Zimmer FJ et al. (1988), The function of the nuclear envelope in nuclear...

XB-ART-27513

J Cell Biol 1988 May 01;1065:1435-44.

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The function of the nuclear envelope in nuclear protein accumulation.

Zimmer FJ , Dreyer C , Hausen P .

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The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.

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Species referenced: Xenopus laevis
Genes referenced: gnl3 npm1 tbx2
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	Figure 1. Nuclear proteins ofX. laevis oocytes. Three hand-isolated nuclei were subjected to two-dimensional microgel analysis, and the polypeptides were stained with silver. Arrows indicate the nuclear proteins nucleoplasmin (NP), N1, N4, and NS, which are highly enriched in oocyte nuclei. Proteins N1 and N4, as described by De Robertis et al. (1978), were identified by their position in two dimensional gels.
	Figure 2. Immunostaining of sections of punctured X. laevis oocytes with mAb b7-1A9 directed against nucleoplasmin. The animal poles of oocytes were punctured 30 times with a glass needle as described in Materials and Methods. 3 and 8 h after puncturing, the oocytes were fixed in Romeis fixative and processed for immunostaining. K,' section of nonpunctured oocyte. Bar, 0.1 mm.
	Figure 3. Immunostaining of oocyte sections after removal of part of the nucleus. Part of the nucleus of each oocyte was removed and 10 rain and 4.5 h later the oocytes were fixed in Romeis fixative and processed for immunostaining with mAbs b7-1A9 (directed against nucleoplasmin) (a) and b6-6E7 (directed against N8) (b). Bar, 0.22 mm.
	Figure 4. Immunostaining of sections ofX. laevis ooeytes injected with nucleoplasm from X. borealis oocytes and fixed either immediately (0 h) or 0.5, 1.5, 3, 8, 19, and 67 h later with 2% TCA. Two oocytes were punctured 1 h after injection 30 times with a glass needle into the animal poles. One oocyte was fixed 7 h later (P8 h) and the other one 18 h after puncturing (P19 h). Oocyte sections were stained with mAb b6-3BT, which is directed against X. borealis nuclear protein NI. Dark areas that are seen in the injected nucleoplasm (see 0 h) are paraffin droplets. Bar, 0.2 mm.
	Figure 5. Comparison of the distribution of antigen b6-3B7 with that of three other nuclear antigens. X. laevis oocytes were microinjected with nucleoplasm from X. borealis oocytes and fixed either immediately (0 h) or 1, 1.5, 3, 8, 19, and 44 h later in 2 % TCA. Four neighboring sections in every oocyte were stained with mAbs b6-3B'/(directed against N1 in X. borealis oocytes), b6-6E7 (directed against N8), b7-2H4 (directed against N4), and b2-2B10 (directed against NI). Staining with mAb b7-2H4 44 h after injection is background (see text). Bar, 0.22 mm.
	Figure 6. Nuclear uptake of purified nucleoplasmin after microinjection into the cytoplasm of X. laevis oocytes. (a) Dependence of nuclear accumulation of nucleoplasmin on site of injection. Oocytes were injected with ,,02.5 ng of iodinated protein either "~45 Â° from the animal pole (as shown in the inset), into the equator, or into the vegetal pole, and incubated in MBS-H at 20Â°C. The percentage of radioactivity found in the nucleus, relative to the total amount injected, is plotted against time after injection. (b) Nuclear accumulation of nucleoplasmin during 3 d of incubation. Oocytes were injected with â¢2.5 ng of iodinated protein into the equator. The nuclear-to-cytoplasmic concentration ratio (N/C) of the iodirutted protein was calculated from the formula (% cpm nuclear/ 12)/(% cpm cytoplasmic/88), assuming that the nucleus contains 12% of the accessible aqueous volume of an oocyte (Bonner, 1975a). The percentage of the total radioactivity remaining in the oocyte, which is found in the nucleus, is plotted on the left. Note that the N/C ratio remains approximately constant after 24 h, despite degradation of injected nucleoplasmin (see text for details). NP, nucleoplasmin.

References [+] :

Bonner, Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. 1975, Pubmed, Xenbase