Structural dynamics within mixed populations of microtubules and protofilament ribbons.

In the presence of glycerol, microtubule proteins reassemble into both microtubules and protofilament ribbons with C- and S-shaped cross-section profiles. By means of electron micrographs of cross-sectioned assemblies, we have demonstrated that, during the steady state, the percentage of ribbons, especially of C-shaped ones, decreases in favour of the formation of microtubules. The following conversion modes are discussed: A, closure of the protofilament wall by increasing its curvature; B, lateral association of C-ribbons; C, completion of C-ribbons to microtubules by lateral association of tubulin; D, disassembly of ribbons and elongation of microtubules. We conclude that ribbon disassembly proceeding in an end-wise fashion and microtubule elongation is the favoured mode of conversion. Microtubule-associated proteins were found to be required for the steady-state conversions of ribbons into microtubules. In the absence of microtubule-associated proteins, C-ribbons associate laterally, forming S-ribbons. It is shown that the protofilaments of the counter-curved parts of S-ribbons have the same polarity.

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.

Induction of cytochrome P450 2B1-mRNA and pentoxyresorufin O-depentylation after exposure of precision-cut rat liver slices to phenobarbital.

Precision-cut rat liver slices were prepared from male Wistar rats with a Krumdieck slicer and cultured in William's medium E for up to 24 h. In untreated control slices, CYP2B1-mRNA concentration, which was quantified by competitive RT-PCR, did not decrease during this time. After exposure of the slices to 100 microM phenobarbital, CYP2B1-mRNA increased by about 10- or 60-fold after 6 or 24 h, respectively. The extent of this in vitro induction was similar to that after in vivo administration of 60 mg/kg phenobarbital. Pentoxyresorufin O-depentylation (PROD) was also inducible in vitro after 24 h, but to a lesser extent than the corresponding CYP-mRNA. Precision-cut liver slices proved to be a simple and reliable in vitro system for the sensitive detection of an induction by phenobarbital.

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices.

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

Use of Fresh and Cryopreserved Human Liver Slices in Toxicology with Special Reference to In vitro Induction of Cytochrome P450.

Human liver slices were prepared with a Krumdieck slicer from macroscopically healthy surgical waste after partial hepatectomy. They were incubated without or with the addition of the inducer beta-naphthoflavone (BNF) (25 mum) either immediately after preparation (fresh slices) for up to 24 hours or after cryopreservation in liquid nitrogen (thawed slices) for up to 6 hours. Potassium concentration was well maintained in fresh and thawed slices over 24 and 6 hours, respectively, but at lower levels than in rat liver slices. Albumin secretion showed relatively large interindividual differences. Both parameters were lower in thawed slices than in fresh ones, but indicated a certain number of viable cells. In untreated fresh slices CYP1A1-mRNA was not detectable; however, it increased distinctly within 6 hours of exposure to BNF. The amounts of induced CYP1A1-mRNA differed by a factor of more than 100 among six human livers and were lower than in fresh rat liver slices. Even in thawed human slices, CYP1A1-mRNA expression could be induced in vitro by BNF, although at a very low level and preferentially in those specimens with comparably high inducibility already before freezing.

Application of cryopreserved precision-cut liver slices in pharmacotoxicology--principles, literature data and own investigations with special reference to CYP1A1-mRNA induction.

Principle steps necessary for cryopreservation of precision-cut liver slices as currently applied by different groups are summarized including own results concerning mode of freezing. Now we use rapid freezing by immersion in liquid nitrogen after exposure to 10% DMSO as the cryoprotectant for rat liver slices. The results indicate well-maintained cytochrome P450 (CYP)-dependent deethylation rates in slice homogenate after short-term incubation. ECOD rate in intact thawed slices was even higher than in fresh ones after 2 h incubation. In contrast to fresh slices all parameters except protein content decreased to marginal levels during long-term incubation of thawed slices for 24 h. The first preliminary experiments on albumin secretion by thawed rat liver slices, measured between the 2nd and the 4th hour of incubation, showed partial maintenance of this liver specific differentiated function. Trials to induce CYP1A1 in thawed rat liver slices in vitro by beta-naphthoflavone (BNF) resulted in increased expression of CYP1A1-mRNA within 6 h as shown by RT-PCR and quantified by competitive RT-PCR. The decline of deethylation rates, determined in slice homogenates, and of viability within 24 h incubation was not prevented by exposure to BNF or DMSO. The results derived from one sample of cryopreserved human liver slices indicate a quite acceptable maintenance of function up to 6 h, if the same protocol as developed for rat liver slices was used.

Monooxygenation, cytochrome P450-mRNA expression and other functions in precision-cut rat liver slices.

Precision-cut rat liver slices (KRUMDIECK slicer, slice thickness 200-250 microm) were incubated in rollers containing modified William's medium E at 37 degrees C for 2, 24 and 48 hrs. Protein, DNA, potassium and glutathione concentrations did not decrease during 48 hrs. Lactate dehydrogenase (LDH) leakage into the medium was relatively marked during the first 2 hrs of incubation, from the 2nd to the 48th hr LDH leakage was very low. The same is true of the release of thiobarbituric acid-reactive substances. Albumin synthesis and transport into the medium decreased to about 70% after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation rate was relatively stable up to 48 hrs, whereas testosterone hydroxylation decreased significantly without alterations of the proportions of the 7 quantified hydroxylated metabolites. After exposure of the slices to beta-naphthoflavone for 6 hrs CYP1A1-mRNA expression, measured by competitive RT-PCR, was increased by a factor of at least 1000. Precision-cut liver slices are a useful tool for the study of various hepatic functions, drug metabolism and its induction in vitro.

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.

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 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.

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+.

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.