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E M Mandelkow

Publications and source records attributed to E M Mandelkow.

At least 55 records · Page 3Linked to original sources
PubMed1995

The microtubule lattice--dynamic instability of concepts.

In the February 1995 issue of trends in CELL BIOLOGY, Linda Amos presented her view of our current understanding of the lattice structure of microtubules, 20 years after publication of the original paper describing the A- and B-lattices for flagellar microtubules. However, the question of the lattices of flagellar and cytoplasmic microtubules remains a matter for debate. In this article, Eckhard Mandelkow, Young-Hwa Song and Eva-Maria Mandelkow argue that the B-lattice is predominant, implying structural asymmetry for most microtubules.

Comment↗
PubMed1995

Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells.

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.

Animals↗
PubMed1995

Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules.

The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.

Amino Acid Sequence↗
PubMed1994

Domains of tau protein and interactions with microtubules.

The role of the neuronal microtubule-associated protein tau has been studied by generating a series of tau constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N- or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N-terminal inserts. The interaction of the mutant tau proteins with microtubules was judged by several independent methods. (i) Direct binding assays between tau and taxol-stabilized microtubules yield dissociation constants and stoichiometries. (ii) Light scattering and X-ray scattering of assembling microtubule solutions reflect the capacity of tau to promote microtubule nucleation, elongation, and bundling in bulk solution. (iii) Dark field microscopy of assembling microtubules allows one to assess the efficiency of nucleation and bundling separately. The repeat region alone, the N-terminal domains alone, or the C-terminal tail alone binds only weakly to microtubules. However, binding is strongly enhanced by combinations such as the repeat region plus one or both of the flanking regions which could be viewed as "jaws" for tau on the microtubule surface (the proline-rich domain P upstream of the repeats and the "fifth" repeat R' downstream). Such combinations make tau's binding productive in terms of microtubule assembly and stabilization, while the combination of the flanking regions without repeats binds only unproductively. Efficient nucleation parallels strong binding in most cases, i.e., when a construct binds tightly to microtubules, it also nucleates them efficiently and vice versa. In addition, the proline-rich domain P in combination with the repeats R or the flanking domain R' causes pronounced bundling. This effect disappears when the N-terminal domains (acidic or basic) are added on, suggesting that the tau isoforms are not "bundling proteins" in the proper sense. In spite of the wide range of binding strength and nucleation efficiency, the stoichiometries of binding are rather reproducible (around 0.5 tau/tubulin dimer); this is in remarkable contrast to the effect of certain types of phosphorylation which can strongly reduce the stoichiometry.

Amino Acid Sequence↗
PubMed1994

A sequence of cytoskeleton changes related to the formation of neurofibrillary tangles and neuropil threads.

Frontal sections of the temporal lobe including the transentorhinal/entorhinal region, amygdala, and/or hippocampus from human adult brains are studied for cytoskeleton changes using immunostaining with the antibodies AT8 and Alz-50 and selective silver impregnation methods for neurofibrillary changes of the Alzheimer type. For the purpose of correlation, the two methods are carried out one after the other on the same section. Layer pre-alpha in the transentorhinal/entorhinal region harbours nerve cells which are among the first nerve cells in the entire brain to show the development of neurofibrillary changes. This presents the opportunity for study of both early events in the destruction of the cytoskeleton in individual neurons, and to relate changes which occur in the neuronal processes in the absence of alterations in their immediate surroundings to those happening in the soma. Immunoreactions with the AT8 antibody in particular reveal a clear sequence of changes in the neuronal cytoskeleton. Group 1 neurons present initial cytoskeleton changes in that the soma, dendrites, and axon are completely marked by granular AT8 immunoreactive material. These neurons appear quite normal and turn out to be devoid of argyrophilic material when observed in silver-stained sections. Group 2 neurons show changes in the cellular processes. The terminal tuft of the apical dendrite is replaced by tortuous varicose fibres and coarse granules. The distal portions of the dendrites are curved and show appendages and thickened portions. Intensely homogeneously immunostained rod-like inclusions are encountered in these thickened portions and in the soma. A number of these rod-like inclusions are visible after silver staining, as well. Group 3 neurons display even more pronounced alterations of their distal--most dendritic portions. The intermediate dendritic parts lose immunoreactivity, but the soma is homogeneously immunostained. Silver staining reveals in most of the distal dendritic parts neuropil threads, and in the soma a classic neurofibrillary tangle. Group 4 structures are marked by accumulations of coarse AT8-immunoreactive granules. Silver staining provides evidence that the fibrillary material has become an extraneuronal, "early" ghost tangle. Finally, group 5 structures present "late" ghost tangles in silver-stained sections but fail to demonstrate AT8 immunoreactivity. It is suggested that the altered tau protein shown by the antibody AT8 represents an early cytoskeleton change which eventually leads to the formation of argyrophilic neurofibrillary tangles and neuropil threads.

Adult↗
PubMed1994

Tau protein and Alzheimer's disease.

The etiology of Alzheimer's disease is still unknown. Because the disease is specific for human brain, a rational search for early diagnosis or prevention is very difficult. This calls for the development of cellular models that mimick the degeneration of neurons in AD. The brains of AD patients contain markers whose composition and location is characteristic of the disease; one of the most reliable markers is tau protein in its pathologically phosphorylated and aggregated form. This form of tau can serve as a guide to the origins of the pathology. One goal of research that should be feasible within the near future is to construct a cell that induces the same abnormal changes in tau protein in response to defined stimuli (extracellular signals, toxins, stress, etc.). This model can then be used to identify possible substances that might cause the disease, or identify strategies for preventing it. Once they are defined on a cellular level, the next step would be to test them on (transgenic) animal models which are being developed at present.

Alzheimer Disease↗
PubMed1994

Phosphorylation of microtubule-associated proteins MAP2a,b and MAP2c at Ser136 by proline-directed kinases in vivo and in vitro.

The microtubule-associated protein 2 (MAP2) and its juvenile splicing variant MAP2c contain a phosphorylation site at Ser136 which is part of a Ser-Pro motif. This site lies within the N-terminal region common to MAP2b and MAP2c. It has been mapped by site-directed mutagenesis of recombinant MAP2c and by a monoclonal antibody AP18 whose epitope contains the phosphorylated Ser136. In vitro this site is phosphorylated by proline-directed kinases such as MAP kinase, GSK-3, or members of the cdk family, but not by other kinases such as PKA, PKC, or CaMK-II. MAP2a,b or MAP2c isolated from brain is found to be endogenously phosphorylated at Ser136. After microinjection into several cell lines dephosphorylated MAP2 isoforms or recombinant MAP2c become also phosphorylated at Ser136 in vivo. Injection of MAP2a,b or MAP2c into living cells causes reorganization of microtubules, including bundle formation. This effect is independent of the phosphorylation at Ser136. The specificity of the phosphorylation reaction provides a tool for analyzing the role and posttranslational processing of MAP2 in nerve cell development.

3T3 Cells↗
PubMed1993

Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5.

We have shown earlier that certain proline-directed kinases such as MAP kinase or GSK-3 can phosphorylate tau protein in an abnormal manner reminiscent of tau from Alzheimer paired helical filaments [Drewes et al. (1992); Mandelkow et al. (1992)]. Both kinases are abundant in brain tissue and associate physically with microtubules through several cycles of assembly and disassembly. In this report we show that cdk2/cyclin A incorporates = 5 Pi into recombinant tau, and that it also induces the MR shift and antibody reactivity typical of Alzheimer tau. However, since there is no cdk2 in brain [Meyerson et al. (1992)] we looked for other members of this family of kinases. Using an antibody against the conserved N-terminus we isolated a cdk-like kinase from brain which was capable of inducing the Alzheimer-like characteristics in tau by phosphorylation. Its size (31 kDa), target specificity (proline-directed), chromatographic behavior, and abundance in brain suggest that this kinase is similar or identical to the neuronal cdc2-like kinase nclk alias PSSARLE or cdk5 [Hellmich et al. (1992); Meyerson et al. (1992); Xiong et al. (1992); Tsai et al. (1993)]. This was confirmed by an antibody specific for cdk5. Like MAP kinase and GSK-3, this kinase is physically associated with microtubules and can be enriched by cycles of microtubule assembly and disassembly. Thus, cdk5 should be regarded as another kinase that could be held responsible for the changes in tau protein during Alzheimer disease progression.

Alzheimer Disease↗
PubMed1993

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A.

We have shown previously that brain tissue contains protein kinases which can phosphorylate tau protein to a state reminiscent of the pathological state of Alzheimer paired helical filaments (PHFs); these include proline-directed kinases which phosphorylate SP or TP motifs (such as MAP kinase and GSK-3) [Drewes et al. (1992); Mandelkow et al. (1992)], as well as a novel kinase which phosphorylates S262 of tau protein and thereby strongly reduces the binding of tau to microtubules [Biernat et al. (1993)]. Here we report on the corresponding phosphatases in brain which normally keep the 'pathological' sites free of phosphate. The major phosphatases acting on tau are calcineurin and PP-2A, but not PP-1. Both are present and active in brain extracts, they can dephosphorylate recombinant tau after prior phosphorylation with either MAP kinase, GSK-3, or brain extract, and the course of dephosphorylation can be monitored with antibodies diagnostic of the pathological state of tau. Both phosphatases also act directly on PHF tau isolated from Alzheimer brains.

Alzheimer Disease↗
PubMed1993

Microtubule-associated protein tau, paired helical filaments, and phosphorylation.

This paper summarizes our recent studies on microtubule-associated protein tau and its pathological state resembling that of the paired helical filaments of Alzheimer's disease. The Alzheimer-like state of tau protein can be identified and analyzed in terms of certain phosphorylation sites and phosphorylation-dependent antibody epitopes. It can be induced by protein kinases which tend to phosphorylate serine or threonine residues followed by a proline; this includes mitogen-activated protein kinase (MAPK) and glycogen-synthase kinase 3 (GSK-3). Both of these are tightly associated with microtubules as well as with paired helical filaments. Structurally, tau appears as a rod-like molecule; it tends to self-associate into dimers whose monomers are antiparallel. Constructs of truncated tau made up of antiparallel dimers of the microtubule binding domain can be assembled into paired helical filaments in vitro.

Alzheimer Disease↗
PubMed1993

Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding.

Tau protein, a component of Alzheimer paired helical filaments, can be phosphorylated by several kinases. Of particular interest is the phosphorylation at Ser/Thr-Pro motifs because the resulting state of tau is similar to that found in Alzheimer's disease, as judged by its immunoreactivity. This state can be mimicked by a brain extract kinase activity and by MAP kinase. We have now studied the effect of these modes of phosphorylation on the interaction between tau and microtubules. Although MAP kinase efficiently phosphorylates many Ser/Thr-Pro motifs of tau, its effect on microtubule binding is only moderate. By contrast, phosphorylation of a single residue, Ser262, has a major effect on binding. Ser262 is not phosphorylated by MAP kinase or other proline-directed kinases, but is phosphorylated by a 35/41 kd kinase in brain, whose purification is described.

Alzheimer Disease↗
PubMed1993

Tau as a marker for Alzheimer's disease.

Alzheimer's Disease (AD) is an age-related dementia that has received increasing attention in recent years. The disease appears to be specific for humans and, moreover, its causes are unknown. Studies of the disease are complicated by the fact that a reliable diagnosis depends on post-mortem analysis of brain tissue, and useful animal or cell models are not yet available. This article discusses the possibility of using tau (a protein associated with the neurofibrillary tangles that are an indicator of AD) as a tool for further pursuing the diagnosis and treatment of the disease.

Alzheimer Disease↗
PubMed1993

Dynamics of microtubules from erythrocyte marginal bands.

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.

Animals↗
PubMed1992

Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau.

The Alzheimer-like state of tau protein includes phosphorylation by a proline-directed Ser/Thr kinase present in normal or pathological human brain. Extending earlier results on MAP kinase, we show here that the proline-directed kinase, GSK3, can induce an Alzheimer-like immune response involving several distinct and phosphorylatable epitopes at Ser-Pro motifs, as well as a gel mobility shift, similar to MAP kinase. Both kinases behave like microtubule-associated proteins in that they co-purify through cycles of assembly and disassembly, and both kinases are directly associated with paired helical filaments.

Alzheimer Disease↗
PubMed1992

The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs.

Tau protein can be transformed into an Alzheimer-like state by phosphorylation with a kinase activity from brain [Biernat et al. (1992) EMBO J. 11, 1593-1597]. Here we show that the phosphorylation at Ser-Pro motifs strongly decreases tau's affinity for microtubules. The major reduction occurs during the first of the three main stages of phosphorylation. The data explain the lower stability of microtubules resulting from the pathological tau phosphorylation.

Alzheimer Disease↗
PubMed1992

Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.

We have studied the phosphorylation of tau protein from Alzheimer paired helical filaments, of tau from normal human brain, and of recombinant tau isoforms. As a tool we used monoclonal antibodies against neurofilament protein [Sternberger, N., Sternberger, L. & Ulrich, J. (1985) Proc. Natl. Acad. Sci. USA 82, 4274-4276] that crossreact with tau in a phosphorylation-dependent manner. This allowed us to deduce the state of phosphorylation in normal and pathological tau, as well as antibody epitopes. The epitope of antibody SMI33 is at the first Lys-Ser-Pro sequence motif (residues 234-236) and requires an unphosphorylated Ser-235. Antibody SMI31 binds between Ser-396 (in the second Lys-Ser-Pro motif) and Ser-404, both of which must be phosphorylated. SMI34 has a conformational epitope that depends on the interaction between regions on either side of the microtubule-binding region; it also requires phosphorylation. The phosphorylatable serines detected by the SMI antibodies are part of Ser-Pro motifs and can be phosphorylated by a protein kinase activity that can be used to induce a paired helical filament-like state in human brain tau in vitro. The phosphates are incorporated in several stages that can be identified by antibody reactivity and gel shift. This suggests a role for the phosphorylation sites in Alzheimer disease, as well as the involvement of a Ser-Pro-directed protein kinase.

Alzheimer Disease↗
PubMed1992

The juvenile microtubule-associated protein MAP2c is a rod-like molecule that forms antiparallel dimers.

We have developed a procedure to isolate the microtubule-associated protein 2c (MAP2c), a juvenile form of MAP2 occurring in mammalian brain. The shape, size, self-association, and antibody interactions of MAP2c were studied. Monomeric MAP2c is an elongated molecule with a length approximately 48 nm, considerably shorter than the higher molecular weight forms MAP2a or b of adult brain. Two monoclonal antibodies whose epitopes are near the N or C terminus, respectively, are located close to the opposite ends of the MAP2c rods. This places constraints on the types of internal folding of the molecule. MAP2c self-associates into dimers and fibrous aggregates. The dimers are predominantly antiparallel and nearly in register, as judged by antibody labeling.

Animals↗