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1.
Disruption of microtubules in living cells and cell models by high affinity antibodies to beta-tubulin. 总被引:2,自引:0,他引:2 下载免费PDF全文
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. 相似文献
2.
The structural transitions occurring during the assembly and disassembly of pig brain microtubule protein were investigated by time-resolved X-ray scattering using synchrotron radiation. The reactions were introduced by a slow temperature scan (2 deg.C/min) from 0 °C to 37 °C and back. Several structurally distinct states could be resolved during one cycle of assembly/disassembly. During the temperature rise, one observes four main phases: prenucleation events, microtubule nucleation, growth, and postassembly events.Heating from 0 °C to 22 °C results in a biphasic breakdown of rings and other aggregates, while the apparent mean diameter increases from 38 to 41 nm. Parallel time-resolved electron microscopic observations suggest that the initial solution contains several types of aggregates, mostly double concentric and single rings, but also rod-like particles, clusters of rings and other aggregates. All of these tend to break down with increasing temperature. Double concentric rings seem to dissociate into large and small single rings before both types of rings break down into protofilament fragments and tubulin subunits. From the breakdown products, associations of several protofilament fragments are formed, which are important for initiating microtubule nucleation. Assembly of nuclei begins around 22 °C. Microtubule elongation takes place between 25 and 30 °C. They grow mainly by addition of tubulin subunits but not via rings.During the reverse temperature scan, microtubules shorten by the release of subunits and/or small protofilament fragments from their ends. The degree of disassembly is strongly increased below 22 °C. Below about 10 °C rings are reformed, probably from the fragments, but their final number is much less than initially.Conditions that prevent microtubule nucleation such as GDP or Ca2+ also stabilize rings, even at 37 °C. Thus, rings are viewed as storage aggregates of tubulin and microtubule associated proteins, whose breakdown is a prerequisite for microtubule formation, and whose reformation is independent of microtubule breakdown.The midpoints of microtubule growth and breakdown differ by about 12 deg.C so that the system shows hysteresis-like behavior. It is dependent on microtubule formation and is not seen when the temperature is cycled below that required for nucleation. Thus, even during a slow temperature scan, microtubule assembly is kinetically limited by nucleation. By contrast, depolymerization proceeds close to equilibrium.The radius of gyration of the tubulin heterodimers is 3.1 nm. The weight average diameter of rings in cold solutions is 38 nm, that of microtubules is 24.5 nm.At radiation dose rates of about 100 rad/s. radiation damage is of minor importance, as judged by the criterion of polymerizability. Total doses of up to 500,000 rad can be applied.Some concepts of analyzing time-resolved X-ray scattering data are presented. They make use of the fact that the scattering intensities vary continuously both with scattering angle and time. Cross-correlation of different regions of the pattern, and comparison of their temperature derivatives, reveals structural transitions not seen by other techniques. 相似文献
3.
Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro 总被引:5,自引:0,他引:5 下载免费PDF全文
H Wille G Drewes J Biernat E M Mandelkow E Mandelkow 《The Journal of cell biology》1992,118(3):573-584
Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein. 相似文献
4.
Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility. 相似文献
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7.
The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity 下载免费PDF全文
Astrid Sydow Frank JA Dennissen Zuzana Siskova Eckhard Mandelkow Eva‐Maria Mandelkow 《EMBO reports》2016,17(4):552-569
We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTauAT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTauAT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate. 相似文献
8.
The beta-propensity of Tau determines aggregation and synaptic loss in inducible mouse models of tauopathy 总被引:1,自引:0,他引:1
Eckermann K Mocanu MM Khlistunova I Biernat J Nissen A Hofmann A Schönig K Bujard H Haemisch A Mandelkow E Zhou L Rune G Mandelkow EM 《The Journal of biological chemistry》2007,282(43):31755-31765
Neurofibrillary lesions are characteristic for a group of human diseases, named tauopathies, which are characterized by prominent intracellular accumulations of abnormal filaments formed by the microtubule-associated protein Tau. The tauopathies are accompanied by abnormal changes in Tau protein, including pathological conformation, somatodendritic mislocalization, hyperphosphorylation, and aggregation, whose interdependence is not well understood. To address these issues we have created transgenic mouse lines in which different variants of full-length Tau are expressed in a regulatable fashion, allowing one to switch the expression on and off at defined time points. The Tau variants differ by small mutations in the hexapeptide motifs that control the ability of Tau to adopt a beta-structure conformation and hence to aggregate. The "pro-aggregation" mutant DeltaK280, derived from one of the mutations observed in frontotemporal dementias, aggregates avidly in vitro, whereas the "anti-aggregation" mutant DeltaK280/PP cannot aggregate because of two beta-breaking prolines. In the transgenic mice, the pro-aggregation Tau induces a pathological conformation and pre-tangle aggregation, even at low expression levels, the anti-aggregation mutant does not. This illustrates that abnormal aggregation is primarily controlled by the molecular structure of Tau in vitro and in the organism. Both variants of Tau become mislocalized and hyperphosphorylated independently of aggregation, suggesting that localization and phosphorylation are mainly a consequence of increased concentration. These pathological changes are reversible when the expression of Tau is switched off. The pro-aggregation Tau causes a strong reduction in spine synapses. 相似文献
9.
Alzheimer's disease is characterized by aggregates of tau protein. Attempts to study the conditions for aggregation in vitro have led to different experimental systems, some of which appear mutually exclusive (e.g., oxidative vs reductive conditions, induction by polyanions vs fatty acids). We show here that different approaches and pathways can be viewed in a common framework, and that apparent differences can be explained by variations in the kinetics of subreactions. A unified view of PHF aggregation should help to analyze the causes of PHF aggregation and devise methods to prevent it. 相似文献
10.
Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias 总被引:7,自引:0,他引:7
Barghorn S Zheng-Fischhöfer Q Ackmann M Biernat J von Bergen M Mandelkow EM Mandelkow E 《Biochemistry》2000,39(38):11714-11721
We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease. 相似文献