免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金所特异标记。本文结果表明,结合于核孔复合体上     

波形纤维蛋白与Nup 180的体外结合

本文作者采用大肠杆菌表达的波形纤维蛋白与大鼠肝细胞分离的核孔蛋白进行体外结合实验,以分析波形纤维与核孔的关系。实验结果显示,细菌表达的波形纤维蛋白在体外能组装成10 nm纤维,在体外反应体系中加入核孔蛋白后,室温反应30 min,SDS-聚丙烯酰胺凝胶电泳及免疫印迹法检测,结果表明180 kD核孔蛋白(Nup 180)与波形纤维蛋白有亲和反应。结合免疫胶体金标记与电镜负染色方法显示,核孔蛋白结合于体外装配的10 nm波形纤维上。本文结果提示在细胞内波形纤维可能通过与Nup 180的结合锚定于核孔复合体上。     

波形纤维蛋白与Nup180的体外结合

本文作者采用大肠杆菌表达的波形纤维蛋白与大鼠肝细胞分离的核孔蛋白进行体外结合实验,以分析波形纤维与核孔的关系。实验结果显示,细菌表达的波形纤维蛋白在体外能组装成１０ｎｍ纤维,在体外反应体系中加入核孔蛋白后,室温反应３０ｍｉｎ,ＳＤＳ－聚丙烯酰胺凝胶电泳及免疫迷法检测,结果表明１８０ｋＤ核孔蛋白（Ｎｕｐ１８０）与波形纤维蛋白有亲和反应。结合免疫胶体金标记与电镜负染色方法显示,核孔蛋白结合于体外装与的     

BHK_(21)细胞的中间纤维-lamina-核骨架体系

以系列选择性抽提技术与显示细胞骨架的整装电镜技术为基础,应用免疫胶体金标记与蛋白质成份的双向电泳分析技术,研究了BHK_(21)细胞的中间纤维-lamina与核骨架(核基质)结构体系及其主要的蛋白成份。BHK_(21)细胞的中间纤维-lamina与核骨架是在结构上相互联系,贯穿于核与质的网络体系。中间纤维单丝直径为10nm,能很好地被抗波形蛋白抗体-金颗粒所标记,生化分析同样说明BHK_(21)细胞中间纤维的主要成份是波形蛋白(vimentin),其分子量为55KD,等电点为5.6。中间纤维网在胞质内呈极性分布,与lamina密切联结。BHK_(21)细胞的lamina能被抗lamin A与C的单克隆抗体-金颗粒标记。双向电泳分析证明,lamina含有三种蛋白成份,即lamin A,B,C,其分子最分别为68KD,70KD与62KD,lamin A,C等电点均为6.9—7.2,而lamin B偏酸,其等电点为5.8。BHK_(21)细胞核骨架纤维网也可以被清晰的显示,其蛋白成份较为复杂,在双向电泳谱上经常出现多个清晰的斑点,很可能含有肌动蛋白(actin)。298KD核基质蛋白的单克隆抗体-金颗粒能准确的标记核骨架纤维。     

一种抗不同类型中等纤维的混合型兔自发抗体

本文报道了存在于未免疫家兔血清中的一种自发抗体(SRI)。使用间接免疫荧光法,这种自发抗体能在鸡肌原纤维上染出-线和Z-盘,在CHO细胞和HeLa细胞中能染出不同排列式样的胞质纤维结构。在秋水仙素处理的CHO细胞中,SRI血清仅能染出具有典型凝聚式样的波形纤维。而在秋水仙素处理的:HeLa细胞中,SRI血清同时能染出对秋水仙素不敏感的前角质蛋白纤维和对该药敏感的波形纤维。细胞松弛素B处理和Trito X-100抽提对:HeLa细胞的纤维染色无影响。根据所染纤维的特征,我们认为SRI自体免疫抗血清是一个至少含有抗前角质蛋白抗体和抗波形纤维蛋白抗体的混合抗体。     

辛德毕斯病毒装配及其6K蛋白与中间纤维的关系

用温和的选择性抽提方法与整装细胞电镜技术,DGD包埋-去包埋剂电镜技术相结合,对辛德毕斯病毒的装配与宿主细胞中间纤维的关系进行了探讨。电镜观察清晰地显示了病毒“装配中心”被中间纤维所网络,病毒的装配过程显然是以中间纤维为支架;正在装配的核壳体与已装配的核壳体紧密结合在中间纤维丝上。根据电镜照片分析,核壳体可能是沿中间纤维由装配中心向外扩散。应用人工合成6K蛋白所得抗体进行胶体金标记,证明辛德毕斯病毒非结构性6K蛋白也与中间纤维紧密结合。     

植物细胞核纤层的研究

应用细胞成分选择性抽提方法,结合非树脂包埋去包埋电镜技术显示悬浮培养的胡萝卜细胞和银杏雄性生殖细胞均具有核纤层结构,免疫印迹反应证明这两种细胞的核纤层由A型和B型核纤层蛋白组成;至少分别含有66ku,84ku和66ku,86ku多肽,免疫胶体金标记将这些蛋白定位在核周缘,光镜和电镜原位分子杂交显示植物细胞具有与动物细胞核纤层蛋白cDNA同源的序列存在;其mRNA分选的部位主要分布在靠近核膜周围的胞质部分,实验结果证明植物细胞确实存在核纤层.     

铁线蕨中间纤维的研究及某些植物类角蛋白的比较分析

应用整装电镜制样技术,结合选择性抽提方法在铁线蕨（ＡｄｉａｎｔｕｍｐｈｉｌｉｐｐｅｎｓｅＬ．）叶细胞中观察到直径１０ｎｍ的纤维网络结构。免疫印迹结果显示：经选择性抽提得到的纤维蛋白与动物角蛋白抗体有免疫交叉反应,间接免疫荧光标记也得到类似结果,而且此类蛋白能在体外自组装为１０ｎｍ或更粗的纤维。说明蕨类植物细胞中存在类角蛋白中间纤维网络。免疫印迹结果表明,螺旋藻（ＳｐｉｒｕｌｉｎａｓｕｂｔｉｌｌｉｓｉｎａＫｕｔｚ．）细胞,地钱（ＭａｒｃｈａｎｔｉａｐｏｌｙｍｏｒｐｈａＬ．）叶状体,铁线蕨（Ａ．ｐｈｉｌｉｐｐｅｎｓｅＬ．）、银杏（ＧｉｎｋｇｏｂｉｌｏｂａＬ．）、白菜（ＢｒａｓｉｃａｐｅｋｉｎｅｎｓｉｓＲｕｐｒ．）的叶组织经选择性抽提后得到的蛋白均与动物角蛋白抗体有免疫交叉反应。其中,螺旋藻仅含有两种类酸性角蛋白,而其余４种植物材料均含有３种类酸性角蛋白及３种类碱性角蛋白。结合以往实验结果,我们认为类角蛋白在植物细胞中是普遍存在的。     

兔网织红细胞与K562细胞胞杂交体（K—RRneo）核基质—中间纤维体系…

本文利用脂质体转基因技术与细胞融合相结合的方法所建立的杂交细胞为研究对象,采用选择性多步抽提配合整装电镜及Ｗｅｓｔｅｒｎ印迹分析等技术,系统观察了兔网织红细胞,人红白血病Ｋ５６２细胞及两者融合形成的胞质杂交体Ｋ－ＲＲｎｅｏ细胞的核基质－中间纤维体系结构,着重分析比较了它们之间的胞质波形蛋白纤维成分的变化。实验结果表明：Ｋ５６２细胞的中间纤维为放射状分布,核纤层为网层状,兔网织红细胞胞质中间纤维胞质     

兔网织红细胞与K_(562)细胞胞质杂交体(K-RRneo)核基质-中间纤维体系的研究

本文利用脂质体转基因技术与细胞融合相结合的方法所建立的杂交细胞为研究对象,采用选择性多步抽提配合整装电镜及Western 印迹分析等技术,系统观察了兔网织红细胞、人红白血病K_(562)细胞及两者融合形成的胞质杂交体K-RRneo 细胞的核基质-中间纤维体系结构,着重分析比较了它们之间的胞质波形蛋白纤维成分的变化。实验结果表明:K_(562)细胞的中间纤维为放射状分布,核纤层为网层状,兔网织红细胞胞质中间纤维为细网格状,其间存有不规则的致密物。胞质体杂交细胞(K-RRneo)的核纤层结构稀薄,核基质较亲代K_(562)细胞致密,中间纤维构型呈现与网织红细胞相似的网格状。中间纤维蛋白电泳及Western 印迹结果亦显示K-RRneo 细胞与网织红细胞的带型类似,即缺乏聚合型波形蛋白及聚合前体物波形蛋白单体,仅检测到解聚前、后而与细胞其它组分结合的波形蛋白复合物。这一生化和超微结构特征提示,红细胞排核可能与波形蛋白纤维的解聚及Vimentin 基因关闭有关。实验结果为排核前细胞内原已装配好的Vimentin 趋于解聚,引起中间纤维瓦解,造成核偏位、固缩而最终排出细胞外的排核机制提供了证据。     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

本文用间接免疫荧光法和电镜术观察了分别来自人表皮(PcaSE-1)、复层上皮(CNE)和单层上皮(SPC-A-1)的3个上皮细胞系的细胞在有丝分裂过程中中等纤维的行为。结果表明,CNE细胞和SPC-A-1细胞表达两种不同类型的中等纤维系统:角蛋白纤维和波形纤维,而PcaSE-1细胞仅表达角蛋白纤维。当细胞进入有丝分裂时,PcaSE-1细胞的角蛋白纤维维持完整的形态且将有丝分裂纺锤体围绕在细胞中央。相反,在CNE细胞和SPC-A-1细胞中,在细胞有丝分裂时,角蛋白纤维解聚成无定形的胞质小体,然而它们的波形纤维始终保持完整的形态。我们认为(1)在分裂上皮细胞中,角蛋白纤维的解聚与细胞的恶性程度有关,而与间期上皮细胞中是否含有丰富的角蛋白纤维无明显关系。(2)在上皮细胞有丝分裂时,中等纤维可能参于纺锤体的定位和趋中。(3)在分裂CNE细胞中,波形纤维的可能功能是染色体的定位和定向。     

人体肝癌细胞和HeLa细胞中等纤维的比较研究

本文用兔抗角蛋白抗体、豚鼠抗波形纤维蛋白抗体和抗角蛋白单抗AE1的间接免疫荧光抗体法比较了两个人体肝癌细胞系(BEL-7402和BEL-7404)和HeLa细胞中等纤维的分布式样,同时用SDS-PAGE法分析了上述细胞的中等纤维抽提物的多肽组成。结果表明:三种上皮细胞均含有两套不同类型的中等纤维系统:角蛋白纤维和波形纤维。但是,人体肝癌细胞和HeLa细胞的中等纤维分布式样和角蛋白多肽组成均有明显的差别。其中最明显的差别是HeLa细胞具有丰富的桥粒-张力纤维复合物和分子量为40 kd的角蛋白多肽,而在两个人体肝癌细胞系中看不到。     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

Vimentin Filaments Follow the Preexisting Cytokeratin Network during Epithelial–Mesenchymal Transition of Cultured Neonatal Rat Hepatocytes

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial–mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell–cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colce- mide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell–cell contact and cell shape.     

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.     

上皮细胞分裂过程中中等纤维的变化

Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis.     

Intermediate Filaments of Schwann Cells

Abstract: Intermediate filaments were prepared from distal stumps of rabbit sciatic nerve 5 weeks after nerve section, at which time Schwann cells account for 85–90% of the cell area. A polypeptide of molecular weight 58,000 was the main component of this fraction. An antiserum raised in guinea pig against this polypeptide stained all cells present in the distal stump, as well as Schwann cells and 3T3 cells in culture. The identity of the molecular weight 58,000 polypeptide obtained from distal stumps with vimentin was proved with one and two-dimensional sodium dodecyl sulfate pol yacrylamide gel electrophoresis and with immunoautoradiography. It is concluded that the intermediate filament subunit of undifferentiated Schwann cells is vimentin. The possibility that Schwann cells in normal nerve may have another type of intermediate filament besides vimentin cannot be ruled out.     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.