Lipopolysaccharide-caused fragmentation of individual microtubules in vitro observed by video-enhanced differential interference contrast microscopy.

Microtubule disassembly is commonly believed to be a process of endwise tubulin dimer release. The present study demonstrates by video interference contrast microscopy that Escherichia coli lipopolysaccharide (LPS) caused microtubule disassembly in vitro by both endwise shortening and fragmentation. In contrast, the microtubules were only shortened from their ends in the presence of DNA, used as another example of a macromolecular microtubule effector. LPS-caused microtubule fragmentation was confirmed by transmission electron microscopy. Because of its ability to induce both fragmentation and endwise shortening, LPS, which is involved in sepsis pathogenesis, has to be regarded as a highly active microtubule-destabilizing agent.

Tubulin assembly in the presence of calcium ions and taxol: microtubule bundling and formation of macrotubule-ring complexes.

It has been confirmed that taxol is able to prevent Ca(2+)-induced inhibition of microtubule formation from tubulin in the presence of microtubule-associated proteins. However, by means of electron microscopy and scanning force microscopy it could be demonstrated that assembly in the presence of Ca2+ and taxol leads to structural aberrations. The kind of aberration depends on the order of addition of taxol and Ca2+ to tubulin. When taxol was added first, microtubules were formed preferentially. But, these microtubules typically associated with each other by close wall-to-wall alignments or they formed complexes with some C-shaped protofilament ribbons, resulting in microtubule bundles or doublet- and triplet-like microtubule structures, respectively. When Ca2+ was added first, macrotubules, rings, and ring crystals were the dominant assembly products. Mostly, the macrotubules were also bundled or they enclosed rings in their lumen. The findings clearly demonstrate the potency of Ca2+ to induce different polymorphic assemblies with additional protofilament associations, not realized in microtubules.

Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion-dependent motility.

We studied the effect of alkaline-earth metal ions on the kinesin-driven gliding of microtubules, using a narrow glass chamber enabling the exchange of buffer components without interrupting microscopic observation. Under standard conditions (0.5 mM Mg2+), microtubules were found to glide at a mean velocity of about 0.6 micron/s. Motility was widely ceased after removing Mg2+. Subsequent addition of Ca2+ restored motility (maximal mean gliding velocity measured: 0.26 micron/s at 2.5 mM Ca2+). Also in the presence of Sr2+ or Ba2+ a slow gliding could be observed (0.025 micron/s and 0.014 micron/s, respectively, at 0.5 mM). After removal of Ca2+, Sr2+, or Ba2+ and re-addition of Mg2+, the gliding velocities reached approximately the values determined under standard conditions. Motility was not changed when 0.5 mM Ca2+, Sr2+, or Ba2+ were applied together with Mg2+. Microtubule gliding stopped after substitution of 0.5 mM BeCl2 for Mg2+. When both BeCl2 and Mg2+ were present, the mean gliding velocity was reduced to 0.29 micron/s. In addition, many microtubules were released from the kinesin coated glass surface, indicating that the beryllium salt disorders the binding between kinesin and microtubules. Our results confirm that Mg2+ is the most suitable cofactor for kinesin driven microtubule motility. However, they also demonstrate that brain kinesin can generate motility when Ca2+ was substituted for Mg2+.

Scanning force microscopy of microtubules and polymorphic tubulin assemblies in air and in liquid.

We have investigated microtubules (MTs) and polymorphic assemblies, formed in vitro from isolated microtubule protein, by scanning force microscopy (SFM) in air and in liquid. Immobilization of MTs was achieved by placing a drop of the assembly solution on a polylysine-coated coverslip. After washing with taxol and air drying, the characteristic microtubular fibrous morphology appeared in the SFM. The MTs formed a network similar to that obtained by transmission electron microscopy (TEM). A height of approximately 9.5 nm for dried MTs was computed from the surface topography. Glutaraldehyde fixation of the MTs yielded higher structures (approximately 14 nm), which swelled to approximately 20 nm after rehydration, a value close to the MT diameter of approximately 25 nm determined from TEM images of ultrathin sections. The protofilament pattern of the MTs and surface attached MT-associated proteins were not apparent from SFM, although the height along the long axis of the MTs appeared slightly modulated. In addition to MTs, various polymorphic tubulin assemblies including ribbons, hoops and double-walled MTs were visualized by SFM.

Effects of the fluorescence dye DAPI on microtubule structure in vitro: formation of novel types of tubulin assembly products.

It has been found that the DNA fluorescence dye 4',6-diamidino-2-phenylindole (DAPI) is able to stain also microtubules. However, electron microscopy revealed that DAPI changed microtubule structure and induced the formation of a broad spectrum of polymorphic tubulin assembly products. Upon addition of DAPI to microtubules assembled from 10 to 15 mumol tubulin (molar DAPI/tubulin ratios of 10 to 40) in the presence of microtubule-associated proteins, most of the microtubules were decorated with additional protofilaments usually running parallel to the protofilaments of the microtubule wall (microtubule-protofilament complexes). When DAPI was already present during assembly, curved C- and S-shaped protofilament ribbons and microtubule-ribbon complexes with 6-shaped profiles were the most prominent products, beside microtubules. Additionally, protofilament bundles, some flat sheets, and hoops occurred. Electrophoresis revealed that DAPI lowered the amount of associated proteins, especially of tau-proteins, bound to the assembly products. Nevertheless, DAPI stimulated the assembly, enabled pure tubulin to assemble even at concentrations as low as 10 mumol, and stabilized the assembly products against cold. The microtubule-protofilament complexes, observed for the first time, are interpreted as the result of DAPI-induced protofilament linking as well as of activation of an additional tubulin-tubulin binding site which is possibly identical to that involved in the formation of microtubule doublets.

Fluorenone-azomethines, a novel class of microtubule inhibitors that specifically affect cell proliferation.

The effects of various azomethine derivatives on microtubule (MT) assembly in vitro as well as on cell proliferation, cell shape, and the cytoskeleton of some cultured murine cell lines were studied. 3 of them, the alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl)-amino]-phenyl)-nit rone (DHPN), alpha-diphenylene-N-(p-[N-(hydroxyethyl)-N-(gamma-hydroxypropyl)- amino]-phenyl)-nitrone, and alpha-diphenylene-N-(p-diethylaminophenyl)-nitrone, strongly inhibit the assembly of microtubules (MTs) in vitro (50% inhibition at 4 to 7 mumol/l). The same compounds are also able to disrupt preformed microtubules. Moreover, they were found to inhibit proliferation of leukaemia L 1210, melanoma B16 K, fibroblast L 929, and embryo fibroblast cells down to 1 to 10 mumol/l, completely. Immunofluorescence microscopy revealed that DHPN, used as a representative of the active azomethines, causes a reversible destruction of the microtubule part of the cytoskeleton. Apparently resulting from microtubule disruption, the intermediate filament system collapsed whereas the microfilament system remained unaffected. The results indicate that the antiproliferative action of the azomethines is based, at least partially, on their ability to attack microtubules.

Measuring of the in-vitro assembly of microtubules by dynamic light scattering.

The kinetics of in-vitro assembly of microtubule protein (MTP) to microtubules (MTs) was followed under various conditions (temperature, GTP, ultrasonic treatment) by dynamic light scattering (DLS) and turbidimetric measurements. The results of both methods roughly coincide, but DLS additionally allows to differentiate between MTs and aggregates and to follow their growth. The complexity of these investigations, however, causes serious restrictions in the interpretation of the DLS data.

Can coated vesicles bind directly to microtubules?

Using an ultrathin-sectioning electron microscopic procedure, no efficient binding of coated vesicles to microtubules (both purified from brain tissue) could be achieved, independently of the presence of microtubule-associated proteins. Addition of the ATP analogue AMP-PNP or the inorganic tripolyphosphate, known to cause tight associations of (uncoated) vesicles to microtubules by means of specific motor proteins, could not enhance the binding efficiency. Moreover, crude preparations of clathrin, the major protein of the coat, did not affect the turbidity course of microtubule assembly. These results were confirmed by electrophoresis indicating that within the preparations of microtubule protein, obtained by temperature-dependent cycles of disassembly/reassembly, constituents of coated vesicles were not present. Beside this, coated vesicles have never been found among microtubules reconstituted in vitro. Vice versa, preparations of coated vesicles were completely free of microtubules. Our results suggest that further proteins should be involved in the binding of coated vesicles to microtubules, if there is indeed a biologically relevant interaction.

Synergistic cytotoxicity of human recombinant tumour necrosis factor alpha combined with microtubule effectors.

Combinations of human recombinant tumour necrosis factor alpha (rhTNF alpha) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azo-methine derivative alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl-amino]-phenyl)- nitrone (DHPN) on the rhTNF alpha cytotoxicity was studied. Applying a novel computer-based isobole method [Suehnel J (1990) Antiviral Res 13:23-40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF alpha with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF alpha and DHPN.

Influence of 1,4-benzoquinone derivatives and azomethines on microtubule formation in vitro and experimental leukemias.

Using two groups of substances (derivatives of 1,4-benzoquinone and azomethines) it was compared their effect on the microtubule formation in vitro and on experimental leukemias. 9 from the 28 substances tested acted cancerostatically, 4 substances inhibited microtubule assembly. 3 compounds (fluorenoneazomethines) revealed both effects.

Inhibition of microtubule assembly in vitro by anti-tubulin monoclonal antibodies.

Five monoclonal antibodies against N-terminal domains of alpha- or beta-tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU-13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N-terminal domains of tubulin are involved in the formation of microtubules.

Nucleation of macrotubules on double-walled microtubule seeds.

It is known that histone H1 is able to cause the formation of double-walled microtubules from microtubule protein. Now, we demonstrate that in dependence on the mass ratio H1/microtubule protein upon addition of tubulin to short pieces of double-walled microtubules either their inner or their outer wall elongates resulting in normal microtubules or in macrotubules, respectively. Because of their genesis we suggest that macrotubules like double-walled microtubules (see Unger et al., Eur. J. Cell Biol. 46, 98-104 (1988)) expose those sides of tubulin dimers at their surface which usually form the lumen face of microtubules.

Effect of MAP 1, MAP 2, and tau-proteins on structural parameters of tubulin assemblies.

When temperature-dependent recycling procedures for purification were used, tubulin is usually accompanied by a mixture of microtubule-associated proteins (MAPs) primarily comprising MAP 1, MAP 2, and the tau-proteins. Formerly we reported that microtubules formed from tubulin in the presence of these MAPs have more protofilaments than those formed without MAPs. Furthermore, these MAPs suppress the formation of aberrant assemblies (Böhm et al., BBA 800, 119, 1984). Now, we report that each single MAP fraction is able to influence the protofilament number of microtubules and the ratio between aberrant assemblies and microtubules in the same manner as the MAP mixture. This implies that all the MAPs investigated play a certain role in lateral association of tubulin dimers.

[Mechanism of action of alloxan on pancreatic B-cells with special regard to the alloxan-metal-complex theory. II. Actions of alloxan, alloxanic acid, Zn2+ and ethyleneglycol-bis-(beta-aminoethylether)-N,N'tetraacetic acid (EGTA) on the assembly of microtubule proteins (MTP) into microtubules or MPT sheets in vitro]. / butersuchungen zum Wirkungsmechanismus des Alloxans auf B-Inselzellen unter besonderer Berücksichtigung der Alloxan-Metall-Komplex-Theorie. II. Wirkungen von Alloxan, Alloxansäure, Zn2+ und Ethylenglycol-bis-(beta-aminoethyl-ether)-N,N'-tetraessigsäure (EGTA) auf die Assemblierung von microtubulären Proteinen (MTP) zu Microtubuli oder MTP-Sheets in vitro.

Analyses of the cell structures in the islets of Langerhans revealed the presence of 2 predominant cations, calcium (beta-granules and saccules, mitochondria, sac membranes, and cell membranes) and zinc (secretory granules, encasing membraneous sacs) in association with organelles which involve directional secretion. Both elements are known to interact with microtubules influencing their structural and functional properties, e.g. movement of secretory granules. Influences of Zn2+ on microtubules are investigated with a view to interactions in vitro with and without diabetogenic substances. Turbidimetry and electron microscopic investigations showed that under the conditions mentioned above, alloxan of a concentration of 2 x 10(-5) mol/l (alloxan/tubulin 1:1) inhibits the formation of microtubules and increases the portion of microtubules stabilized of 4 degrees C. The Zn2(+)-induced formation of MTP sheets is not influenced by alloxan and the metal complex forming agent EGTA, if the molar concentrations of the substance and Zn2+ are equally high. With a molar proportion of 2:1 (EGTA:Zn2+), the formation of sheets does not longer occur and only microtubules are formed, whereas neither sheets nor microtubules were assembled by alloxan with this molar proportion. However, neither the assembly of microtubules nor the formation of Zn2(+)-induced sheets are influenced by alloxanic acid in both molar proportions. It is shown that the diabetogenic alloxan influences the formation of microtubules and that performed microtubules are destroyed. This result of alloxan corresponds to its antimitotic activity analogously to the wellknown effect of colchicine (s. Schmidt et al. 1990).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the protofilament number in microtubules by templates.

We have used microtubules (MTs) and double-walled microtubules (dwMTs), both fragmented, as templates for MT formation from phosphocellulose-purified tubulin. In both cases the mean protofilament number of the nucleated MTs corresponds to that of the templates. The results confirm the observations of Scheele et al. (J. Mol. Biol. 154, 485, 1982) that templates determine the protofilament number of nucleated MTs.

Effect of sodium chloride on the structure of tubulin assemblies.

By use of a taxol-containing assembly medium, it has been demonstrated that the mean protofilament number of microtubule populations is significantly lower at elevated NaCl concentrations. Assembly of microtubule protein, i.e. tubulin plus microtubule-associated proteins (MAPs), at 80 and 580 mM NaCl results in microtubule populations with mainly 13 and 10 protofilaments whereas microtubules formed from tubulin alone have 12 and 10 protofilaments, respectively. Moreover, when MAPs are prevented from assembly, the formation of taxol-induced aberrant assemblies (C- and S-shaped protofilament ribbons) is suppressed at high NaCl concentrations in favour of microtubules. The described effects are obviously caused by both weakening of MAP binding to tubulin and alterations in tubulin-tubulin association.

Structural changes within mixed populations of microtubules and protofilament ribbons caused by dilution and cold incubation.

By means of electron microscopy we have investigated the influence of dilution and cold incubation (0 degree C) on mixed populations of tubulin assemblies consisting of microtubules (MTs) and protofilament ribbons with C- and S-shaped profiles formed in the presence of glycerol. Dilution results in a partial disappearance of ribbons, whereas cold incubation causes a decrease of the percentage of MTs in favour of C-ribbons, probably produced by splitting of MT ends. In the case of dilution, we assume a lower dynamic stability of ribbons compared with MTs, which was already observed during long-time incubation of mixed population (Böhm et al., Biochem. Biophys. Acta 929, 154, 1987), whereas the splitting effect in the cold should be caused by the deficiency of MT-bound microtubule-associated proteins (MAPs), which is observed in the presence of glycerol.

Structural diversity and dynamics of microtubules and polymorphic tubulin assemblies.

Tubulin, the main protein of microtubules (MTs), has the potency of forming a variety of other assembly products in vitro: rings, ring-crystals, C- and S-shaped ribbons, 10 nm fibres, hoops, sheets, heaped sheets, MT doublets, MT triplets, double-wall MTs, microtubules, curled ribbons, and paracrystals. The supramolecular subunits of all of them are the protofilaments which might be arranged either parallel to the axis (e.g., in MTs, ribbons) or curved (e.g., in hoops, microtubules). There is strong evidence that in the second case the protofilaments have an inside-out orientation compared to MTs. All assembly products mentioned are described structurally and their relevance to the in vivo situation is considered. Moreover, MTs and the other assemblies undergo permanent changes. These dynamics occurring in both individual assemblies and assembly populations are discussed from the structural point of view.

Structural comparison of assemblies from brain microtubule proteins of different vertebrates.

Formerly, we reported for microtubule protein (MTP), i.e. tubulin plus microtubule-associated proteins (MAPs), from porcine brain that the protofilament number of microtubules and the percentage of aberrant assemblies depend on taxol and MAP activity (Böhm et al., BBA 800, 119, 1984). Now, it is demonstrated that these effects can be also observed when MTPs from other higher vertebrate brains (Guinea pig, mouse chicken) were used. Comparing the structural features of assemblies formed from these kinds of MTP with each other no significant differences were found, excepted chicken MTP which produces more aberrant assemblies in the presence of taxol.