The microtubule (MT)-associated protein tau is important in neuronal development and in Alzheimer's and other neurodegenerative diseases. Genetic analyses have established a cause-and-effect relationship between tau dysfunction͞misregulation and neuronal cell death and dementia in frontotemporal dementia and parkinsonism associated with chromosome 17; several mutations causing this dementia lead to increased ratios of four-repeat (4R) to three-repeat (3R) wild-type tau, and an attractive hypothesis is that the abnormally high ratio of 4R to 3R tau might lead to neuronal cell death by altering normal tau functions in adult neurons. Thus, we tested whether 3R and 4R tau might differentially modulate the dynamic instability of MTs in vitro using video microscopy. Although both isoforms promoted MT polymerization and decreased the tubulin critical subunit concentration to approximately similar extents, 4R tau stabilized MTs significantly more strongly that 3R tau. For example, 4R tau suppressed the shortening rate, whereas 3R tau had little or no detectable effect. Similarly, 3R tau had no effect on the length shortened during a shortening event, whereas 4R tau strongly reduced this parameter. Further, when MTs were diluted into buffer containing 4R tau, the MTs were stabilized and shortened slowly. In contrast, when diluted into 3R tau, the MTs were unstable and shortened rapidly. Thus, 4R tau stabilizes MTs differently and significantly more strongly than 3R tau. We suggest a ''dosage effect'' or haploinsufficiency model in which both tau alleles must be active and properly regulated to produce appropriate amounts of each tau isoform to maintain MT dynamics within a tolerable window of activity.M any neurodegenerative diseases exhibit abnormal pathological fibers composed primarily of the microtubule (MT)-associated protein, tau (for a recent review, see ref. 1). These disorders, termed ''tauopathies,'' include Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), Pick's disease, and progressive supranuclear palsy. In 1998, several groups reported a direct genetic linkage between mutations in the tau gene and FTDP-17 (2-5). Although some tau mutations are structural and others are regulatory, all exhibit dominant phenotypes. Thus, both dysfunction and misregulation of tau are causally related to neuronal cell death, neurodegenerative disease, and dementia.Tau is normally present predominantly in the cell bodies and axons of neuronal cells and is necessary for the establishment of neuronal cell polarity and axon outgrowth, axonal transport, and maintenance of axonal morphology (6-10). Tau is also expressed in glial cells (11). Mechanistically, tau stimulates MT polymerization, stabilizes MTs, and suppresses MT dynamics (12-15). Because MT dynamics must be tightly regulated for cells to function and remain viable (e.g., ref. 16), it follows that the action of tau must also be finely regulated.Although there is only a single tau gene, alternative splicing of tau mRNA p...

The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low-to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo exchange with the bulk medium readily, suggesting that the taumicrotubule interaction is essentially irreversible. Given this dilemma, we investigated the mechanism of interaction between tau and microtubules in kinetic detail. Stopped-flow kinetic analysis reveals moderate binding affinity between tau and preassembled microtubules and rapid dissociation͞association kinetics. In contrast, when microtubules are generated by copolymerization of tubulin and tau, a distinct population of microtubule-bound tau is observed, the binding of which seems irreversible. We propose that reversible binding occurs between tau and the surface of preassembled microtubules, whereas irreversible binding results when tau is coassembled with tubulin into a tau-microtubule copolymer. Because the latter is expected to be physiologically relevant, its characterization is of central importance.T he microtubule-associated protein (MAP) tau is a critical regulator of microtubule dynamics, and defects in microtubule dynamics have been shown to result in cell death (1). Furthermore, a number of mutations in the tau gene that are associated with frontotemporal dementia with parkinsonism linked to chromosome 17 have proven that tau dysfunction can play a causal role in the death of neurons (2). A characteristic defect associated with these mutations is a weaker interaction between tau and microtubules. Thus, knowledge of the detailed mechanism by which tau binds to microtubules is of crucial importance.To date, no information is available on the kinetic steps involved in the binding process; rather, all studies on the mechanism of tau binding to microtubules have been thermodynamic in nature. Studies by Weingarten et al. (3) demonstrated that tau does not interconvert between differently radioactively tagged tubulin fractions through multiple cycles of polymerization and depolymerization. Thus, binding is inferred to be tight, perhaps irreversible. On the other hand, purified tau binds to microtubules with moderate as opposed to high affinity (4-10), and consequently the microtubule-bound and free forms of tau are expected to be readily exchangeable. Thus, not only is kinetic information lacking, but the available thermodynamic data also seem to be conflicting.To address these issues, we carried out rapid-mixing stoppedflow kinetic experiments as well as equilibrium-competition binding studies to investigate the mechanism of interaction between tau and microtubules. Our data suggest that tau can bind to two distinct sites on microtubules: one that displays ...

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wildtype ϳ50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a "microtubule misregulation" model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.

A strategy employing a combination of peptide nucleic acid (PNA) probes, an optically amplifying conjugated polymer (CP), and S1 nuclease enzyme is capable of detecting SNPs in a simple, rapid, and sensitive manner. The recognition is accomplished by sequence-specific hybridization between the uncharged, fluorescein-labeled PNA probe and the DNA sequence of interest. After subsequent treatment with S1 nuclease, the cationic water soluble CP electrostatically associates with the remaining anionic PNA͞ DNA complex, leading to sensitized emission of the labeled PNA probe via FRET from the CP. The generation of fluorescent signal is controlled by strand-specific electrostatic interactions and is governed by the complementarity of the probe͞target pair. To assess the method, we compared the ability of the sensor system to detect normal, wild-type human DNA sequences, and those sequences containing a single base mutation. Specifically, we examined a PNA probe complementary to a region of the gene encoding the microtubule associated protein tau. The probe sequence covers a known point mutation implicated in a dominant neurodegenerative dementia known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), which has clinical and molecular similarities to Alzheimer's disease. By using an appropriate PNA probe, the conjugated polymer poly[(9,9-bis(6 -N,N,Ntrimethylammoniumhexylbromide)fluorene)-co-phenylene] and S1 nuclease, unambiguous FRET signaling is achieved for the wildtype DNA and not the mutant sequence harboring the SNP. Distance relationships in the CP͞PNA assay are also discussed to highlight constraints and demonstrate improvements within the system. biosensor ͉ energy transfer ͉ hybridization probes ͉ polyelectrolyte ͉ fluorescence

Historically, sodium azide has been used to anesthetize the nematode Caenorhabditis elegans; however, the mechanism by which it survives this exposure is not understood. In this study, we report that exposure of wild-type C elegans to 10 mM sodium azide for up to 90 minutes confers thermotolerance (defined as significantly increased survival probability [SP] at 37 degrees C) on the animal. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed enhanced Hsp70 expression, whereas Western blot analysis revealed the induction of Hsp16. We also tested the only known C elegans Hsp mutant def-21 (codes for Hsp90), which constitutively enters the stress-resistant state known as the dauer larvae. Daf-21 mutants also acquire sodium azide-induced thermotolerance, whereas 3 non-Hsp, constitutive dauer-forming mutants exhibited a variable response to azide exposure. We conclude that the ability of C elegans to survive exposure to azide is associated with the induction of at least 2 stress proteins.