Filament proteins in rat optic nerves undergoing Wallerian degeneration: localization of vimentin, the fibroblastic 100-A filament protein, in normal and reactive astrocytes.
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Animals↗
Biomedical subjects
K Weber
Publications and source records attributed to K Weber.
Conditions for pox virus-specific microvilli formation studied during synchronized virus assembly.
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Animals↗
Acyl bis(monoacylglycero)phosphate, assumed to be a marker for lysosomes, is a major phospholipid of vaccinia virions.
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Chemical Phenomena↗
F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end.
We have studied the mechanism of Ca++-dependent restriction of actin filament length by villin, one of the major actin-associated proteins of intestinal microvilli microfilament bundles. Villin acts, even at a ratio of 1 to 1000 with respect to actin, very efficiently as a Ca++-dependent nucleation factor on actin assembly. This gives rise to unidirectional assembly, with the morphologically defined "barbed" end of the resulting filament being capped. Consequently, at steady state treadmilling of actin monomers through the filament is inhibited. Increase of the villin-to-actin ratio enhances the number of nucleated filaments necessarily shorter in length. This results finally in nonsedimentable F actin and a low molecular weight complex of one villin and three monomeric actins, which itself is a potent nucleator. Thus restriction of actin assembly by villin is not due to a direct inhibition of assembly but arises as the consequence of strongly enhanced nucleation followed by unidirectional elongation at the pointed end of the nucleated filaments. In addition, in the presence of Ca++-villin, but not the villin-actin complex, seems able to "break" or "sever" preformed F actin filaments. Thus a variety of cellular phenomena-nucleation, unidirectional assembly, filament end capping, nonpolymerizable actin and F actin bundles-can be observed in vitro in a two-protein component system modulated by the concentration of free Ca++.
Actins↗
Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin.
Smooth muscle cells of the digestive, respiratory, and urogenital tracts contain desmin as their major, if not exclusive, intermediate-size filament constituent and also show a predominance of gamma-type smooth muscle actin. We have now examined smooth muscle tissue of different blood vessels (e.g., aorta, small arteries, arterioles, venules, and vena cava) from various mammals (man, cow, pig, rabbit, rat) by one- and two-dimensional gel electrophoresis of cell proteins and by immunofluorescence microscopy using antibodies to different intermediate-sized filament proteins. Intermediate-sized filaments of vascular smooth muscle cells contain abundant amounts of vimentin and little, if any, desmin. On gel electrophoresis, vascular smooth muscle vimentin appears as two isoelectric variants of apparent pI values of 5.30 and 5.29, shows the characteristic series of proteolytic fragments, and is one of the major cell proteins. Thus vimentin has been demonstrated in a smooth muscle cell present in the body. Vascular smooth muscle cells are also distinguished by the predominance of a smooth muscle-specific alpha-type actin, whereas gamma-type smooth muscle actin is present only as a minor component. It is proposed that the intermediate filament and actin composition of vascular smooth muscle cells reflects a differentiation pathway separate from that of other smooth muscle cells and may be related to special functions and pathological disorders of blood vessels.
Actins↗
Calcium control of microfilaments: uncoupling of the F-actin-severing and -bundling activity of villin by limited proteolysis in vitro.
Villin is a major F-actin-bundling protein present in the microfilament bundle underlying the plasma membrane of the microvilli present on intestinal epithelial cells. Mild in vitro proteolysis converts villin (Mr, 95,000) into a large fragment, the villin core (apparent Mr, 90,000). Villin core has lost the F-actin-bundling activity expressed by villin in the absence of calcium but retains the micromolar Kd calcium-binding site and the calcium-dependent restriction of actin filament length (F-actin severing) of intact villin. This finding suggests a common structural and functional relatedness between the known calcium-dependent F-actin-severing proteins from different cell types, even though not all of them reveal F-actin-bundling activity.
Actins↗
Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: desmin and vimentin.
Although all intermediate-size filaments (10-nm filaments) seem to show similar morphology and share a number of biochemical properties, different cell- and tissue-specific subclasses have been distinguished by immunological experiments and by differences in apparent molecular weights and isoelectric points of the major constituent proteins. In order to understand the degree of possible homology between these proteins, we have begun amino acid sequence analysis of the polypeptides. Here we characterize a large fragment of chicken gizzard and pig stomach desmin as well as the corresponding fragment from porcine eye lens vimentin. The fragments are situated at the carboxyl end and consist of 138-140 amino acid residues--i.e., some 28% of the corresponding polypeptide chains. The results show that the two immunologically distinct porcine proteins are different gene products. They show a related amino acid sequence but differ in 36% of the residues present in the carboxy-terminal region. Thus tissue specificity overrides species divergence. These results are discussed in the light of previous immunological experiments. They lend further support to the hypothesis that intermediate filaments belong to a multigene family, which is expressed in line with certain rules of differentiation during embryogenesis.
Amino Acid Sequence↗
Localization and organization of actin in melanophores.
Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.
Actins↗
Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.
Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre-prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.
Chromosomes↗
Expression of glial and vimentin type intermediate filaments in cultures derived from human glial material.
Several cultures established from biopsies of apparently normal adult human glial material showed no cells positive for glial fibrillary acidic protein (GFA) when examined after seven or more cumulative population doublings (CPD), although the established glioma line U251 MG showed approximately 3% GFA-positive cells, and U333 CG/343 MG clone 3 showed greater than 98% GFA-positive cells. Both the human glia delivered cultures and the glioma lines were positive when assayed with sera specific for vimentin. We therefore investigated the expression of GFA as a function of cumulative population doublings after the establishment of primary cultures. Under our experimental conditions, although GFA-positive cells were clearly present in the primary cultures accounting for some 3%-14% of the cells present, the GFA marker was subsequently lost, and the proliferating cultures expressed only the vimentin type of intermediate filament. Those cells that were GFA-positive also appeared to be vimentin-positive. GFA expression was not reinduced in cultures that had lost the GFA marker by treatment with dibutyryl cyclic AMP. We discuss two alternate hypotheses for the origin of the GFA-negative cells: (1) the cultures area of astrocyte origin but lost the ability to express GFA on culturing; (2) the cultures originate from cells of nonastrocyte origin present in the primary material.
Cell Division↗
Heterogeneity of intermediate filament expression in vascular smooth muscle: a gradient in desmin positive cells from the rat aortic arch to the level of the arteria iliaca communis.
The display of the two distinct intermediate filament proteins, desmin and vimentin, in rat vascular smooth muscle tissue was studied by immunofluorescence microscopy on frozen sections of aorta and other blood vessels. Vascular smooth muscle cells present in these vessels always appeared rich in vimentin. However, staining of sections covering six distinct but contiguous parts of the aorta showed that the number of desmin containing cells was low distal to the truncus brachiocephalicus, but increases until in distal parts of the aorta and in the arteria iliaca communis almost all cells appear positive for desmin. Thus blood vessels show heterogeneity of intermediate filament expression not only in cross-section but can also display heterogeneity along their length. Muscular arteries such as the renal artery femoralis, as well as arterioles and veins including the vena jugularis and the vena cava also contain desmin. Thus it may be that low numbers of desmin-positive cells are typical of elastic arteries, while muscular arteries and other blood vessels are characterized by large numbers of desmin-positive cells. We discuss whether desmin-positive and desmin-negative vascular smooth muscle cells may perform functions and raise the possibility that desmin expression may coincide with the turn on of a specially regulated contractility program.
Animals↗
Actin typing on total cellular extracts: a highly sensitive protein-chemical procedure able to distinguish different actins.
Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactivity labeled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actions in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic action present in Schneider L-2 Drosophila melanogaster cells. Although the method is currently used routinely on 10(5) cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.
Actins↗
Characterization of intracellular and extracellular vaccinia virus variants: N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine interferes with cytoplasmic virus dissemination and release.
Infectious vaccinia virus can be purified from whole cells by experimentally induced lysis (intracellular virus) or from supernatant growth medium (extracellular virus). Extracellular virus and intracellular virus differed by buoyant density (1.237 versus 1.272 g/cm3), phospholipid content and composition, and polypeptide pattern. Differences in structural polypeptides on the virus surface could be detected by lactoperoxidase-catalyzed radioiodination or Brij treatment. Characteristic of extracellular virus was an additional polypeptide, with a molecular weight of 37,000 (37K), which represented 5 to 7% of the total particle protein. Antibodies to the 37K protein detected only some of the cell-associated particles late in normal infection. Upon treatment of infected cultures with N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine, a drug which prevents vaccinia virus release, no particle-associated 37K protein could be detected. In all other properties tested so far, except for a slight difference in phospholipid composition, the virus obtained in the presence of the drug resembled the normal intracellular virus. N1-Isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine prevented vesicularization of intracellular viral particles. Lack of vesicularization was accompanied by the absence of particle-associated 37K viral protein and seemed to correlate with an inhibition of virus dissemination to the cell periphery.
Animals↗
Serological study with rickettsial antigens in erythema chronicum migrans.
21 sera of 13 patients with erythema chronicum migrans (ECM) and related diseases were examined for the presence of antibodies against 14 rickettsial antigens by using 3 serological techniques in 2 laboratories. Most tests were negative. This indicates that ECM and related diseases are not due to an hitherto known rickettsia. 5 out of 158 tests showed, however, positive reactions in low titers against Rickettsia akari and Coxiella burnetii. Thus, an antigenic relationship may exist between one or more rickettsia(e) and the etiologic agent of ECM.
Antibodies, Bacterial↗
Ultrastructure of multiple microtubule initiation sites in mouse neuroblastoma cells.
Morphologically undifferentiated and differentiated mouse neuroblastoma N115 and N18 cells were examined after serial sectioning by electron microscopy. A sizeable percentage of the cells revealed multiple centrioles, usually clustered together in the perinuclear area with 2 preferential locations, i.e. above and below the largest nuclear diameter. These results indicate that the multiple microtubule-organizing centres previously visualized by immunofluorescence microscopy with tubulin antibody in neuroblastoma cells recovering from Colcemid poisoning are most likely in majority related to multiple centrioles. This interpretation is further strengthened by experiments in which cells are first recorded in the fluorescence microscope and then after serial sectioning in the electron microscope. The results show that under optimal conditions immunofluorescence microscopy is able to visualize single centrioles. The possible biological significance of the combined electron and immunofluorescence microscopical results is discussed.
Animals↗
Treatment of Lyme disease.
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Anti-Bacterial Agents↗
Antibodies to different intermediate filament proteins. Cell type-specific markers on paraffin-embedded human tissues.
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Animals↗
Arrangement of neurofilaments, microtubules and microfilament-associated proteins in cultured dorsal root ganglia cells.
Using the indirect immunofluorescence technique, a study was made of the distribution of the major cytoskeletal proteins in cultures of cells derived from chicken embryo and newborn rat dorsal root ganglia. An antibody raised against the 200 000 molecular weight neurofilament "triplet" polypeptide isolated from rat sciatic nerve strongly stained some, but not all neurones in these cultures. In contrast filamin and vimentin antibodies stained Schwann cells and fibroblasts but not neurones. Antibody to fibronectin only stained material associated with fibroblasts. Thus these four antibodies can be used to distinguish between neurones, Schwann cells and fibroblasts, as well as to detect a heterogeneity in the neuronal population. In addition these antibodies, plus antibodies to actin, myosin, alpha-actinin, tropomyosin, fimbrin and tubulin allow a more detailed description of the cytoskeleton of cultured neurones at the light microscopic level.
Animals↗