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。     

The Existence of Intermediate Filament in Arabidopsis thaliana Cell

The network structure of cytoplasmic filaments of 10 nm in diameter was detected from callus cells of Arabidopsis thaliana (L.) Heynh by selective extraction combined with whole mount electron microscopy. Western blot analysis showed that the major filament components were 6 polypeptides, which reacted with keratin monoclonal antibody of AE1 or AE3 respectively. By indirect immunofluorescence technique, the AE1 and AE3-reactive antigens were localized throughout the cytoplasm in a diffused pattern. The 10 nm-plant filaments could be reassembled in vitro. These results demonstrated that keratin-like intermediate filaments exist in the cytoplasm of A. thaliana. Using conservative sequence of animal IF genes as primer, a cDNA fragment was further cloned from this model material by RT-PCR, which might shed more light on molecular characterization of IF existence in higher plant.     

拟南芥细胞中存在中间纤维的研究

利用整装电镜制样与选择性抽提技术,在拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ （Ｌ．） Ｈｅｙｎｈ） 愈伤组织细胞质中观察到直径１０ ｎｍ 左右的纤维网络结构。免疫印迹分析表明纤维的主要成分是６ 种多肽,它们分别与动物角蛋白单克隆抗体ＡＥ１ 、ＡＥ３ 有免疫交叉反应。利用间接免疫荧光技术,与ＡＥ１ 和ＡＥ３ 反应的抗原呈弥散状定位于整个细胞质中,而且１０ ｎｍ 纤维可以在体外重新组装。以上结果表明,在拟南芥细胞质中存在类角蛋白的中间纤维。以动物中间纤维基因的保守序列为引物,采用ＲＴ＿ＰＣＲ技术,进一步从这一模式植物中克隆到一个ｃＤＮＡ片段,这可能为从分子水平上证明植物中间纤维的存在提供了一个线索     

The keratin intermediate filament—like system in maize protoplasts

The application of Penman‘s method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.     

Fine structure and assembly in vitro of nuclear lamina in plant cells

Ｔｈｅｎｕｃｌｅａｒｌａｍｉｎａ(ＮＬ)ｉｎａｎｉｍａｌｃｅｌｌｓｉｓａｍｅｓｈｗｏｒｋｓｔｒｕｃｔｕｒｅｃｏｍｐｏｓｅｄｏｆｉｎｔｅｒｍｅｄｉａｔｅｆｉｌａｍｅｎｔｐｒｏｔｅｉｎｓ,ｔｅｒｍｅｄｌａｍｉｎｓ.Ｉｔｕｎｄｅｒｌｉｅｓｔｈｅｉｎｎｅｒｎｕｃｌｅａｒｍｅｍｂｒａｎｅａｎｄｃｏｎｆｅｒｓｍｅｃｈａｎｉｃａｌｓｔａｂｉｌｉｔｙｔｏｔｈｅｎｕｃｌｅａｒｅｎｖｅｌｏｐｅ[1].Ｉｎａｄｄｉｔｉｏｎ,ａｎｕｍｂｅｒｏｆｐｕｔａｔｉｖｅｒｏｌｅｓｈａｖｅｂｅｅｎｓｕｇｇｅｓｔｅｄｆｏｒｌａｍｉｎｓｂｏｔ…     

The Keratin-Like Intermediate Filament Network Exists in the Chlamydomonas sp.Pyrenoid

The 10 nm diameter filement network in the Chlamydomonas pyrenoid was investigated using selective extraction and diethylene glycol distearate embedding-free electron microscopy. The pyrenoid was cross reacted with animal keratin-antibody using electron microscopic immunogold labeling. Indirect immunofluorescence observation showed the same result. These suggest that a keratin-like intermediate filament network does exist in the Chlam ydomonas pyrenoid.     

Fine structure and assemblyin vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro.     

不同条件对植物中间纤维体外装配的影响(简报)

细胞骨架的研究是当今细胞生物学中最为活跃的领域之一,而中间纤维是三种主要骨架纤维中研究较少的一种。从60年代发现至今,人们对动物细胞中间纤维的研究已经比较深入,近来又发现它在基因表达等重要生命活动中起一定的作用。中间纤维有一个显著的特征,就是能够在体外进行自我装配,不需要核苷酸和结合蛋白参加,也不依赖于蛋白质的浓度。植物细胞中是否存在中间纤维一直是未解决的问题。从80年代起,有一些研究发现在高等植物细胞中存在能与动物细胞中间纤维抗体进行     

Fine structure and assembly in vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro. Project supported by the National Natural Science Foundation of China (Grant No. 3500073).     

Assembly characteristics of plant keratin intermediate filamentsin vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments. Project supported by the National Natural Science Foundation of China (Grant No. 39370352) and the Doctor Foundation of Minishy of Education of China.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

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

Nuclear matrix (NM) and intermediate filament (IF) scaffold in primitive eukaryote Crypthecodinium cohnii were shown using selective extraction together with embedment-free electron microscopy, whole mount cell preparation and immunoblot techniques. There exists a delicate NM-IF network spreading over cytoplasm and nucleus in dinoflagellate cells, however, nuclear lamina is undeveloped. The diameter of NM fiber is about 3-5 nm and IF is 10 nm. Chromosomes are connected with NM filament network. Immunoblot analysis showed that dinoflagellate contained keratin-like polypeptides (63 kD and 67 kD) while mammalian lamin antibodies did not crossreact with dinoflagellate total protein. Our experiment results demonstrated that a framework similar to NM-IF scaffold in mammalian cell appeared in primitive eukaryote. We propose that: (1) NM-IF scaffold is not restrict to vertebrate cell, and it may be originated from early stages of eukaryote evolution; (2) Keratin is probably very conservative; (3) Compared with IF, lamina might appear late in evolution, and some of primitive characteristics of dinoflagellate nucleus may be related to the lack of lamina.     

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

Summary In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with M_rs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the -IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.Dedicated to Professor Sir Henry Harris on the occasion of his 70th birthday     

Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Daucus carota L.,and their partial amino acid sequences were determined.The homological analysis showed that the sequence from the 64 ku protein was highly homological to b -glucosidase,and that from the 55 ku protein had no significant homologue in GenBank.Using conservative sequence of animal IF proteins as primer,we cloned a cDNA fragment from Daucus carota L.Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves.Homological analysis revealed that it had moderate homology to a variety of a -helical proteins.Our results might shed more light on molecular characterization of IF existence in higher plant.     

Intermediate filament protein partnership in astrocytes.

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.     

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2–4 nm diameter, and intermediate filament protein is included in numerous, variously sized (2–15 μm) spheroidal aggregates containing densely stained granular particles of 5–16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.     

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Immunocytochemical demonstration of a new vimentin-associated protein in 3T3 fibroblasts

Summary Using a xanthophore cytoskeletal preparation as immunogen, we have produced a monoclonal antibody, A2, which recognized a 160 kDa protein in 3T3 fibroblasts. This protein makes up a cytoplasmic filamentous system, which colocalizes with vimentin filaments. When microtubules and actin filaments are dissolved by high salt extraction, staining with antibody A2 is unaffected. Immunoblot analysis confirms that the 160 kDa protein is co-isolated with vimentin duringin vivo high salt extraction. Following vinblastine treatment, both the 160 kDa protein and vimentin become localized to perinuclear caps, as do other intermediate filaments and their associated proteins; after vinblastine removal, the immunostaining produced by A2 becomes filamentous. Immunoelectron microscopy demonstrates that antibody A2 stains a filament system with a diameter of about 10 nm. Our observations suggest that the 160 kDa protein may be a new vimentin-associated protein which differs from the intermediate filament-associated proteins previously reported, and is widely distributed in several cell types.     

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin‐like proteins

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled‐coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross‐react with anti‐intermediate filament and anti‐lamin antibodies, form filaments 6–12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin‐like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin‐like proteins by co‐immunoprecipitation and co‐localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with M_r and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin‐like proteins. Its similarities with some of the proteins described as onion lamin‐like proteins suggest that they are highly related or perhaps the same proteins.