tation assays and electron microscopy were performed to study Gas2- cytoskeletal interactions in vitro. Our results suggest that the Gas2 protein plays a role in cell division and that its function is mediated by bundling microtubules. Results The Gas2 protein is conserved during evolution Bioinformatics comparisons of the Gas2 protein sequences among 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] reveal that Gas2 is conserved during evolution (Fig. 1A and B), which suggests that Gas2 has a conserved biological function. The Gas2 protein contains two cytoskeletal binding domains: a putative actin- binding calponin homology (CH) domain near its N-terminus (Fig. 1C for mouse Gas2 [P11862] amino acids#: 36-158), and a tubulin-binding Gas2 domain near its C-terminus (amino acids#: 201–274). There are two low complexity domains (amino acids#: 167–178 and 181–198) between the CH and Gas2 domains, and the second low complexity domain contains 4 proline-serine (P-S) repeats (amino acids#: 183–190 shown in the boxed region in its sequence), which gives this region more structural flexibility. The N-terminus GFP-tagged full-length mouse Gas2 protein [P11862] and its CH and Gas2 domains were cloned to study their functions in Xenopus laevis (Fig. 1C), and the protein expression in Xenopus oocytes was verified by Western blot analysis (Fig. 1D). Exogenous expression of the full-length Gas2 protein in Xenopus embryos inhibits cell division The Gas2 protein is up-regulated upon serum starvation in NIH 3T3 cells and it also results in cells arresting at G0 phase of cell cycle [9]. Conversely, the Gas2 protein level is down-regulated with serum and growth factor stimulation, allowing for cell cycle progression [6]. These two observations imply a relationship between cell cycle progression and the Gas2 protein level. To test this hypothesis, Xenopus laevis embryos were used to investigate whether the over-expression of the Gas2 protein could inhibit cell division. The first cell division of Xenopus embryo occurs approximately 90 minutes post-fertilization at room temperature, and subsequent cell divisions occur at 30 minute intervals. Xenopus embryos are relatively large, approximately 1 mm in diameter, facilitating the microinjection of the Gas2 protein and the observation of cell morphological changes. The fertilized eggs were injected after they completed their first cell division. One cell of the 2-cell stage embryo was microinjected with either 25 ng bovine serum albumin (BSA) in phosphate buffered saline (PBS) solution or the bacterial purified full-length mouse Gas2 protein into the cytoplasm of a cell. The non-injected cell of the 2-cell embryo acts as an internal homo-genomic negative control (Fig. 2A). The BSA-injected embryos are shown in Fig. 2B–D (Video S1) and Gas2-injected embryos are shown in Fig. 2E–G9 (Video S2). BSA-injected embryos proceed through normal cell division and the number of cells increased 2-fold every 30 minutes. However, cells that were injected with 25 ng Gas2 protein divided once and then arrested in subsequent cell divisions while the non-injected control cells of the same embryo divided normally (Fig. 2H smaller cells on the left). In Gas2-injected cells, cell division arrested after approximately 30 minutes, consistent with the time required for Gas2 to diffuse in the cytoplasm of the injected cell (approximately 3.5 mm/second under the injection needle pressure). Statistical analysis of the microinjection exper- iments showed that 7.264.2% BSA-injected embryos arrested in Gas2 Inhibits Cell Division cell division, likely as a result of unsuccessful fertilization and/or microinjection damage to the embryos. In contrast, 79.265.5% Gas2-injected embryos arrested in cell division, which was a significantly higher percentage than the BSA control group (Fig. 2K). The non-100% arresting effect by Gas2 in cell division is thought to be caused by the Gas2 protein clotting in the injection needle. BSA-injected embryos succeed in developing into tadpoles, but Gas2-injected embryos all died within 24 hours, presumably due to the interference of the arrested cells with the embryo development. In 32-cell embryos, the cortical microtu- bules were significantly longer in the Gas2-injected arrested cells than non-injected control cells (Fig. 2I). The two large Gas2 arrested cells were connected together with microtubules (arrow in J). Two-cell stage Xenopus embryos were microinjected with increasing amounts of the Gas2 protein to investigate the minimum amount of Gas2 required to arrest their cell division. Dosage dependent analysis was done by microinjecting the same volume (5 nl) of a serial dilution of Gas2 protein, and observing the cell division arresting phenotype. The calculated lethal dose 50% (LD50) was 5.5 ng or 31 mM for one cell of the 2-cell stage Xenopus embryo (Fig. 2L). Gas2 co-localizes with microtubules in arrested cells and over-expression of either the full-length Gas2 protein or the Gas2 domain alone results in multinucleated cells To further investigate the mechanism of how Gas2 arrested Xenopus embryo cell division, cryo-confocal microscopy was performed with Gas2-injected embryos that were fixed and stained for Gas2, tubulin and DNA by DAPI (Fig. 3A–D). Gas2- injected cells that failed division were larger than non-injected control dividing cell. Gas2 localizes to the cortex in arrested cells and co-localizes with cortical microtubules (arrow in A). The Gas2-injected cell has no DAPI staining presumably due to the fact that the injected cell arrested in the early embryo developmental stage when the cell is relatively large; therefore, it is difficult to maintain the integrity of all cellular organelles (such as the cell nucleus) during cryo-cutting (Fig. 3C shows no DAPI staining in the large arrested cell). The same observations were obtained with repeated experiments. To examine the exogenous Gas2 during cell division, GFP control, full-length GFP-Gas2, GFP-CH domain and GFP-Gas2 domain constructs were injected into the cytoplasm of one cell of the 2-cell stage embryos, and embryos were fixed at either Stage 10 or 11. Cryo-confocal microscopy was performed to examine post-injected embryos. Embryos expressing the GFP control (Fig. 3E–H) and GFP-CH domain (Fig. 3I–L) are approximately the same size as neighboring non-expressing control cells, which is an indication of normal cell division. Some GFP and GFP-CH domain expressing cells can be recognized in anaphase of mitosis on the basis of their microtubule morphology (arrows in F and J) and their separating chromosomes (arrows in G and K). However, cells expressing both full-length GFP-Gas2 (Fig. 3M–P) and GFP- Gas2 domain (Fig. 3Q–T) are larger than control cells, and have multiple nuclei, indicative of a failure in cell division (arrows in O and S). The full-length GFP-Gas2 (Fig. 3M) and GFP-Gas2 domain (Fig. 3Q) localize to the cell cortex and co-localize with microtubule spindles. Gas2 stabilizes microtubules via its Gas2 domain in Xenopus oocytes To test the hypothesis that Gas2 regulated microtubule dynamics/stability leading to cell division arrest, Xenopus laevis oocyte wound healing contractile array assay, which mimics PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24698