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Biomedical subjects

B M Jockusch

Publications and source records attributed to B M Jockusch.

At least 91 records · Page 5Linked to original sources
PubMed1987

Labeling of structural elements at the ventral plasma membrane of fibroblasts with the immunogold technique.

We investigated the organization of two structural elements (microfilaments and clathrin coated structures) at the ventral plasma membrane in fibroblasts. The methods employed included exposure of the inner face of the ventral plasma membrane by lysis-squirting, labeling the exposed structures with specific antibodies conjugated with colloidal gold, and platinum-carbon replica techniques. The results demonstrate that this regimen combines good ultrastructural preservation with unambiguous identification of structural elements associated with the plasma membrane. This method is therefore reliable and can be generally used to explore in detail the ultrastructural organization of the plasma membrane.

Actin Cytoskeleton↗
PubMed1987

Differential response of stress fibers and myofibrils to gelsolin.

The actin-severing activity of human platelet gelsolin was analyzed on embryonic skeletal and cardiac myofibrils, and on stress fibers in non-muscle cells. These subcellular structures, although in all three cell types composed of contractile proteins arranged in sarcomeric units, were found to respond differently to gelsolin. The myofibrils in permeabilized myotubes or cardiac cells, as well as in living, microinjected muscle cells proved resistant to a wide concentration range of gelsolin. The same was found for the "mini-sarcomeres" which are seen in developing muscle cells. In contrast, stress fibers in microinjected fibroblasts or epithelial cells, as well as in permeabilized cells, were broken down rapidly by the platelet gelsolin. We conclude from these results that the mini-sarcomeres in embryonic myotubes and cardiac myocytes are not identical with stress fibers.

Actin Cytoskeleton↗
PubMed1986

Actin-severing activity copurifies with phosphofructokinase.

Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers). The mode of disruption as well as of recovery of stress fibers was very similar to that found previously in experiments performed with the actin-severing protein brevin, an extracellular variant of gelsolin. PFK, like brevin, was also capable of disrupting stress fibers in detergent-extracted cells and in ethanol-fixed cells, in a Ca2+-dependent manner. When compared with heart muscle gelsolin, PFK comigrated with the 85- to 90-kDa band. Antibodies against PFK crossreacted with gelsolin from the same species. These results point to a tight association between polypeptides with similar biochemical and immunological parameters present in both preparations. They suggest hitherto unexpected cellular control mechanisms for both microfilament functions and glycolysis.

Actin Cytoskeleton↗
PubMed1985

Disruption of microtubules in living cells and cell models by high affinity antibodies to beta-tubulin.

Polyclonal antibodies with high affinity for beta-tubulin were found to disrupt cytoplasmic microtubules efficiently after microinjection into tissue culture cells. The degree of microtubular fragmentation was directly proportional to the amount of the injected antibody. At molar ratios of 1 antibody per 100 tubulin dimers, most microtubules were disrupted within 90 min after injection. In contrast, the time course of disintegration was relatively independent of the antibody concentration. Within the range of 1 antibody per 10(2)-10(4) tubulin dimers, the maximal values for microtubular disintegration were reached approximately 1-1.5 h after injection. Mitotic microtubules were found to be resistant to all antibody concentrations used. In living cells, microtubules recovered within a few hours after antibody-induced decay. The time course of recovery, like the extent of disintegration, was a function of the antibody concentration. The antibody acted also on microtubules in detergent-extracted cell models and on microtubules polymerised in vitro. When added to microtubular protein, the bivalent antibody as well as its Fab fragments prevented polymerisation. The data suggest that these antibodies disrupt microtubules because their affinity to tubulin is at least 100 times higher than the affinities found for tubulin:tubulin interaction. Fragmented microtubules are probably unstable and decompose into smaller units.

Animals↗
PubMed1985

Vinculin and 36 kDa protein are not tyrosine-phosphorylated in Rous sarcoma virus infected cells which have been treated with interferon.

The expression of membrane-associated transformation-specific parameters was analyzed in de novo Rous sarcoma virus (strain SR-RSV-D) infected chicken embryo fibroblasts pretreated with homologous interferon. Cellular morphology, hexose transport, microfilament organization, and tyrosine-phosphate content of two primary substrates of the transformation-generating viral kinase, pp60src, were found indistinguishable from non-infected controls. These observations support the hypothesis that vinculin and possibly 36 kDa protein are involved in microfilament organization and that tyrosine-phosphorylation of these structural proteins is a prerequisite for the rearrangement of microfilaments during transformation. In de novo infection, interferon pretreatment reduces viral protein synthesis and pp60src activity as compared to non-treated, SR-RSV-D infected cells. However, the phosphotyrosine content of total cellular proteins as measured under steady state conditions is as high in interferon-pretreated as in nontreated transformed cells.

Animals↗
PubMed1984

Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes.

Nuclei of amphibian oocytes contain large amounts of actin, mostly in unpolymerized or short-polymer form. When antibodies to actin or actin-binding proteins (fragmin and the actin modulator from mammalian smooth muscle) are injected into nuclei of living oocytes of Pleurodeles waltlii, transcription of the lampbrush chromosomes, but not of the rRNA genes, is inhibited. When transcription is repressed by drugs or RNA is digested by microinjection of RNAase into oocyte nuclei, an extensive meshwork of actin filament bundles is seen in association with the isolated lampbrush chromosomes. These observations indicate a close relationship between the state of nuclear actin and transcriptional activity and suggest that nuclear actin may be involved in transcriptional events concerning protein-coding genes.

Actins↗
PubMed1984

Herpes simplex virus-induced cell surface protrusions.

Cell surface alterations following herpes simplex virus infection were studied by scanning electron microscopy at different times after infection of chick embryo fibroblasts and Vero cells. Beginning at 4 h, an increasing number of cells showed numerous microvillus protrusions 0.12-0.18 micron in diameter and 0.3 micron in length. These structures could also be stained on Vero cells with fluorescent phalloidin, indicating the presence of filamentous actin within them. At 12 h, the number of chick embryo fibroblasts with virus-induced microvillus-like structures began to decrease. There was an increase in virus titer in the medium beginning at 24 h, and it was therefore considered unlikely that the microvilli induced earlier had any function in virus release. Similar to poxvirus-induced microvilli formation, DNA and protein synthesis inhibitors prevented the induction of microvilli, indicating the involvement of a late viral function. N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine inhibited the formation of herpesvirus-induced microvilli in chick embryo fibroblasts but not in Vero cells. As this inhibitor had no effect on virus replication in either cell type, it is likely that the inhibition of microvillus formation was due to an anticellular activity of the drug.

Animals↗
PubMed1983

Cytoskeletal organization in locomoting cells of the V2 rabbit carcinoma.

The relationship between the organization of cytoskeletal elements and locomotory activity was studied in single cells of the V2 rabbit carcinoma. Like migratory fibroblasts, and unlike colony-forming epithelial cells, these cells show a pronounced horizontal polarization, and develop a large lamella at their leading front. With affinity-purified antibodies and a combination of light and electron microscopic techniques, actin and alpha-actinin (but not myosin and tropomyosin) were found highly concentrated within the marginal region of the leading lamella, both in ruffles and in the underlying zone of contacts with the substratum. Close contacts prevailed in the locomotory cells and small focal contacts developed only in cells detaching from others. Focal contacts always contained small microfilament bundles. Reorganization of actin filaments is suggested as the fundamental event for the dynamic contact formation of the leading lamella. Large microfilament bundles (stress fibers) were absent in all stages of locomotion. Since locomotory behavior and shape changes of V2 cells are the same on glass as on the surface of a natural membrane, the rabbit mesentery, organization and distribution of contractile elements of cultured V2 cells probably reflect the in vivo situation.

Actinin↗
PubMed1983

Lectin-resistant variants of mouse melanoma cells. II. In vitro characteristics.

Lectin-resistant variants of B-16 melanoma cells were selected with wheat-germ agglutinin, ricin and concanavalin A. They exhibited altered metastasizing capacity and tumorigenicity in C57BL mice. Several in vitro properties were defined and compared including homotypic adhesiveness, microfilament organization, melanin release, activity of tyrosine hydroxylase, DNA content, and karyotypes. The possible relevance of these properties in vitro for the malignant behavior displayed in vivo is discussed. The usefulness of this approach of selecting surface variants to study the problem of metastasis is also discussed.

Animals↗
PubMed1983

Organization and function of structural elements in focal contacts of tissue culture cells.

The role of structural elements in the organization and maintenance of focal contacts was studied by microinjecting into tissue culture cells specific probes which interfere with filamentous actin or with vinculin: actin interaction. Injection of actin capping proteins from Physarum and brain resulted in breakdown of microfilament bundles starting at their distal ends and in loss of focal contacts. This process was fully reversible. Injection of a high affinity antibody against chicken gizzard vinculin led to partial breakdown of microfilament bundles con-concomitant with disruption of focal contacts with vinculin remaining at the plasma membrane. This process was irreversible.

Actinin↗
PubMed1983

Congenital nemaline myopathy. II. Quantitative changes in alpha-actinin and myosin in skeletal muscle.

Skeletal muscle obtained from 2 patients with congenital nemaline myopathy (CNM) and from a healthy control was analyzed by 1- and 2-dimensional gel electrophoresis. In total extracts, an increase of alpha-actinin by a factor of 2:3 was found for CNM muscle as compared with the control. One- and two-dimensional gels revealed the presence of LCF3, the smallest light chain associated with type 2 (fast) myosin in total extracts of normal control of mixed fiber type. Both CNM samples showed the absence of this polypeptide. This result is consistent with the finding that muscle of the 2 patients exhibited nearly exclusively the ATPase activity indicative of type 1 (slow) myosin.

Actinin↗
PubMed1983

Differences in the stress fibers between fibroblasts and epithelial cells.

In the stress fibers of two types of nonmuscle cells, epithelia (PtK2, bovine lens) and fibroblasts (Gerbil fibroma, WI-38, primary human) the spacing between sites of alpha-actinin localization differs by a factor of about 1.6 as determined by indirect immunofluorescence and ultrastructural localization with peroxidase-labeled antibody. Both methods reveal striations along the stress fibers with a center-to-center spacing in the range of 0.9 mum in epithelial cells and 1.5 mum in fibroblasts. Periodic densities spaced at comparable distances are seen in PtK2 and in gerbil fibroma cells when they are treated with tannic acid and examined in the electron microscope. In such cells, densities are found not only along stress fibers but also at cell-cell junctions, attachment plaques, and foci from which stress fibers radiate. These latter three sites all stain with alpha-actinin antibody on the light and electron microscope level. Stress fibers in the two cell types also vary in the periodicity produced by indirect immunofluorescence with tropomyosin antibodies. As is the case for alpha-actinin, the tropomyosin center-to-center banding is approximately 1.6 times as long in gerbil fibroma cells (1.7 mum) as it is in PtK2 cells (1.0 mum). These results suggest that the densities seen in the electron microscope are sites of alpha-actinin localization and that the proteins in stress fibers have an arrangement similar to that in striated muscle. We propose a sarcomeric model of stress fiber structure based on light and electron microscopic findings.

Actinin↗
PubMed1983

Differential response of three types of actin filament bundles to depletion of cellular ATP levels.

The effect of low levels of ATP on actin filament bundles in PtK2 cells was investigated by using 2-deoxyglucose, together with either sodium cyanide, sodium azide, or 2,4-dinitrophenol. Three actin filament systems were examined: stress fibers, cleavage rings, and dimethyl-sulfoxide (DMSO)-induced actin bundles in the nucleus. Of the three, only stress fibers disassembled when the ATP production was inhibited. The disassembly progressed slowly with the cells losing all stress fibers after about 90 min, but remaining in flat interconnected sheets. Mitotic cells that had progressed as far as metaphase when inhibitors were added, assembled cleavage rings. The process of cytokinesis took place in these cells but at a rate 5 to 10 times slower than normal, and disassembly of the cleavage ring was inhibited after the completion of cytokinesis. DMSO-induced nuclear actin bundles did not disassemble in cells depleted of ATP even when DMSO was eliminated from the medium. The peripheral aggregates of contractile proteins present in these cells became redistributed, however, and the cells flattened in the low ATP environment when DMSO was removed. Nuclear actin bundles did not form in DMSO-treated cells if the ATP inhibitors were present for as little as 5 min prior to DMSO exposure. Thus, the three types of actin filament bundles are affected in different ways by low intracellular levels of ATP. Stress fibers are most sensitive and cleavage rings, the least.

Actins↗
PubMed1982

Peripheral and integral proteins of human blood platelet membranes. alpha-Actinin is not identical to glycoprotein III.

Isolation of human platelet membranes on polylysine beads and selective solubilization of membrane proteins allowed classification of membrane-associated proteins into integral and peripheral proteins. No major integral protein was found that was not exposed on the surface. Glycoprotein Ic was the only surface-exposed protein that behaved as a peripheral protein. The localization and identification of alpha-actinin was performed with an antibody against porcine skeletal muscle alpha-actinin. Human platelet alpha-actinin had an apparent molecular weight of 100 000 and a pI of 5.7-6.3. It was membrane-associated and behaved as a peripheral protein. Immunoisolation on protein A beads, as well as the 'Western Blot' technique applied to two-dimensional gels, demonstrated that alpha-actinin is not identical to GP III as was previously suggested (Gerrard, J.M., Schollmeyer, J.V., Phillips, D.R. and White, J.G. (1979) Am. J. Pathol. 94, 509-528).

Actinin↗