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Biochem Biophys Res Commun
2010 Sep 17;4002:200-6. doi: 10.1016/j.bbrc.2010.08.032.
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An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development.
Aoki R
,
Inui M
,
Hayashi Y
,
Sedohara A
,
Okabayashi K
,
Ohnuma K
,
Murata M
,
Asashima M
.
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Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.
Fig. 1.
Expression patterns of HP1-GFP and H1-GFP proteins in Xenopus embryos at different stages of development. Images of animal cap cells at stage 9 (A–F), whole embryos at stage 20 (G–L), and tadpoles at stage 38 (M–R). (A–C, G–I, M–O) Bright-field images; (D–F, J–L, P–R) fluorescence images. Embryos were injected with HP1-GFP mRNA (A, D, G, J, M, P), H1-GFP mRNA (B, E, H, K, N, Q) or GFP mRNA (C, F, I, L, O, R). Scale bars: 100 μm (A–F); 1 mm (G–R).
Fig. 2.
HP1 and H1 fluidities in cytoplasmic extracts at three different developmental stages (A) Schematic of the experimental procedure. Nuclei that expressed HP1-GFP or H1-GFP were isolated from the tailbuds (red rectangle) of stage 32 embryos. Cytoplasmic extracts from eggs, stage 9 embryos, or stage 15 embryos were added and FRAP analyses were conducted. (B) Representative image of an isolated nucleus (arrowhead) that expresses HP1-GFP in balanced salt solution with Texas Red-conjugated dextran. (BF) Bright-field image; (GFP) green fluorescence image; (TexRed-Dex) red fluorescence image; and (Merge) merged image of green and red fluorescence images. The nuclei of intact cells, which were not disrupted during the nuclear isolation procedure (arrows), are not visible due to the yolk-rich cytoplasm. Texas Red-conjugated dextran does not permeate the cellular membranes or nuclear envelopes. (C–F) FRAP experiment with HP1-GFP (C, E) and H1-GFP (D, F). Images taken at 0, 10, and 60 s (C) or at 0, 60, and 360 s (D) in the FRAP sequences. The foci (C, arrowheads) and the lower half (D) of each nucleus were bleached by the laser at time zero. Scale bar: 5 μm. Each inset in (C) contains a higher-magnification image. (E, F) Normalized FRAP recovery curves for the foci in (C) and the lower half of (D), respectively. The fluorescence intensities before and just after bleaching were defined as 1 and 0, respectively. Error bars, SE; ∗P < 0.05; ∗∗∗P < 0.0005 (t-test; n = 45 for egg extract, n = 38 for stage 9embryo extract, and n = 39 for stage 15embryo extract in (E); n = 9 for egg extract, n = 11 for stage 9embryo extract, and n = 13 for stage 15embryo extract in (F)).
Fig. 3.
HP1-GFP fluidities in three types of isolated nuclei treated with two types of cytoplasmic extracts. (A) Schematic of the experimental procedure. Nuclei that express HP1-GFP were collected from the animal cap cells of stage 10 embryos, neural plates cells of stage 20 embryos, and tailbud cells of stage 32 embryos. Extracts from eggs or stage 15 embryos were added, and FRAP measurements were conducted. (B–D) Normalized FRAP curves of the HP1 fluidities in egg extracts and stage 15embryo extracts in nuclei of stage 10animal cap cells (B), stage 20neural plate cells (C), and stage 32tailbud cells (D). (E, F) Normalized FRAP curves of the HP1 fluidities in 10 animal cap cell nuclei, stage 20neural plate nuclei, and stage 32tailbud nuclei in egg extract (E) and in stage 15embryo extracts (F). (G) Recovery rates (see Section 2) for each condition. Error bars represent the SE. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005 (two-sample t-test; n = 31 for the egg extract, n = 24 for the stage 15embryo extract (B); n = 33 for the egg extract, n = 28 for the stage 15embryo extract (C); n = 34 for the egg extract, n = 36 for the stage 15embryo extract (D)).