Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells

The monoclonal antibody (anti-IFA) raised (Pruss et al., 1981, Cell 27:419-428) against an intermediate filament antigen, which is widespread throughout phylogeny, has been shown here to cross-react with higher plants. On immunoblotting, anti-IFA cross-reacted with proteins in homogenates of carrot suspension cells and of meristematic cells from onion root tips. A 50-kD cross-reactive protein was enriched in a fraction that consisted of detergent-insoluble bundles of 7-nm fibrils from carrot protoplasts (Powell et al., 1982, J. Cell Sci. 56:319-335). By use of indirect immunofluorescence, anti-IFA stained formaldehyde-fixed onion meristematic cells and carrot protoplasts in patterns approximating those obtained with monoclonal anti-tubulins. That anti-IFA was not recognizing plant tubulins was established by use of immunoblots of two-dimensional gels on which the proteins that comprised isolated fibrillar bundles and taxol-purified carrot tubulins had been separated. The two groups of proteins had different positional coordinates: anti-IFA recognized the fibrillar bundle proteins, and anti-tubulins recognized plant microtubule proteins with no cross-reaction to the heterologous proteins. Likewise, formaldehyde-fixed taxol microtubules from carrot cells could be stained with anti-tubulin but not with anti-IFA. It is concluded that an epitope common to intermediate filaments from animals co-distributes with microtubules in higher plant cells.     

Neuron-like derivatives of cultured F9 embryonal carcinoma cells express characteristics of parietal endoderm cells

Murine F9 embryonal carcinoma cells exposed to retinoic acid and dibutyryl cyclic AMP gradually arborize and acquire a neuron-like morphology in monolayer culture. Whether F9 cells can be induced to differentiate into cells with features specific to neural cells is controversial. We analyzed the intermediate filament content and pericellular matrix proteins of F9 cells after exposing them to retinoic acid, dibutyryl cyclic AMP, and nerve growth factor. In long-term cultures, a great majority of the cells appeared neuron-like, but showed intra- and pericellular laminin and type IV collagen, and frequently cytokeratin filaments as well. Several monoclonal antibodies to neurofilaments did not react with these cells in immunofluorescence or immunoblotting, though they recognize either all or individual mouse neurofilament triplet proteins. Polyclonal antibodies to neurofilament proteins gave a diffuse, nonfibrillar, vinblastine-resistant fluorescence in the morphologically neuron-like cells, but in immunoblotting failed to reveal polypeptides compatible with neurofilament triplet proteins. In long-term cultures, most of the cells appeared to have partially or totally lost the intermediate filaments. This was confirmed with anti-IFA antibodies which normally react with all intermediate filament proteins. The F9-derived cells did not respond to nerve growth factor in any detectable way. We conclude that the morphologically neuron-like derivatives of F9 cells display characteristics of modified parietal endoderm-like cells and do not show unequivocal features of neural cells.     

Novel filamentous bundles in the cytoplasm of a unicellular eukaryote,Crithidia fasciculata

Summary Trypanosomes, an evolutionarily ancient group of unicellular eukaryotic parasites, appear to lack both microfilaments (actin) and intermediate filaments (IFs): the major cytoskeletal component common to all trypanosomes consists of a stable microtubular array intimately associated with the plasma membrane. We present here evidence of bundles of trans-cytoplasmic filaments ca. 10 nm in diameter, seen by transmission electron microscopy, that are formed in stationary cultures of an insect trypanosome,Crithidia fasciculata. Immunofluorescent labelling with an antibody raised against plant fibrillar bundles (AFB) and Western blotting with an antibody that cross-reacts with a broad range of IFs (anti-IFA) as well as with fibrillar bundles, indicates that these filaments appear to share antigenic determinants common to animal IFs and to fibrillar bundles of plant origin.Abbreviations AFB anti-fibrillar bundle antibody - anti-IFA anti-intermediate filament antibody - IF intermediate filament - SEM scanning electron microscope - TEM transmission electron microscope - YOL 1/34 anti--tubulin antibody     

Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes.

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.     

Evidence for intermediate filaments in squirrelfish erythrophores of Holocentrus ascensionus (Rufus)

We have documented the presence of intermediate filaments (IF) in cultured erythrophores of the squirrelfish Holocentrus ascensionus (Rufus). SDS-PAGE and Western blots with monoclonal antibodies T11 and R12 demonstrated that isolated IF consisted of a pair of polypeptides of 54 and 52 kDa. Immunofluorescent studies revealed that the two proteins formed prominent radially oriented IF networks in erythrophores. Immunoelectron microscopic studies showed that the IF were distributed in a "spider-web"-like network of filaments which occasionally intersected with the microtubule surfaces. The IF proteins also were found in fish iridiphores but not in fish epithelial cells which cocultured with the chromatophores.     

Immunodetection of spectrin antigens in plant cells

The occurrence of spectrin in plant cells was studied by immunoblotting of extracts, and its localization by immunolabelling of cells, using two polyclonal antibodies raised against spectrin from human and chicken erythrocytes. A variety of plant cells were studied. The two antibodies gave the same results on blots as well as on cells. Western blots of extracts showed weak immunolabelling at 220 kD, where spectrin can be expected, but bands at 85 kD stained more heavily. Because the latter bands were also seen on blots with commercially purified spectrin, we conclude that they were breakdown products of spectrin. Native plant extracts on blots from IEF gels showed a band at pI 4.8, where the purified animal spectrin is also found. Immunolocalizations done on whole cells, PEG-, BMM-, or cryo-sections gave similar data. In most cells the labelling was localized predominantly at the plasma membrane, especially of thin-walled cells. Labelling was also seen in the periphery of a particular class of organelles, probably plastids. This labelling was tissue specific in maize somatic embryos. During carrot somatic embryogenesis cytoplasmic labelling was observed depending on the developmental stage. Many cells with cytoplasmic labelling also had stained nuclei. Labelled nuclei had more condensed chromatin than non-labelled nuclei.     

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     

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.     

Monoclonal antibodies to intermediate filaments in chick muscle cell cultures

Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 degrees C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.     

Regeneration and post-metamorphic development of the central nervous system in the protochordate Ciona intestinalis: a study with monoclonal antibodies

In this study, we use three monoclonal antibodies that recognise antigens present in the central nervous system of the ascidian Ciona intestinalis to study regeneration and post-metamorphic development of the neural ganglion. We have also used bromodeoxyuridine labelling to study generation of the neuronal precursor cells. The first antibody, CiN 1, recognises all neurones in the ganglion, whereas the second, CiN 2, recognises only a subpopulation of the large cortical neurones. Western blotting studies show that CiN 2 recognises two membrane-bound glycoproteins of apparent Mr 129 and 100 kDa. CiN 1 is not reactive on Western blots. Immunocytochemical studies with these antibodies show that CiN 1-immunoreactive neurone-like cells are present at the site of regeneration as early as 5–7 days post-ablation, a sub-population of CiN 2-immunoreactive cells being detected by 9–12 days post-ablation. The third antibody, ECM 1, stains extracellular matrix components and recognises two diffuse bands on Western blots of whole-body and ganglion homogenates. The temporal and spatial pattern of appearance of CiN 1 and CiN 2 immunoreactivity both during post-metamorphic development and in regeneration occurs in the same sequence in both processes. Studies with bromodeoxyuridine show labelled nuclei in some neurones in the regenerating ganglion. Plausibly these originate from the dorsal strand, an epithelial tube that reforms by cell proliferation during the initial phases of regeneration. A second population of cells, the large cortical neurones, do not incorporate bromodeoxyuridine and thus must have been born prior to the onset of regeneration. This latter finding indicates a mechanism involving trans-differentiation of other cell types or differentiation of long-lived totipotent stem cells.     

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.     

Binding of Bodian''s Silver and Monoclonal Antibodies to Defined Regions of Human Neurofilament Subunits: Bodian''s Silver Reacts with a Highly Charged Unique Domain of Neurofilaments

Cleavage at cysteine and chymotrypsin digestion were applied to two human neurofilament (NF) subunits, low- and high-molecular-weight NF (NF-L and NF-H), to locate the regions reacting with Bodian's silver stain and with several monoclonal antibodies, including NF-specific antibodies and one that recognizes all intermediate filaments (anti-IFA). Our findings indicate that whereas anti-IFA recognizes the highly conserved rod domain, all the NF-specific antibodies, as well as Bodian's silver, react with the carboxy-terminal tailpiece of NF subunits. The silver binding sites in NF-L are located in a carboxy-terminal 12-Kd chymotrypsin fragment, a highly charged, unique domain of NF.     

Spectrin-like proteins in green algae (Desmidiaceae)

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.     

Localization of a myosin‐like protein to plasmodesmata

Myosin has been localized to plasmodesmata in root tissues of Allium cepa, Zea mays and Hordeum vulgare using a polyclonal antibody to animal myosin in both fluorescence and electron microscopy. Labelling was also observed throughout the cytoplasm, mainly associated with the endoplasmic reticulum and plasma membrane. On Western blots, bands of 180 and 110 kDa were consistently labelled in all three species. These bands were also labelled when the blot was incubated in actin prior to staining with antibodies to actin, raising the possibility that either of these proteins (180 kDa or 110 kDa) may be present in plasmodesmata. Pre-treatment of the tissue with 2,3-butanedione monoxime (BDM), an inhibitor of actin–myosin motility, resulted in a strong constriction of the neck region of plasmodesmata. These results indicate that a myosin-like protein may be present in plasmodesmata and may also play a role in the regulation of transport at the neck region.     

The Plant Nucleoskeleton: Ultrastructural Organization and Identification of NuMA Homologues in the Nuclear Matrix and Mitotic Spindle of Plant Cells

In the present work we investigate the structural organization of the nucleoskeleton ofAllium cepameristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.     

Reconstitution of intermediate filaments from a higher plant.

Immunological studies have shown that plants contain intermediate-filament antigens, but it is not known whether these proteins are capable in themselves of forming filaments. To address this problem, a detergent-resistant and high-salt-insoluble fraction from carrot (Daucus carota L.) suspension cells was solubilized with 9 M-urea and then subjected to a two-step dialysis procedure, devised for the reconstitution of animal intermediate filaments. This induced the self-assembly of 10 nm filaments and large bundles of filaments. The predominant components of reconstituted material were polypeptides with apparent molecular masses between 58 and 62 kDa. These polypeptides immunoblotted with two monoclonal antibodies known to show broad cross-reactivity with intermediate filaments across the phylogenetic spectrum. This establishes that the antigens are able to self-assemble into intermediate-sized filaments.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.     

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。     

Study of Intermediate Filaments in Adiantum philippense and Comparative Analysis of Karetin-like Proteins in Some Plant Species

Network of filaments, 10 nm in diameter, was detected in the frond cells of Adiantum philippense L. by selective extraction combined with whole mount electron microscopy. Western blot analysis showed that the major filament components were cross-reacted with monoclonal antibodies against animal keratin. The phenomenon was in concert with the result of indirect immunofluoreseenee observation. The filaments with 10 nm in diameter could be reassembled in vitro. Using selective extraction, proteins were prepared from cells of Spirulina subtillissima Kutz., thalli of Marchantia polymorpha L., and leaf blades of A. philippense L. , Ginkgo biloba L. and Brassica pekinensis Rupr. Western blot showed that S. subtillissima Kutz. has two acidic keratin-like pro- teins and all of the other four plant species have three basic keratin-like proteins and three acidic keratin-like proteins. These results may suggest the existance of an intermediate filament network with keratin-like proteins in the cells of pteridophyte and their existance in plant cells is popular.     

Polyclonal and monoclonal antibodies to human lysyl hydroxylase and studies on the molecular heterogeneity of the enzyme.

Human placental lysyl hydroxylase gave two bands in SDS/polyacrylamide-slab-gel electrophoresis: a broad, diffuse, major band corresponding to an apparent Mr of 80,000-85,000, and a sharp minor band with Mr 78,000. Mouse and chick-embryo lysyl hydroxylases gave only the broad, diffuse band, whereas the sharp band could not be detected. Polyclonal antibodies were prepared to the two bands of the human enzyme separately, and monoclonal antibodies were prepared to the whole purified enzyme preparation. Both types of polyclonal antibody inhibited and precipitated the enzyme activity, and both stained the two polypeptide bands in immunoblotting after SDS/polyacrylamide-gel electrophoresis. Only one out of five monoclonal antibodies inhibited the enzyme activity, whereas they all precipitated the activity when studied with antibody coupled to Sepharose. All five monoclonal antibodies stained the whole broad band in immunoblotting, and at least three of them also stained the sharp band. Peptide maps produced from the two polypeptide species by digestion with Staphylococcus aureus V8 protease were highly similar. Experiments with endoglycosidase H demonstrated that the Mr-80,000-85,000 polypeptide contains asparagine-linked carbohydrate units, which are required for maximal lysyl hydroxylase activity. The data suggest that the lysyl hydroxylase dimer consists of only one type of monomer, the heterogeneity of which is due to differences in glycosylation.