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Biomedical subjects

M Bärmann

Publications and source records attributed to M Bärmann.

13 recordsLinked to original sources
PubMed2000

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.

Binding Sites↗
PubMed2000

Shape instability of a biomembrane driven by a local softening of the underlying actin cortex.

We present a theory showing that local shape instabilities of composite biological membranes, consisting of a lipid bilayer and an underlying actin cortex, can be triggered by a local softening of the membrane-associated cytoskeleton. A membrane containing such cortical defects can form blisters or invaginations, depending on external conditions. The theoretical predictions agree with observations provided by two sets of experiments: (i) microscopic observations of shape changes of giant vesicles with underlying shells of a thin actin network show the formation of local blisters and (ii) micropipet aspiration experiments of Dictyostelium discoideum cells in which we observed the formation of blisters in the aspirated cell part. In the latter experiments, the existence of a hole in the underlying cortex is confirmed by observation of the entrance of cell organelles into the blister. Our model may also be applied to the formation of lobopodia, fast-growing cell protrusions that play an important role in the locomotion and spreading of biological cells.

Actins↗
PubMed1997

Energy filtered electron tomography of ice-embedded actin and vesicles.

Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in "phantom cells," i.e. lipid vesicles. The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode. Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control. This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope. In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation. In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than approximately 2 microns tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.

Actins↗
PubMed1996

Determination of the inelastic mean free path in ice by examination of tilted vesicles and automated most probable loss imaging.

Using electron microscopy, the thickness of ice-embedded vesicles is estimated examining tilted and untilted views and assuming an ellipsoidal shape of the vesicles that appear to be circular in the untilted view. Another thickness measure is obtained from the ratio of the unfiltered and zero-loss-filtered image intensities of the vesicle. From these two measurements, the mean free path A for inelastic scattering of electrons in ice is calculated as 203 +/- 33 nm for 120 kV acceleration voltage. It is found that vesicles in thin ice films (< or = 1.5 lambda) significantly protrude out of the ice film. Due to surface tension the shape becomes an oblate ellipsoid. In holes covered with a thick ice film (> or = 3 lambda) and strong thickness gradients, vesicles are predominantly found in regions where the ice thickness is appropriate for their size. Also, a way of imaging the most probable loss under low-dose conditions involving thickness measurement is proposed. Even at large ice thicknesses zero-loss filtering always gives better image contrast. Most probable loss imaging can only help where there is no intensity in the zero-loss image, at very large thicknesses (lambda > 8).

Dimyristoylphosphatidylcholine↗
PubMed1993

Actin binding to lipid-inserted alpha-actinin.

The interaction of alpha-actinin with lipid films and actin filaments was investigated. First alpha-actinin was incorporated in lipid films at the air/water interface. Injection of alpha-actinin into the subphase of a lipid monolayer led to a significant increase of the surface pressure only for lipid films consisting of a mixture of a negatively charged lipid with a high proportion of diacylglycerol. These alpha-actinin-containing films were transferred onto silanized quartz slides. Photobleaching experiments in the evanescent field allowed quantification of the lateral number density of the lipid-bound alpha-actinin. In combination with the area increase from the monolayer experiments, the photobleaching measurements suggest that alpha-actinin is incorporated into the lipid film in such a way that actin binding sites are accessible from the bulk phase. Binding experiments confirmed that the alpha-actinin selectively binds actin filaments in this configuration. We also showed that, in contrast to actin filaments which are adsorbed directly onto planar surfaces, the alpha-actinin-bound actin filaments are recognized and cleaved by the actin-severing protein gelsolin. Thus we have constructed an in vitro system which opens new ways for investigations of membrane-associated actin-binding proteins and of the physical behavior of actin filaments in the close neighborhood to membranes.

Actinin↗
PubMed1991

Talin binds to actin and promotes filament nucleation.

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.

Actins↗
PubMed1989

Botulinum C2 toxin treatment increases the G-actin pool in intact chicken cells: a model for the cytopathic action of actin-ADP-ribosylating toxins.

Botulinum C2 toxin ADP-ribosylates actin in intact chicken embryo cells in a concentration-dependent manner. This effect correlates with an enhancement in the inhibitory potency of the respective cell lysates on DNAse I activity, indicating an increase in the cellular G-actin content of toxin-treated cells. The data support our view, that ADP-ribosylation of cellular actin with subsequent depolymerization of cytoskeleton-associated F-actin to monomeric G-actin is involved in the cytotoxic effects of botulinum C2 toxin. A model of the cytopathic action of actin-ADP-ribosylating toxins is presented.

Actins↗
PubMed1988

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin.

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.

ADP Ribose Transferases↗
PubMed1988

Botulinum C2 toxin ADP-ribosylates cytoplasmic beta/gamma-actin in arginine 177.

Isolated cytoplasmic actin of human platelet and pig liver actin, but not rabbit skeletal muscle actin, was ADP-ribosylated by botulinum C2 toxin in the presence of [32P]NAD. Tryptic digestion of the [32P]ADP-ribosylated platelet actin generated two labeled peptides: a soluble peptide covering residues 174-183 and an insoluble fragment containing residues 148-183. Further digestion of these two peptides with thermolysin yielded the same radioactive peptide, which was in both cases peptide 175-177. Amino acid sequence analysis of peptides 174-183 and 175-177 located the ADP-ribosylation on Arg177.

Actins↗
PubMed1987

Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177.

Clostridium perfringens iota toxin ADP-ribosylates actin. Substrates of C. perfringens toxin are both non-muscle beta/gamma-actin and skeletal muscle actin. This finding suggests that C. perfringens iota ADP-ribosylates the same amino acid in skeletal muscle and non-muscle actin as does C. botulinum C2 toxin in non-muscle actin. Protein chemical analysis involving thermolysin cleavage on [32P]ADP-ribosylated actin or tryptic digestion followed by a secondary thermolysin cleavage of the radiolabelled fragments showed one major site of ADP-ribosylation. From its amino acid composition and sequence, the radiolabelled peptide was identified as peptide 175-177, locating the acceptor ADP-ribosyl amino acid as Arg-177.

ADP Ribose Transferases↗
PubMed1986

A 50 kDa, actin-binding protein in plasma membranes of rat hepatocytes and of rat liver tumors.

Plasma membranes from normal rat livers and rat liver tumors were compared by SDS-gel electrophoresis, and analyzed for actin-binding proteins by an 125I-labelled actin gel-overlay assay and by actin-affinity blotting. After treatment of rats with alpha-hexachlorocyclohexane and after induction of liver tumors by combined treatment with N-nitrosomorpholine and phenobarbital, liver plasma membranes prepared from these animals were found to be highly enriched in an actin-binding, 50 kDa polypeptide. This polypeptide seemed to be an integral protein of the plasma membrane as judged by Triton X-114-phase separation. Microsomes did not contain an actin-binding polypeptide in the 50 kDa region. Therefore, the 50 kDa protein is a candidate for interaction of actin with the liver cell plasma membrane. A possible relationship of this protein with the multi-specific, cholate transporting system of the rat liver plasma membrane is discussed.

Actins↗
PubMedDate not supplied

Botulinum C2 toxin ADP-ribosylates actin.

ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.

Actins↗