Gas2 Inhibits Cell Division gives this region more structural flexibility. (D) Western blot analysis of GFP Gas2 constructs expression. GFP = 27 kDa, full-length GFP-Gas2 = 62 kDa, GFP-CH domain = 49 kDa and GFP-Gas2 domain = 44 kDa. doi:10.1371/journal.pone.0024698.g001 cytokinesis, was used to study Gas2 interactions with the cytoskeleton in vivo [10]. The relatively large size of Xenopus Stage VI oocytes (approximately 1 mm in diameter) provides a useful tool for studying cytoskeleton dynamics under the cell surface [11]. The nuclei of Stage VI oocytes were injected with either full-length GFP-Gas2, GFP-CH domain or GFP-Gas2 domain constructs, and incubated for 48 hours to allow for protein expression. A micro-capillary glass tube pulled needle was used to wound the animal poles of the oocytes. After wounding, the oocytes were allowed to recover for approximately 5 minutes in oocyte ringer-2 (OR-2) solution, and were then fixed and stained for F-actin and tubulin (Fig. 4A). The non-injected control oocyte forms a single actin contractile ring around the wound; and microtubules radially distribute around the wound (Fig. 4B–D). In oocytes pre-treated for one hour with 10 mM taxol to stabilize microtubules, actin assembles into two separate rings surrounding the wound (Fig. 4E– G). The internal ring is composed of the contractile F-actin, while the outer ring is formed by de novo actin polymerization (arrows in E) [2]. Please see the discussion for further details. Oocytes expressing full-length GFP-Gas2 have a similar phenotype to taxol-treated oocytes. The GFP-Gas2 rings (arrows in 4H) co-localize with the two separate actin rings (arrows in 4I) during wound healing (Fig. 4H–K). This observation implies that Gas2 mimics taxol-treatment and therefore may stabilize micro- tubules. When full-length GFP-Gas2-expressing oocytes were pre- treated with 20 mM nocodazole to destabilize microtubules for one hour prior to wounding, GFP-Gas2 co-localized with a single actin ring instead of double rings (Fig. 4L–O); therefore, microtubule stabilization by Gas2 was sensitive to nocodazole treatment. Gas2 has an actin-binding CH domain near its N-terminus, which may explain the observed co-localization of GFP-Gas2 and actin rings. Oocytes expressing GFP-CH domain have a single actin ring during wound healing (Fig. 4P–S). This result is also observed in the control (Fig. 4B–D) and nocodazole-treated oocytes expressing full-length GFP-Gas2 (Fig. 4L–O). GFP-CH domain co-localizes with the actin ring since it is the actin-binding domain of Gas2. Oocytes expressing the GFP-Gas2 domain alone form double actin rings (arrows in 4U), and these double actin rings are similar to the taxol pre-treated (Fig. 4E) and full-length GFP-Gas2 expressing oocytes (Fig. 4I). GFP-Gas2 domain forms a ring-like structure and radially distributes around the wound (Fig. 4T), but it does not overlap with either actin ring. This series of experiments showed that the tubulin-binding Gas2 domain alone is sufficient for eliciting the double actin rings phenotype, suggesting that Gas2 binds microtubules via its Gas2 domain and stabilizes them during oocyte wound healing. The Gas2 protein co-sediments with F-actin and microtubules Cytoskeleton co-sedimentation assays were performed to investigate the cytoskeletal binding properties of Gas2 in vitro. Full-length GFP-Gas2, GFP-CH domain and GFP-Gas2 domain constructs were injected into Xenopus oocytes and the expressed proteins were used for co-sedimentation assays (Fig. 5A). As expected, the CH domain co-sedimented with polymerized F-actin in the pellet (Fig. 5B). When F-actin was de-polymerized into actin monomers with latrunculin B, the CH domain was detected in the supernate (Fig. 5B: SL and PL). Similarly, the tubulin binding Gas2 domain co-sedimented with microtubules in the pellet (Fig. 5C). When microtubules were de-polymerized with nocodazole, more Gas2 domain was found in the supernate compared with taxol- treated samples (Fig. 5C: SN and PN vs. ST and PT). Full-length Gas2 co-sedimented with both F-actin and microtubules (Fig. 5D). However, when F-actin was de-polymerized with latrunculin B, Gas2 remained in the supernate (Fig. 5D: SL and PL). It was surprising to note that Gas2 did not co-sediment with microtubules in the pellet in the latrunculin B treated sample. The full-length Gas2 protein bundles microtubules in vitro Gas2 interactions with F-actin and microtubules were studied at high resolution by electron microscopy (EM). The phalloidin- stabilized polymerized F-actin alone appears as non-organized long filaments (Fig. 6A). In the presence of the purified full-length Gas2 protein, F-actin remains as non-organized long filaments (Fig. 6B). Therefore, the full-length Gas2 does not possess F-actin organizing properties in vitro. Similarly, the taxol-stabilized polymerized microtubules appear as randomly non-organized long cables (Fig. 6C). However, in the presence of the purified full- length Gas2 protein, microtubules appear as distinct, well- organized bundles (Fig. 6D). Therefore, the full-length Gas2 protein has microtubule-organizing ability. Since the Gas2 protein has only one tubulin-binding domain, we believe Gas2 must form a protein complex in order to bundle microtubules. When F-actin, microtubules and full-length Gas2 are mixed together, Gas2 bundles microtubules, but F-actin still appears as non-organized long filaments (Fig. 6E). Similar experimental results were obtained by mixing actin or tubulin with purified full-length Gas2 protein at the beginning of their polymerization. Discussion The data reported here identify Gas2 as a microtubule-bundl- ing protein that inhibits cell division in Xenopus oocytes. Over- expression of the full-length Gas2 protein arrested Xenopus embryo cell division and resulted in multinucleated cells, indicating a failure of cytokinesis. A similar phenotype was observed upon the over-expression of the tubulin-binding Gas2 domain alone, but not when the actin-binding CH domain was over-expressed. We propose, therefore, that Gas2 inhibits cell division via its C- terminal tubulin-binding Gas2 domain. The cytoskeleton plays a key role in cell division and cytokinesis. To investigate the cytoskeleton-binding activity of Gas2, we used a cytoskeleton co-sedimentation assay and showed that the Gas2 protein indeed co-sedimented with both F-actin and microtubules in vitro (Fig. 5D). Interestingly, when F-actin was de-polymerized, full-length Gas2 did not sediment with microtubule polymers in the pellet as would be expected considering the binding interaction between Gas2 and microtubules (Fig. 5D: SL and PL). One explanation is the presence of a flexible linker region connecting the CH and Gas2 domains (Fig. 1C). This structural flexibility could permit masking of the Gas2 domain by the N-terminal region of the protein and would inhibit the microtubule-binding activity of full-length Gas2. Another possibility is that, due to the high affinity of Gas2 for F-actin, the full-length Gas2 protein binds to small, incompletely de-polymerized F-actin filaments that are too light to be pelleted and thus remain in the supernate. Dynamic microtubules that are able to switch between polymerizing and de-polymerizing states are a necessary require- ment for cell division [12]. Using electron microscopy we have PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24698