of Gas2-like 3 protein is not essential for its localization to microtubules and does not directly interact with microtubules in vitro, this may be due to the large C-terminus domain which is present in Gas2-like 3 protein, but not in the Gas2 protein [16]. This domain may directly impact on protein functions including the ability of Gas2-like 3 to bind microtubules. Several studies have provided evidence suggesting that Gas2 activity is mis-regulated in cancer. Human gas2 is located on the short arm of Chromosome 11 and genetic rearrangements or deletions of this region are frequently found in tumors and sporadic human cancers [17,18,19,20]. The analysis of oncogene transformed cells v-fos, v-myc, v-ras and v-src showed that the expression of Gas2 fails to increase in response to serum starvation [6]. Furthermore, proteomic studies have identified Gas2 in normal rat liver cells, but not in the rat liver tumors [21]. Finally, translation initiation factor eIF4E binding proteins have been shown to control p53-dependent senescence by regulating Gas2 translation [22]. In light of this, a better understanding of the role of Gas2 functions in cell division and how Gas2 is regulated will aid the development of improved cancer therapies. Materials and Methods Experimental animals Xenopus laevis frogs were bought from Nasco (Fort Atkinson, WI, USA). The experiments were conducted by trained skilled personnel and were approved by McGill University Animal Care Committee (Protocol #: 4858 to C.A.M.). Bioinformatics analysis Homo sapiens (human) [O43903], Canis familiaris (Dog) [F1PBV2], Mus musculus (house mouse) [P11862], Bos taurus (Bovine) [A8E4Q5], Gallus gallus (Chicken) [F1NSM4] and Xenopus tropicalis (Silurana tropicalis) [ENSXETP00000005555] Growth-arrest- specific protein 2 (Gas2) amino acid sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov), Uniprot (http:// www.uniprot.org/) and Ensembl (http://www.ensembl.org/ Xenopus_tropicalis) websites. They were compared the similarity with ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index. PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24698