Wild-type Tau Research Articles

The tau mutation (val337met) disrupts cytoskeletal networks of microtubules.

The missense point mutation found in the tau gene, which was segregated in a family with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), has proved to be the causal molecule for widely spread dementia diseases. Here we examined the effects of the tau mutation using confocal analysis. When wild-type tau cDNA was introduced into cells, extensive cell processes and well-developed thick bundles of microtubules were induced. On the other hand, when altered tau cDNA with the mutation (valine337-methionine) was introduced, cell lost processes and microtubule networks resulted in more round cell shape but showed intact mitochondria or endoplasmic reticulum. We conclude that the tau mutation primarily affects the microtubules and resultantly causes the loss of cellular organization and function due to microtubule disruption.

Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules.

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at approximately 20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.

Phosphorylation of tau by glycogen synthase kinase 3β affects the ability of tau to promote microtubule self-assembly

To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules.

Conversion of Serine to Aspartate Imitates Phosphorylation-induced Changes in the Structure and Function of Microtubule-associated Protein Tau

Microtubule-associated protein tau is a neuronal phosphoprotein that promotes microtubule assembly in vitro and has been shown to play a role in the development of axonal morphology. Tau can be phosphorylated in vitro by several kinases, some of which cause a change in the conformation and activities of tau. Here we report the consequences of converting two of the protein kinase A phosphorylation sites (positions 156 and 327), first to alanine to eliminate phosphorylation, and second to aspartate, to mimic phosphorylation. We show that a serine to aspartate mutation at position 327 results in a conformational change similar to that caused by phosphorylation of this residue. This mutation does not affect the activities of tau in microtubule assembly as compared with wild-type tau. However, an additional mutation at position 156 to aspartate drastically decreases the microtubule nucleation activity of tau but does not affect the activity of tau to promote microtubule growth. All constructs are similarly bound to microtubules and promote process formation when expressed in cytochalasin-treated PC12 cells. We conclude that serine to aspartate mutations provide a useful system for analyzing the effect of individual phosphorylation sites on the conformation and function of tau in vitro and in cells. The results provide evidence that microtubule growth and nucleation can be differentially affected by phosphorylation of individual residues in a region amino-terminally flanking the microtubule binding domain of tau.

Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle.: IV. ATP-BINDING SITE MUTANTS IDENTIFY THE CLAMP LOADER

The gamma complex (gamma delta delta' chi psi) and tau complex (tau delta delta' chi psi) clamp loaders require ATP hydrolysis to load beta sliding clamps onto DNA. The beta sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis. Pol III* contains both gamma and tau, but only one each of the delta, delta', chi, and psi subunits. Hence, there is ambiguity with respect to which clamp loader, the gamma or tau complex, exists in the Pol III* replicase structure. In this study, ATP-binding site mutants of gamma and tau have been prepared, and these mutants, when assembled into either the gamma or tau complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol III*. The nine-subunit Pol III* has been assembled using either mutant gamma or tau in place of wild-type gamma or tau. The results show that mutation of gamma inactivates Pol III* activity, but mutation of tau does not, indicating that the gamma complex (and not the tau complex) is the clamp loader of Pol III*. The tau subunit carries the task of dimerizing the core polymerase, and it is this association of tau with core that appears to direct the single copy subunits away from tau and onto gamma.

Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404.

The microtubule-associated protein tau is hyperphosphorylated in the paired helical filaments (PHFs) of Alzheimer's disease. Immunological and direct chemical studies have identified Ser396 and Ser404 as two of the phosphorylated sites. Previously, we have demonstrated, using synthetic tau peptides containing phosphorylated Ser396, that this site is recognized by the monoclonal antibody PHF-1. The present study extends this observation by showing that PHF-1 recognizes tau peptides containing either individually phosphorylated Ser396 or Ser404, but that there is a > 10-fold increase in the sensitivity of detection of tau peptides by PHF-1 when both serines are phosphorylated. The recognition of singly or doubly phosphorylated Ser396 and Ser404 in tau by PHF-1 can also be demonstrated in Chinese hamster ovary cells transfected with full-length wild-type tau constructs or mutant constructs with Ala substituted for Ser396 or Ser404. We conclude that the PHF-1 epitope contains both phosphorylated Ser396 and Ser404.

Multiple pathways of copy control of gamma replicon of R6K: mechanisms both dependent on and independent of cooperativity of interaction of tau protein with DNA affect the copy number.

The ability of a replication initiator protein to promote intermolecular pairing of two replication origins resulting in the turning off of the origin pair has been called handcuffing. We have endeavored to test the validity of the handcuffing model by isolating two mutant forms of the tau initiator protein of R6K that elicit high copy number phenotype. We have discovered that one mutant called tau 113 yielded a 3.6-fold increase in copy number of a gamma replicon with a concomitant impairment of its ability to loop DNA and to pair binding sites (iterons) in comparison with normal tau, thus supporting the handcuffing model. A second mutant called tau 108, on the other hand, elicited a 3-fold increase in copy number without showing any measurable loss in its ability to loop and pair gamma iterons. Both mutant forms of the wild-type tau protein showed no detectable differences in their affinity of binding to the gamma iterons. Thus, the phenotype of tau 108 is consistent with the proposition that copy number control involves macromolecular interactions other than cooperativity at a distance of tau or interaction of tau with the primary binding sites at gamma. Taken together, the results are consistent with the notion that tau-mediated handcuffing is a mechanism, but not the only mechanism, of copy control in R6K. Interaction of tau with host proteins is likely to provide additional facets of the copy control mechanism.

Apoptotic cell death in Alzheimer's disease : Carl W. Cotman, Ph.D., David Cribbs, Ph.D., Christian J. Pike, Ph.D., and John Watt, Ph.D., Irvine Research Unit in Brain Aging, University of California, Irvine, CA 92717, USA

Conditional expression systems for 4-repeat wild-type (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 × g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.