PubMed Health⌕ Search

Biomedical subjects

E M Mandelkow

Publications and source records attributed to E M Mandelkow.

At least 91 records · Page 5Linked to original sources
PubMed1988

Structure and elasticity of microtubule-associated protein tau.

Tau is one of the diverse group of microtubule-associated proteins that bind to microtubules and may thereby influence their structure and function. It occurs in the mammalian brain, mainly in axons, and is a component of the neurofibrillary tangles of Alzheimer's disease. Tau was recently sequenced, but there remains a short-age of structural data on the protein. We have now prepared paracrystals of tau suitable for electron microscopy and image processing. They show distinct transverse banding and polarity, indicating that the protein subunits are aligned with the same orientations. In contrast to other paracrystals, those of tau protein can stretch or contract continuously by more than three-fold; the axial repeats range from 22 to 68 nm. After scaling to a common period, the density distributions are closely superimposable. This suggests that tau is an elastic molecule.

Chemical Phenomena↗
PubMed1988

Dynamics of the microtubule oscillator: role of nucleotides and tubulin-MAP interactions.

Microtubules can be induced to perform synchronous and periodic cycles of assembly and disassembly at constant temperature. The process depends on GTP hydrolysis. Time-resolved X-ray scattering using synchrotron radiation shows a cyclic interconversion of tubulin subunits, microtubules and oligomers (= short protofilament fragments). Oscillations are correlated with conditions that stabilize polymers and destabilize oligomers, and others of opposite effect. Microtubule stabilizers include GTP, Mg2+ or microtubule-associated proteins (MAPs), destabilizers include GDP or elevated ionic strength. K+ at intracellular concentrations noticeably increases the stability of tubulin-MAP oligomers, in contrast to Na+. ATP and the non-hydrolyzable analogue AMP-PNP enhance oscillations by mechanisms that are not directly linked to the role of nucleotide hydrolysis in assembly. We propose a mechanism of oscillations that include oligomers as microtubule disassembly products which transiently lock the protein in an unpolymerizable state; this may point to a role of oligomers in controlling microtubule assembly cycles in cells.

Adenosine Triphosphate↗
PubMed1987

Tubulin oligomers and microtubule assembly studied by time-resolved X-ray scattering: separation of prenucleation and nucleation events.

This paper describes a time-resolved X-ray scattering study of microtubule assembly by synchrotron radiation. The method is complementary to light scattering but allows a better distinction between oligomeric and polymeric assembly states. With an improved rapid temperature jump device, it is shown that temperature-induced microtubule assembly is preceded by prenucleation and nucleation events involving oligomers of tubulin, in analogy with earlier results from near-equilibrium temperature scans. In general, the two phases closely overlap, but in certain conditions they can be observed separately. The prenucleation events seen by X-rays can be described as a rapid temperature-dependent equilibrium, with ring oligomers dissociating into smaller oligomers and subunits at elevated temperature. Different solution conditions affect mainly the time lag between the prenucleation and nucleation phases; this in turn determines the apparent magnitude of the prenucleation steps. By contrast, the temperature dependence of the equilibrium between the prenucleation oligomers shows little influence on solution conditions. The results suggest that the ring-forming and tubule-forming assembly modes of tubulin are governed by different interactions between subunits, although they may be based on a pool of similar intermediates.

Hydrogen-Ion Concentration↗
PubMed1986

On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness.

The tubulin monomers of brain microtubules reassembled in vitro are arranged on a 3-start helix, irrespective of whether the number of protofilaments is 13 or 14. The dimer packing is that of the B-lattice described for flagellar microtubules. This implies that the tubulin core of microtubules contains at least one helical discontinuity. Neither 5-start nor 8-start helices have a physical significance and thus cannot be implicated in models of microtubule elongation, but the structure is compatible with elongation of protofilaments by dimers or protofilamentous oligomers. The inner and outer surfaces of the microtubule wall can be visualized by propane jet freezing, freeze fracturing, and metal replication, at a resolution of at least 4 nm. The 3-start helix is left-handed, in contrast to a previous study based on negative staining and shadowing. The reasons for this discrepancy are discussed.

Cytoskeleton↗
PubMed1986

Microtubule structure studied by quick freezing: cryo-electron microscopy and freeze fracture.

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryoelectron microscopy of the unstained vitrified samples; (2) freezing by the propane-jet method, followed by freeze fracturing and metal replication. The unstained frozen-hydrated microtubules show a structure in agreement with X-ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2-3 nm); it is limited by low contrast and lack of crystalline order. Propane-jet or cryo-block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross-striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.

Animals↗
PubMed1985

Tubulin domains probed by limited proteolysis and subunit-specific antibodies.

The substructure of the tubulin molecule was studied by limited proteolysis and high affinity polyclonal antibodies specific for alpha or beta-tubulin. Brief enzymatic cleavage separates the tubulin monomer into two domains of unequal size. Trypsin splits alpha-tubulin into components with Mr values of 36 X 10(3) and 14 X 10(3), chymotrypsin splits beta-tubulin into 31 X 10(3) Mr and 20 X 10(3) Mr fragments. The cleavage occurs at Arg339 (alpha) and Tyr281 (beta), as determined by sequencing several N-terminal residues of the small domains, i.e. the small domains are the C-terminal parts of the molecules, the large ones are the N-terminal parts. There is a second cleavage site of chymotrypsin within Mr 10(3) to 2 X 10(3) of the C terminus of beta-tubulin. The fragments can be separated only under denaturing conditions. They copolymerize into microtubules and incomplete microtubule walls joined by a wall junction, forming S-shapes and hooks in cross-section. The antibodies were raised against electrophoretically purified tubulin monomers. Those produced with alpha-tubulin are directed predominantly against the large domains; they are either specific for alpha-tubulin or cross-react with the large domain of beta-tubulin. Conversely, antibodies raised against beta-tubulin are directed predominantly against the small domains (beta-specific and beta-cross-reacting fractions). Thus the antibodies discriminate not only between the tubulin chains but also between the domains generated by the proteases. The complementary antigenicity correlates well with the stability of the domains. Potential sites of antigenic determinants are located within the polypeptide chains by comparing theoretical predictions with the pattern of immunoblots. Two epitopes of the alpha-cross-reacting antibodies have been located approximately. One is very close to the C terminus (within about 20 residues), the other is close to the N terminus (within about Mr 8 X 10(3) ). The epitope of the beta-cross-reacting antibody is also located within Mr 12 X 10(3) of the C terminus. The antibodies prevent microtubule assembly and cause disassembly of preformed microtubules. A variety of breakdown products are observed by electron microscopy. They include fibres of about 10 nm width, sheets with undefined substructure, thick tapered fibrous bundles and wispy filaments.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Unstained microtubules studied by cryo-electron microscopy. Substructure, supertwist and disassembly.

Unstained microtubules embedded in amorphous ice have been studied by cryo-electron microscopy and image reconstruction. The structural integrity is well preserved, judging by the similarity of optical diffraction patterns with X-ray fiber diagrams. Protofilaments are not straight but show a variable right-handed twist around the tubule axis with a pitch of several micron. While rapid freezing of warm solutions (about 37 degrees C) leaves microtubules intact, gradual cooling, followed by rapid freezing, allows one to visualize the time-course of microtubule breakdown. Disassembly proceeds both from the ends and from inside, and short protofilament fragments are among the early breakdown products.

Animals↗
PubMed1985

Tubulin domains responsible for assembly of dimers and protofilaments.

The protein domains responsible for the dimerization and polymerization of tubulin have been determined using chemical cross-linking and limited proteolysis. The intra-dimer bond is formed by the N-terminal domain of alpha-tubulin and the C-terminal domain of beta-tubulin. Conversely, the inter-dimer bond along protofilaments is formed by the N-terminal domain of beta-tubulin (carrying the exchangeable GTP) and the C-terminal domain of alpha-tubulin. The domains of proteolytically cleaved tubulin remain tightly associated in solution. Apart from the monomer, tubulin shows three levels of assembly: the dimer, oligomer and polymer. Several oligomeric species can be visualized by electron microscopy of rotary shadowed phosphocellulose-tubulin, h.p.l.c. and non-denaturing gel electrophoresis. Tubulin's capacity to form the higher level aggregates is not destroyed by enzymatic nicking.

Animals↗
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↗
PubMed1984

Assembly and three-dimensional image reconstruction of tubulin hoops.

The three-dimensional structure of tubulin hoops has been determined by image reconstruction. The surface lattice of hoops is similar to that of microtubules, but in addition hoops possess a superstructure of protofilament triplets. The protofilaments differ mainly in their apparent volumes and lateral spacings. The volumes depend strongly on the orientation on the carbon support, while the spacings do not. The differences of appearance do not reflect changes of intrinsic subunit structure. They are explained by differential staining related to the orientation and packing of protofilament. Microtubule-associated proteins do not contribute to the average subunit structure. All apparent protofilament structures differ from that expected from X-ray patterns of microtubules in terms of subunit tilt and distribution of contrast. It is concluded that the negatively stained structure is a reliable representation of the arrangement of protein subunits, but not of their shape. Tubulin hoops occur in conditions of microtubule assembly near the critical concentration in a stabilizing buffer. Their formation depends on microtubule-associated proteins and on the initial presence of tubulin oligomers, which may associate into short protofilament triplets. If their elongation is rapid compared to lateral aggregation, they form closed hoops. The growth phase is followed by a redistribution phase, during which hoops disappear in favour of microtubules. This behaviour is explained by kinetic overshoot assembly. Each triplet resembles an incomplete microtubule wall so that the junction between two triplets may be compared to a junction between microtubule walls. Such junctions are formed by a closely spaced pair of protofilaments. They are analogous to junctions between microtubules and incomplete microtubule walls, and they have the same clockwise curvature when viewed at the growing end.

Macromolecular Substances↗
PubMed1984

Reconstructions of tubulin protofilaments: different appearances of the same structure.

We compare the structures of tubulin protofilaments obtained by image reconstruction of tubulin sheets and hoops, and by X-ray diffraction of microtubules. Negatively stained specimens yield up to eight different appearances of protofilaments. They all represent the same intrinsic structure. The effects are explained by the packing of the protein subunits and the stain distribution resulting from it. They cannot be interpreted directly in terms of protein structure or composition; in particular, differences in staining cannot be attributed to microtube-associated proteins (MAPs). None of the reconstructions reproduce the X-ray structure faithfully. The discrepancies between the reconstructions are seen at resolutions of 4 nm or less. This appears to be the limit of structural fidelity of negatively stained tubulin specimens, even when the nominal resolution of the micrographs is better. Most of the differences may be defined in terms of the diffraction patterns and are therefore genuine. Additional differences become apparent after computing the 3D reconstruction and may be genuine and/or due to the data treatment. Several factors affecting the appearances are discussed.

Crystallography↗
PubMed1983

Structure of tubulin rings studied by X-ray scattering using synchrotron radiation.

X-ray patterns have been obtained from solutions of microtubules and tubulin rings using synchrotron radiation. They are interpreted on the basis of the oriented fiber X-ray diagram of microtubules, and by comparing them with the calculated scattering traces of model structures. The data are consistent with models in which rings consist of coiled protofilaments. This is in agreement with some published ring models while it excludes others.

Microscopy, Electron↗