Nucleus (blue) and Microtubules (green).

Presentation on theme: "Nucleus (blue) and Microtubules (green)."— Presentation transcript:

1 Nucleus (blue) and Microtubules (green).

2 细胞核的主要结构包括：①核被膜（nuclear envelope）、②核仁（nucleolus）、③核基质（nuclear matrix）、④染色质（chromatin）、⑤核纤层（nuclear lamina）等部分

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6 Nucleus and Internal Membranes
Chinese hamster ovary cells (CHO cells) labeled with Hoechst (Molecular Probes # H-1399) and  DiOC6 (Molecular Probes # D-273). The Hoechst stains the nuclear DNA blue, and the DiOC6 stains  the internal organelles green.  (Omega Optical Multi-dye Filter Set # XF50).      Photographs by Victoria Osborne.

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9 The space between the outer and inner membranes is also continuous with rough endoplasmic reticulum space. It can fill with newly synthesized proteins just as the rough endoplasmic reticulum does. The nuclear envelope is enmeshed in a network of filaments for stability.

10 Nucleus and Microtubules
Chinese hamster ovary cells labeled with Hoechst (Molecular Probes # H-1399) to stain the nuclear DNA blue and by immunofluorescence with an antitubulin antibody and a FITC-conjugated secondary antibody (green). (Omega Optical Multi-dye Filter Set # XF64).

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13 Nucleus and Filamentous Actin
Chinese hamster ovary cells (CHO cells) labeled with Hoechst (Molecular Probes # H-1399) and BODIPY TR-X phallacidin (Molecular Probes # B-7464).  The Hoechst stains the nuclear DNA blue, and BOPIPY TR-X phallacidin stains filamentous actin red. (Omega Optical Multi-dye Filter Set # XF56). 

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17 第一节 核被膜(nuclear membrane)与核孔复合体（Nuclear Pore Complex ）
外核膜outer nuclear membrane 内核膜 Inner nuclear membrane 核周隙perinuclear space 核膜孔nuclear pore

18 (nuclear membrane)，又          称核被膜(nuclear envelope)    核周间隙(perinuclear space)     核孔(nuclear pore)     核纤层(nuclear lamina)     核骨架(nuclear skeleton)     染色质（chromatin)

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21 核被膜由内核膜（inner nuclear membrane）、外核膜（outer nuclear membrane）和核周隙（perinuclear space）三部分构成。核被膜上有核孔与细胞质相通。 外核膜胞质面附有核糖体，并与内质网相连，核周隙与内质网腔相通，可以说是内质网的一部分。外核膜上附着10nm的中间纤维intermediate filament，可见核是被内质网和中间纤维相对固定的。 核周隙宽20~40nm，腔内电子密度低，一般不含固定的结构。 内核膜的内表面有一层网络状纤维蛋白质，叫核纤层（nuclear lamina），可支持核膜

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23 Nuclear Envelope The nuclear envelope has two membranes, each with the typical unit membrane structure. They enclose a flattened sac and are connected at the nuclear pore sites. The outermost membrane is continuous with the rough endoplasmic reticulum (ER) and has ribosomes attached (see figure to the left).

24 The nuclear envelope is shown in an electron micrograph in the figure to the right. The filaments outside the envelope are not visualized with these protocols. Also, the nuclear lamina just inside the nuclear envelope is not shown well (see paragraph below for description).

25 However, one can see ribosomes on the outer membrane and the sac enclosed by the two membranes. Dense patches of Heterochromatin are seen just inside the inner membrane.

26 二、核孔复合体 (一)结构模型 迄今尚未有一个统一的认识，但主要有三种模型 1）纤丝模型 ）Ris的滴漏模型 3）圆柱模型

27 Nuclear Pore Complex: Structure

28 Nucleus with Nuclear Pores (TEM x73,200)
Nucleus with Nuclear Pores (TEM x73,200). The cytoplasm also contains numerous ribosomes

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32 The above figure shows a view of the nuclear pore from the top
The above figure shows a view of the nuclear pore from the top. It contains 8 subunits that "clamp" over region of the inner and outer membrane where they join. Actually, they form a ring of subunits nm in diameter. Each subunit projects a spoke-like unit into the center so that the pore looks like a wheel with 8 spokes from the top. Inside is a central "plug".

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34 This electron micrograph shown in the figure to the right depicts a nuclear pore complex seen with the transmission electron microscope. As is obvious, little detail can be seen. The inner and outer membranes of the nuclear envelope are joined and there appears to be a diaphragm-like structure in the center.

35 However, the intricate detail pictured in the foregoing figure cannot be appreciated. One needs to use different preparative techniques to see the subunits and their organization. These will be discussed and illustrated in the following sections.

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38 Freeze-fracture/freeze-etch

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40 Another preparation shows more details of the structure of the nuclear pore complex. Here we see the subunits forming the rings and their spokes. Note that one of the pores appears to be open in the center, forming a channel. Figure modified from Bloom and Fawcett, A Textbook of Histology, Chapter 1, Figure 1-9, Chapman and Hall, Publishers, 1994

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42 在电镜下观察，核孔是呈圆形或八角形（图12-4、5），一般认为其结构如fish-trap，主要包括以下几个部分：①胞质环（cytoplasmic ring），位于核孔复合体胞质一侧，环上有8跳纤维伸向胞质；②核质环（nuclear ring），位于核孔复合体核质一侧，上面伸出8条纤维，纤维端部与端环相连，构成笼子状的结构；③转运器（transporter），核孔中央的一个栓状的中央颗粒；④辐（Spoke）：核孔边缘伸向核孔中央的突出物

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44 核孔复合体的功能 1通过核孔复合体的被动扩散 扩散速度与分子大小成反比，小于5kD的小分子可以自由扩散，大于60kD的球蛋白几乎不能进入核内。 2 通过核孔复合体的主动运输

45 （1）对运输 颗粒大小的限制 可以运输象RNP大小的颗粒。说明核孔直径的大小可以被调节。
（2）核孔主动运输是一个信号识别和载体介导的过程，需要ATP。 （3）通过核孔的主动运输是双向的。 1、亲核蛋白的核运输 亲核蛋白（P265} 核质蛋白nucleoplasmin（265）具有头尾两个结构域。如此大的蛋白质不能有效的扩散进入核内。那么亲核的输入信号位置在那？

46 A peptide sequence, called nucleoplasmin, was isolated and linked to colloidal gold. It was then injected into an oocyte and traced with electron microscopy. As shown in the figure to the left, the gold particles mark the site of transport of the nucleoplasmin and the studies showed that it was transported into the nucleus. Small gold markers are evident inside the nucleus. Figure was taken from Alberts et al., Molecular Biology of the Cell, Garland Pub., N.Y. 1994, Fig

47 In another test, workers took advantage of the fact that nucleoplasmin has a head region which is not transported into the nucleus and a tail region which is transported. When the heads and tails were separated and each linked to gold, only the gold-labeled tail region was transported. Gold-labeled head regions remained in the cytoplasm.

48 核孔由至少50种不同的蛋白质（nucleoporin）构成，称为核孔复合体（nuclear pore complex，NPC）。一般哺乳动物细胞平均有3000个核孔。细胞核活动旺盛的细胞中核孔数目较多，反之较少。如蛙卵细胞每个核可有37.7X106个核孔，但其成熟后细胞核仅150~300个核孔。

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50 核质蛋白 爪蟾卵母细胞核质蛋白质注射实验 放射性 尾部 头部 细胞质 注如入胞质 细胞核 摄入细胞核

51 有限的水解 注如细胞 滞留在细胞质 放射性头部 摄入细胞核 放射性尾部

52 尾部 胶体金 包裹尾部的胶体金 进入细胞核 注入细胞

53 核定位信号（ NLS ） NLS特异性结合蛋白（NAPs）分布于胞质、核膜、核内 RNA RNP RNP RNA CLS 细胞质定位信号 核仁 CLS 细胞核 NLS 内膜 NLS NAP 外膜 NLS NAP NAP

54 1982年R. Laskey发现核内含量丰富的核质蛋白（nucleoplasmin）的C端有一个信号序列，可引导蛋白质进入细胞核，称作核定位信号（nuclear localization signal，NLS）。第一个被确定的NLS是病毒SV40的T抗原，它在胞质中合成后很快积累在核中。其NLS为：pro-pro-lys-lys-lys-Arg-Lys-val，即使单个氨基酸被替换，亦失去作用。

55 NLS由4-8个氨基酸组成，含有Pro、Lys和Arg。对其连接的蛋白质无特殊要求，并且完成核输入后不被切除。
Karyopherin是一类与核孔选择性运输有关的蛋白家族，相当于受体蛋白。其中imporin负责将蛋白从细胞质运进细胞核，exportin负责相反方向的运输。

56 通过核孔复合体的转运还涉及Ran蛋白，Ran是一种G蛋白，调节货物受体复合体的组装和解体，在细胞核内Ran-GTP的含量远高于细胞质。

57 核质蛋白向细胞核的输入可描述如下：①蛋白与NLS受体，即imporin α/β二聚体结合；②货物与受体的复合物与NPC胞质环上的纤维结合；③纤维向核弯曲，转运器构象发生改变，形成亲水通道，货物通过；

58 ④货物受体复合体与Ran-GTP结合，复合体解散，释放出货物；⑤与Ran-GTP结合的imporin β，输出细胞核，在细胞质中Ran结合的GTP水解，Ran-GDP返回细胞核重新转换为Ran-GTP；⑥imporin α在核内exportin的帮助下运回细胞质（图）。

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