小鼠胚胎干细胞(ES-M_(13))核骨架-核纤层-中间纤维结构体系的研究

本文使用细胞的选择性抽提、DGD包埋去包埋电镜制样、免疫荧光和免疫印迹技术研究了小鼠胚胎干细胞(ES-Ml_(13))的核骨架-核纤层-中间纤维(NM-L-IF)结构体系。在电镜下可以看到,ES细胞存在精细发达的核骨架结构,核骨架纤维同核纤层结构相连接,细胞质中有许多直径为10nm的中间纤维单丝。在免疫荧光分析中,使用角蛋白单克隆抗体有阳性反应,细胞质区域可以看到较强的荧光,没有极性分布现象,也没有观察到纤维状的荧光染色。ES细胞对波形蛋白和结蛋白抗体呈阴性反应,同对照组一样,只能看到非特异性的很微弱的荧光染色。在免疫印迹分析中,使用角蛋白单克隆抗体AF6检测到三条角蛋白多肽,分子量分别为65KD,62KD和52KD。     

北京鸭B淋巴细胞的中间纤维—核纤层—核内骨架体系

以北京鸭腔上囊为实验材料,应用细胞分级抽提方法与非树脂包埋－去包埋剂的电镜制样技术相结合,显示出Ｂ细胞中相互连结的中间纤维－核纤层－核内骨架体系的超结构及其分布,中间纤维交织成网络状,纤维直径在９－１１ｎｍ,其成份是分子量为６７ｋＤ,等电点约为６．２的波形蛋白,核纤民支呈片层状结构环绕在核区周围,其主要成份是分子量为６７ｋＤ,等电点偏酸性的Ｌａｍｉｎ Ｂ。核内骨架由粗细不一的纤维形成网络结构,其上     

PtK2细胞的核骨架—核纤层——中间纤维体系

本文用选择性系列抽提的方法结合整装细胞电镜技术和DGD包埋-去包埋超薄切片技术,在电镜下清晰地显示了PtK 2细胞的核骨架-核纤层-中间纤维体系的精细结构。处于分裂中期的细胞经抽提后可以看到,染色体残余与中间纤维仍然保持一定的联系。用免疫荧光技术对抽提后的PtK 2细胞进行分析结果表明:其中间纤维能同时与AE1和AE3反应;能与Lamin B反应的单抗可以特异地定位于其核周,而Lamin A(C)的单抗除了与其核纤层蛋白有很强的反应外还与中间纤维有交叉反应。此外,在分裂期细胞中可以看到Lamin A(C)可能与染色体能特异结合;与HeLa细胞不一样。PtK 2细胞的核骨架成份不能与280kD的核骨架蛋白单抗反应。双向电泳结果显示出PtK 2细胞的核骨架-核纤层-中间纤维体系的组成成份与HeLa细胞相比有较大的差异,而且这种差异主要反映在核骨架组份上,TdR的处理也能导致其组份发生变化。     

Nuclear Lamina（核纤层）—一种重要的细胞核结构

Nuclear lamina(核纤层)是细胞核内膜下由1至3种纤维蛋白(lamin)所构成的纤维网络或片层结构。Lamina与中间纤维及核内骨架相互联结,形成贯穿于细胞核和细胞质的骨架体系。Lamin与中间纤维蛋白具有同源性;Lamin的表达与细胞分化有关;Lamina在细胞分裂期中发生解聚和重装配。Lamina对核膜和染色质起支架作用,并与细胞核构建有关。     

急纤虫营养细胞和休眠细胞的中间纤维-核骨架体系

利用生化分级抽提、DGD包埋—去包埋透射电镜术和SDS—PAGE凝胶电泳,研究了膜状急纤虫营养细胞和休眠细胞内中间纤维—核骨架体系的分化特征及其蛋白组成。观察到营养细胞中,位于细胞质不同区域的中间纤维形成网状,其网络的密度不同;核骨架中,核纤层位于细胞核周缘,薄层状,厚约50nm;核内骨架由较致密的纤维网络组成。休眠细胞内该结构体系依然存在,但位于细胞内不同层次的纤维网比营养细胞的同种结构要致密得多,这可能与纤毛虫脱分化时细胞大范围的收缩有关;休眠细胞的包囊壁中层壁存在相当于中间纤维的网络结构。SDS—PAGE电泳图谱显示,休眠细胞内该体系的蛋白组成发生了较明显的变化,其中保留了营养细胞的部分蛋白条带,丢失了部分条带,同时还产生了一些特异性条带。分析表明,膜状急纤虫的中间纤维—核骨架体系是细胞在营养条件下和休眠状态下都稳定存在的结构;而纤毛虫形成休眠细胞后中间纤维—核骨架体系及蛋白组成上的变化提示,细胞在休眠状态下,基因的表达水平与营养细胞是不同的。     

核骨架与真核基因复制起点

一、核骨架 核骨架(nuclear scaffold)又名核基质(nuclear matrix,NM),是细胞核内一种动态亚组分结构,其功能是将DNA组织成相对独立的区域(domain),并为其提供专一性的转录、复制及RNA加工的控制位点。它在形态上为一种细胞核内不溶的骨架网络,包括核纤层(lamina)、内层蛋白纤维颗粒、残留的核仁和核孔复合体。它普遍存在于真核细胞中,维持了细胞核的基本形态。     

寇氏隐甲藻(Crypthecodinium cohnii)细胞核骨架与中间纤维的研究

甲藻(涡鞭毛虫,dinoflagellate)的细胞核是现存真核生物中最原始的。我们采用整装细胞制样和非树脂包埋去包埋剂超薄切片电镜技术,结合选择性生化抽提方法显示在寇氏隐甲藻(Cryptheccdinium cohnii)细胞中存在一个以水不溶性纤维蛋白成份为主的,贯穿于细胞核和细胞质的纤维网架系统,即核骨架-中间纤维结构体系,而Lamina结构不明显。免疫印迹法显示,甲藻细胞中存在类角蛋白组分,分子量为63kD和67kD,哺乳动物Lamin抗体与甲藻细胞全蛋白反应阴性。实验结果表明,在原始真核细胞中已经出现了类似于哺乳动物细胞的核骨架和中间纤维,并提示核骨架-中间纤维细胞骨架体系可能起源于真核细胞起源早期。本文对Lamina与中间纤维在进化上的关系及Lamina在真核细胞进化中的功能意义作了讨论。     

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。     

植物细胞中含有类角蛋白多肽及角蛋白cDNA的同源序列

根据中间纤维对去圬剂和高盐溶液的抗性,用类似动物细胞中间纤维抽提的方法,对玉米和烟草的叶肉细胞进行选择性抽提处理,提取中间纤维的蛋白成份。蛋白经SDS聚丙烯酰胺凝胶电泳后,用动物细胞角蛋白的单克隆抗体进行免疫印迹反应,主要显示了分子量为52kD和64kD两种多肽成份。同时,提取玉米和烟草的DNA与α-~(32)P标记的兔K_3cDNA片段RB_3进行点杂交,均呈现较强的阳性反应,说明它们的基因组DNA中存在角蛋白基因的同源序列。     

四膜虫细胞的核骨架及类中间纤维

采用非树脂包埋去包埋剂超薄切片结合选择性生抽提方法显示,原生动物四膜虫细胞大核具有发达的核骨架纤维网络,核周是一层完整的核纤层结构,在四膜虫细胞小核中,亦存在核骨架和核纤层。四膜虫细胞皮层中存在水下溶性纤维网架,其中含有类中间纤维蛋白组分,４９ＫＤ蛋白。     

The intermediate filament-lamina-nuclear matrix system of BHK-21 cells

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diameter. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina. Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there are lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly presented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.     

Human epithelial cell intermediate filaments: isolation, purification, and characterization

Intermediate filaments (IF) isolated from human epithelial cells (HeLa) can be disassembled in 8 M urea and reassembled in phosphate-buffered solutions containing greater than 0.1 mg/ml IF protein. Eight proteins were associated with HeLa IF after several disassembly-reassembly cycles as determined by sodium dodecyl sulfate gel electrophoresis (SDS PAGE). A rabbit antiserum directed against HeLa IF contained antibodies to most of these proteins. The immunofluorescence pattern that was seen in HeLa cells with this antiserum is complex. It consisted of a juxtanuclear accumulation of IF protein and a weblike array of cytoplasmic fibers extending to the cell border. Following preadsorption with individual HeLa IF proteins, the immunofluorescence pattern in HeLa cells was altered to suggest the presence of at least two distinct IF networks. The amino acid composition and alpha-helix content (approximately 38%) of HeLa IF proteins was similar to the values obtained for other IF proteins. One-dimensional peptide maps show extensive homology between the major HeLa IF protein of 55,000-mol- wt and a similar 55,000-mol-wt protein obtained from hamster fibroblasts (BHK-21). HeLa 55,000-mol-wt homopolymer IF assembled under conditions similar to those required for BHK-21 55,000-mol-wt homopolymers. Several other proteins present in HeLa IF preparations may be keratin-like structural proteins. The results obtained in these studies indicate that the major HeLa IF protein is the same major IF structural protein found in fibroblasts. Ultrastructural studies of HeLa cells revealed two distinct IF organizational stages including bundles and loose arrays. In addition, in vitro reconstituted HeLa IF also exhibited these two organizational states.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

植物细胞中间纤维角蛋白性质的分析

角蛋白是植物细胞中间纤维的主要成分。应用选择性抽提和生物化学技术,分离纯化了豌豆根尖细胞58-、52 kD、白菜子叶52kD和胡萝卜悬浮细胞64kD角蛋白,测定了它们的氨基酸组成,结果表明上述角蛋白与动物细胞中间纤维角蛋白的氨基酸组成有较大的相似性。比较了动、植物细胞角蛋白的肽谱,结果显示它们之间存在较大的差异,但是植物细胞间角蛋白的肽谱比较一致,这提示它们属于同一蛋白家族,为植物中间纤维及其角蛋白的存在提供了新的论据。     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin

As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.     

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M _r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations M _r apparent molecular mass - Da dalton - Ig immunoglobulins - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - 2-D gel two-dimensional gel electrophoresis - 2,4-D 2,4-dichlorophenoxyacetic acid     

Cellular distribution of cholesterogenesis-linked, phosphoisoprenylated proteins in proliferating cells

A set of isoprenylated proteins has been detected in rapidly proliferating, suspension-grown murine lymphoma cells. Our evidence indicates that all of these isoprenylated proteins are phosphorylated. Subsequent to a 24 h incubation with mevinolin to deplete the intracellular mevalonate (MVA) level, cells were incubated with [3H]MVA and/or 32Pi and both total cell and subcellular fraction proteins were resolved via 1- and 2-D gel electrophoresis, then assessed via subsequent autoradiography. The phospho-isoprenylated proteins comprise a set spanning a molecular mass range of 21-69 kDa and all dispay acidic pI. MVA-derivatized proteins of 21-24 kDa, which consist of multiple isoforms, are present in both cytosolic and nuclear fractions. Larger phospho-isoprenylated protein species (44-69 kDa) are specifically localized within the nucleus, where applicable extraction protocols indicate that they are part of or closely affiliated with the nuclear matrix-intermediate filament (NM-IF) components. The localization of the 69 kDa prenylated species within the NM-IF fraction, together with evidence of its phosphorylation, supports recent indications that this protein is the nuclear matrix component lamin B.     

衣藻(Chlamydomonas sp)中间纤维及核纤层存在的证实(简报)

衣藻(Chlamydomonas sp)是属于绿藻门的最低等单细胞植物,为典型的真核生物。迄今以衣藻为材料所作的有关细胞骨架方面的研究多集中在微管蛋白(tubulin)。C.J.Miller等曾以衣藻(Chlamydomonas reinhardtii)全蛋白与几种中间纤维抗体进行免疫印迹实验有阳性反应,但是衣藻中是否存在中间纤维与核纤层是不清楚的问题。衣藻中间纤维与核纤层的形态研究更未见报道。目前认为中间纤维-核纤