Gas2 Inhibits Cell Division 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). (D) The full-length GFP-Gas2 co-sediments with both F-actin and microtubules. When F-actin is de-polymerized with latrunculin B, the full- length Gas2 surprisingly remains in the supernate only. ST: Supernate of Taxol treatment, PT: Pellet of Taxol treatment; SN: Supernate of Nocodazole treatment, PN: Pellet of Nocodazole treatment; SP: Supernate of Phalloidin treatment, PP: Pellet of Phalloidin treatment; SL: Supernate of Latrunculin B treatment, and PL: Pellet of Latrunculin B treatment. doi:10.1371/journal.pone.0024698.g005 mounted with ProLong Gold anti-fading reagent (Molecular Probes) and covered with cover slips (Fisher: 12–545M) for microscopy examination. Xenopus laevis ooctyes collection and preparation Oocytes were surgically removed from an anesthetized adult Xenopus laevis female, as previously described [11] and stored at 16uC in oocyte ringer-2 solution (OR-2: 82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM Figure 6. The full-length Gas2 protein bundles microtubules in vitro. (A) F-actin alone appears as non-organized long filaments. (B) In the presence of Gas2, F-actin remains as non-organized long filaments as in A. Bars, 200 nm. (C) Microtubules alone appear as long, randomly distributed cables. (D) Microtubules bundle together when Gas2 is present in the sample. Bars, 0.2 mm. (E) Gas2 bundles microtubules, but does not organize F-actin when microtubules, F- actin and Gas2 protein are mixed together. Bar, 200 nm. The order of Gas2 addition relative to the microtubules or F-actin has no effect on the observed results. HEPES at pH 7.4). Oocytes were treated with 0.2% collagenase (Type 2) OR-2 solution with gentle rotation at room temperature for 1 hour. Oocytes were washed after the treatment until the brown color collagenase was removed. The washed oocytes were kept at 16uC in OR-2 solution for at least 3 hours for recovery. Healthy oocytes have uniform animal and vegetal hemispheres. StageVI oocytes were manually chosen and de-folliculated with Watchmaker’s forceps (Dumont #5) in a Petri dish containing OR-2 solution. This step is critical for cortex wound healing experiments. Pharmacological perturbations and oocyte wounding Oocytes were incubated in fresh 10 mM taxol (Sigma) or 20 mM nocodazole (Calbiochem) OR-2 solution for 1 hour at 16uC. The wounding experiments were performed in the same solutions. Oocytes were placed in a Petri dish fitted with a nylon grid to position the animal pole facing upwards. They were then wounded in the animal pole with a microinjection needle (cut to outside with a diameter of approximate 150 mm). Cells were incubated for about 5 minutes in OR-2 solution for recovering before being placed in fix solution. Xenopus laevis embryos and oocytes immunofluorescence The protocol was the same as previously described [11]. Briefly, embryos or wounded oocytes were fixed and incubated with Alexa Fluor 568 phalloidin (6.6 mM, at 1:200 dilution) (Molecular probes) and monoclonal alpha tubulin antibody (1:200 dilution) (Sigma, DM1A). Samples were then re-probed with Alexa Fluor 568 phalloidin for F-actin and Alexa Fluor 647 goat anti-mouse IgG antibody (1:200 dilution) (Molecular probes) for alpha tubulin. Finally, samples were carefully placed in the center of a high vacuum grease (Dow Corning Co.) mounted ring on a microscope slide plain (Fisher), and covered with a microscope cover glass #1.5 thickness (Fisher) for microscopy examination. Confocal fluorescence microscopy All confocal images were collected using Zeiss 510 LSM Meta confocal microscope at Cell Imaging and Analysis Network at McGill University. The samples were examined with Plan- Neofluar 25X/0.8 N.A. Immersion Correction DIC or Plan- Apochromat 63X/1.4 N.A. oil DIC objective lens. DAPI (Molecular probes) staining was visualized with 405 nm blue diode laser and band pass (BP) 420–480 filter; GFP signal was visualized with 458 nm Ar ion laser and BP 505–530 filter; Alexa Fluor 568 was visualized with 543 nm HeNe green laser and BP 560–615 filter; and Alexa Fluor 647 was visualized with 633 nm HeNe red laser and long pass (LP) 650 filter. Z-sections of varying depths were stacked into projection images with maximum intensity to allow visualization of details. Cytoskeleton co-sedimentation assays The full-length GFP-Gas2, GFP-CH domain and GFP-Gas2 domain constructs were injected into Xenopus oocytes and oocytes were incubated at 16uC for 48 hours to allow for relative proteins expression. The expressing proteins were collected by sonication PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24698