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Amphibian oocytes can rapidly and efficiently reprogram the transcription of transplanted somatic nuclei. To explore the factors and mechanisms involved, we focused on nuclear actin, an especially abundant component of the oocyte's nucleus (the germinal vesicle). The existence and significance of nuclear actin has long been debated. Here, we found that nuclear actin polymerization plays an essential part in the transcriptional reactivation of the pluripotency gene Oct4 (also known as Pou5f1). We also found that an actin signaling protein, Toca-1, enhances Oct4 reactivation by regulating nuclear actin polymerization. Toca-1 overexpression has an effect on the chromatin state of transplanted nuclei, including the enhanced binding of nuclear actin to gene regulatory regions. This is the first report showing that naturally stored actin in an oocyte nucleus helps transcriptional reprogramming in a polymerization-dependent manner.
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Figure 1. Visualization of nuclear F-actin. (A) Schematic diagram of the experimental strategy to visualize nuclear F-actin formed in transplanted nuclei. Injected nuclei cannot be observed without dissecting GVs. Dissected GVs can be maintained alive in mineral oil and are transparent enough to observe their interior structure by confocal microscopy. (B) Nuclear F-actin in a Xenopus oocytenucleus, visualized with GFP-UtrCH probes. A meshwork structure of nuclear F-actin is naturally formed in nucleoplasm. Bar, 5 μm. (C) Nuclear F-actin is observed in injected nuclei. F-actin was labeled with GFP-UtrCH (green) and chromatin of injected nuclei was visualized with Cherry-histone H2B (red). Actin bundles in a transplanted nucleus are marked with white arrows. The image was taken 24 h after NT. Bar, 10 μm.
Figure 4. Toca-1 exists in a transplanted nucleus and enhances nuclear actin polymerization. (A) TOCA1-CHERRY is present in injected nuclei. Toca1-Cherry and GFP-UtrCH mRNA were injected before NT. The injected nuclei were observed 48 h after NT by confocal microscopy using the oil GV method. A nucleus in which TOCA1-CHERRY is present shows nuclear F-actin formation (white arrow). In contrast, nuclear F-actin is not detected in a nucleus without TOCA1-CHERRY (blue arrow). Bar, 20 μm. (B) TOCA1 overexpression increases nuclear F-actin formation. Different amounts of TOCA1 were expressed in oocytes by mRNA injection with different concentrations. These injected oocytes were used for NT. The oocytes were subjected to immunofluorescence analysis and phalloidin staining 48 h after NT. Images were obtained from randomly selected areas. Nuclear F-actin amounts were quantified using the ImageJ software (Materials and Methods). Statistical differences were measured by F and T-tests. Data represent mean ± SEM; Toca-1 1.5 ng: n = 45; Toca-1 4.6 ng and 13.8 ng: n = 55. (C) Actin fractionation experiment indicates that TOCA-1 overexpression increases the F-actin population in GVs. TOCA-1 was expressed in oocytes by mRNA injection. GVs were collected from these injected oocytes or noninjected oocytes, and total actin was fractionated to monomeric and polymeric actin (G- and F-actin, respectively). The amounts of G- and F-actin were measured by Western blot analysis using anti-βactin antibody. CB was added to culture medium at a 1 μg/mL concentration to check that enhanced actin polymerization was diminished with this concentration. F-actin proportion in total actin was compared among three samples, and fold differences relative to the control noninjected oocytes (Toca− and CB−) are shown. Data represent mean ± SEM; n = 2. (D) Actin fractionation experiment indicates that overexpression of a TOCA-1 mutant, W518K, does not increase the F-actin population in GVs. F-actin ratio in total actin is relative to control water-injected oocytes. Data represent mean ± SEM; n = 2.
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