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Originally published In Press as doi:10.1074/jbc.M413490200 on January 25, 2005

J. Biol. Chem., Vol. 280, Issue 14, 13520-13528, April 8, 2005
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Three- and Four-repeat Tau Regulate the Dynamic Instability of Two Distinct Microtubule Subpopulations in Qualitatively Different Manners

IMPLICATIONS FOR NEURODEGENERATION*

Sasha F. Levy{ddagger}, Adria C. LeBoeuf{ddagger}, Michelle R. Massie, Mary Ann Jordan, Leslie Wilson, and Stuart C. Feinstein§

From the Neuroscience Research Institute and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106

Received for publication, November 30, 2004 , and in revised form, January 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 wild-type ~50:50 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The microtubule (MT)1-associated protein tau is necessary for the establishment of neuronal cell polarity, axonal outgrowth, axonal transport, and the maintenance of axonal morphology (1–5). Tau dysfunction has been correlated with a variety of neurodegenerative diseases, including Alzheimer disease, fronto-temporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), Pick disease, and progressive supranuclear palsy. Each of these diseases is characterized by dead neurons filled with abnormal fibers primarily composed of hyperphosphorylated tau (6–8). In 1998, several groups (9–12) reported a direct genetic linkage between mutations in the tau gene and FTDP-17. These mutations all exhibit dominant phenotypes and fall into the following two general classes: structural mutations that alter the encoded sequence of the tau protein, and regulatory mutations that alter the pattern of tau RNA alternative splicing (13). Mutations in the latter class do not affect the primary sequence of tau but rather alter the expression ratios of the different wild-type isoforms. Thus, both tau dysfunction and misregulation of isoform expression can cause neuronal cell death and dementia.

Although there is only a single tau gene, alternative RNA splicing of exons 2, 3, and 10 produces six different tau isoforms in the central nervous system (14–16). Each of the tau isoforms possess either three or four imperfect 18 amino acid repeats separated by 13–14 amino acid inter-repeats near the C terminus (Fig. 1A). The inclusion or exclusion of exon 10, encoding the first inter-repeat and the second repeat, determines whether a molecule will be a "4-repeat tau" (4R) or a "3-repeat tau" (3R). Inclusion or exclusion of exons 2 and 3 near the N terminus results in short (S), medium (M), or long (L) tau.



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  		FIG. 1.
A, schematic of the six alternatively spliced tau isoforms expressed in the central nervous system. Boxes above each line correspond to the 18-amino acid imperfect repeats; boxes below each line correspond to alternatively spliced exons. Tau isoforms are designated as either 3R or 4R, which differ by the presence or absence of exon 10, a 31-amino acid insertion that contains the first inter-repeat and repeat 2. Isoforms are also designated as either S (short), M (medium), or L (long), depending on whether they contain 0, 1, or 2 of the 29-amino acid inserts near their N termini, exons 2 and 3. B, equal masses of a 3RM tau standard (STD), whose concentration had been determined by amino acid analysis, and all HPLC-purified tau isoforms were separated by SDS-PAGE and stained with Coomassie Blue. The band density of each isoform did not vary from the others by more than 10%. C, MT co-sedimentation assay. 4R and 3R tau were incubated with 15 µM tubulin at various tau:tubulin molar ratios until steady-state and co-sedimented with MT polymer. The percent of MT-bound tau was determined by quantitating the pellet (bound) and supernatant (free) fractions by tau-1 Western blots.

 
Because proper MT behavior is essential for numerous cellular functions (17, 18) and tau is an important regulator of MT behavior (19–23), it is not surprising that cells impose extensive regulatory control over tau activity. Indeed, tau activity is finely regulated, both by complex combinatorial patterns of phosphorylation (6) and developmentally regulated alternative RNA splicing. Human fetal brain expresses only the shortest tau isoform (3RS), whereas all six tau isoforms are expressed in the adult brain, with 4R and 3R tau being expressed in an ~50:50 ratio (10, 14–16, 24, 25). The regulatory FTDP-17 mutations cause disease by expressing improper ratios of wild-type tau isoforms, generally resulting in an increase in the expression ratio of 4R:3R tau to ~75:25 (13). The fact that such alterations in isoform ratio are sufficient to induce neuronal cell death highlights the necessity for fine regulation of tau activity and indicates that 4R and 3R tau are likely to exert functionally distinct effects on MTs.

Previous work suggests that 4R and 3R tau differ mechanistically in their interactions with MTs; however, whether the differences are qualitative or only quantitative remains unclear. Four-repeat tau binds to MTs with greater affinity than 3R tau (26–28); 4R tau is also a more potent promoter of MT assembly in vitro (24, 29) and a more potent regulator of MT dynamics (20, 23). However, competition studies of MT binding suggest that 4R and 3R tau may interact with MTs in qualitatively different ways (30). Differences between the short, medium, and long isoforms have been much less intensively investigated. Although these N-terminal inserts do not affect MT binding affinity (27), they do appear to regulate MT spacing and bundling (31–34). However, their ability to regulate MT assembly and dynamics has not been methodically investigated.

Although the genetic linkage between tau mutations and neuronal cell death establishes a clear cause-and-effect relationship between tau dysfunction/misregulation and neurodegenerative disease, the underlying molecular mechanism leading to neuronal cell death and dementia is not well understood. A widely held, gain-of-toxic-function model suggests that tau mutations lead to the formation and accumulation of abnormal intracellular tau fibers, which in turn cause cell death by virtue of their proposed cytotoxicity (10, 35). This model is consistent with the ubiquitous presence of abnormal tau fibers in numerous neurodegenerative diseases (7). However, it has not been established that insoluble tau fibers are in fact cytotoxic. Indeed, numerous animal models have shown that tau-mediated neurodegeneration can occur in the absence of tangles, indicating that they may not necessary for cell death (36–38). An alternative "microtubule misregulation" model suggests that tau mutations interfere with the normal ability of tau to regulate MT dynamic instability, which in turn leads to cell death. This model is consistent with a variety of pharmacological and somatic cell genetics investigations demonstrating that tight regulation of MT dynamics is essential for proper cell function and viability (17, 18, 39, 40).

To assess further the plausibility of the MT misregulation model, we have used differential interference contrast enhanced video microscopy to analyze the dynamic instability behavior of individual MTs stabilized by each of the six human tau isoforms. We show that the N-terminal insertions have no effect on the regulation of MT dynamics. However, we have identified both quantitative and qualitative mechanistic differences between 4R and 3R tau. 4R tau increases the MT growth rate at all concentrations tested, 3R tau decreases the MT growth rate at low tau:tubulin ratios and increases the MT growth rate at high tau:tubulin ratios. Furthermore, we found two subpopulations of growing MTs in both the absence and presence of tau. Both 4R and 3R tau exerted two distinct effects on these subpopulations, which we term "subpopulation shifting" and "subpopulation redistribution," but did so in different ways. These observations provide strong support for the view that abnormal tau action can cause neuronal cell death and dementia by virtue of aberrant regulation of MT dynamics.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of Tau and Tubulin—Expression vectors containing the human cDNA sequences encoding all six tau isoforms expressed in the central nervous system (pET vector containing 3RS tau; pRK vectors containing 3RM, 3RL, 4RS, 4RM, and 4RL) were the kind gifts from Dr. Kenneth Kosik (University of California, Santa Barbara) and Dr. Gloria Lee (Iowa State University). Tau was expressed and purified, as described (23). Briefly, tau was expressed in Rosetta (DE3) pLacI cells (Novagen, Madison, WI). Bacteria were lysed by sonication and boiled for 20 min. Heat-stable proteins were isolated by centrifugation, bound to a phosphocellulose column, and eluted with a salt gradient (0.2 to 1.0 M NaCl). Tau-containing fractions were pooled and further purified using reverse phase-HPLC (DeltaPak-C18; Millipore, Billerica, MA). HPLC fractions containing tau were pooled, lyophilized, and resuspended in BRB-80 buffer (80 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM MgSO4) with 0.1% {beta}-mercaptoethanol. The concentration of each tau sample was determined by SDS-PAGE comparison with a 3RM tau mass standard, the concentration of which was established by amino acid analysis (20). The accuracy of our tau concentration determinations is demonstrated by the gel shown in Fig. 1B.

Tubulin was purified, as described (41). Briefly, microtubule-associated protein-rich bovine brain MT protein was prepared by three cycles of assembly and disassembly. Tubulin was purified from other MT proteins by elution through a Whatman P-11 phosphocellulose column equilibrated in PEM50 (50 mM Pipes, 1 mM MgSO4, 1 mM EGTA, 0.1 mM GTP). Purified tubulin (>99% pure) was drop-frozen in liquid nitrogen and stored at –70 °C.

MT Co-sedimentation Assays—Tubulin (15 µM tubulin dimer) was mixed with 3RS or 4RS tau at 0.19 µM (1:80 tau:tubulin), 0.25 µM (1:60), 0.75 µM (1:20), 1.5 µM (1:10), and 7.5 µM (1:2) in PMEM buffer (87 mM Pipes, 36 mM MES, 1.4 mM MgCl2, 1 mM EDTA, pH 6.8) with 2 mM GTP. Microtubules were assembled at 35 °C until steady-state was achieved (2 h), layered over an 80-µl sucrose cushion (50% sucrose in PMEM, 2 mM GTP) in 5 x20-mm ultraclear centrifuge tubes (Beckman Instruments, Palo Alto, CA), and centrifuged in a Beckman AH650 swinging bucket rotor for 12 min at 35,000 rpm (150,000 x g) at 35 °C. Supernatants and pellets were harvested and solubilized in SDS-PAGE sample buffer. Relative amounts of tau in the supernatants and pellets were determined by SDS-PAGE and immunoblotting with the monoclonal antibody tau-1 (42).

Dynamic Instability Analyses—Purified tubulin (15 µM tubulin dimer) was polymerized at the ends of sea urchin (Strongylocentrotus purpuratus) axonemal seeds at 37 °C in the presence or absence of a purified tau isoform in PMEM buffer (87 mM Pipes, 36 mM MES, 1.4 mM MgCl2, 1 mM EDTA, pH 6.8) and 2 mM GTP. The dynamics of individual MTs were recorded at 37 °C using differential interference contrast-enhanced video microscopy. The ends were designated as plus or minus on the basis of the growth rate, the number of MTs that grew at opposite ends of the seeds, and the relative lengths of the MTs (21, 43). Plus ends were analyzed during the early elongation phase of polymerization (2–10 min after initiation of polymerization). Life histories of individual MTs were collected as described by Panda et al. (21) with modifications. Data points were collected at 1- to 3-s intervals. Recalibration of tracking software revealed a previous error in scaling factors, resulting in previously reported growth and shortening rates being ~1.56-fold below true values. Values reported here have been corrected for this error, and thus growth and shortening classifications have been corrected accordingly. A MT was considered to be growing or shortening if it increased or decreased in length at a rate >0.5 µm/min. MTs exhibiting growth rates of ≤0.5 µm/min over a period greater than 30 s were considered to be in an attenuated state. Average growth rates are the average of independent growth events. The catastrophe frequency was calculated by dividing the number of shortening events by the total time tracked. Growth rate histograms were plotted by grouping individual growth events into 0.4 µm/min bins. Best-fit Gaussian distributions were determined using Kaleidagraph software. To control for experimental error, each condition was filmed over multiple days using 2–5 distinct tau/tubulin/GTP mixtures (3–4 slides each). No gross variation in MT dynamics was observed between mixtures or slides of a given condition. Growth rates of the relatively fast and slow growing subpopulations did not grossly correlate with time after polymerization initiation. Growth rates of a given MT were typically different before and after an attenuation or catastrophe event. MTs were tracked by two independent investigators each of whom obtained approximately equal average growth rates for each condition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Before determining the effects of 4R and 3R tau on in vitro MT dynamic instability, we first sought to determine the efficiency with which each isoform incorporates into growing MTs under the conditions of our standard MT dynamics assay. Therefore, we used a co-sedimentation assay to determine the fraction of tau bound to MTs assembled with varying tau: tubulin dimer ratios (1:80 to 1:2). As shown in Fig. 1C, >95% of both 4R and 3R tau was MT-associated at ratios of 1:80, 1:60, and 1:20. At 1:10 and 1:2, an increasing fraction of the tau was not MT-associated, indicating saturation of the MTs. Based on these data, all dynamics studies were performed at tau:tubulin molar ratios between 1:20 and 1:55. Thus, any differences in the abilities of 4R and 3R to regulate MT dynamics can be attributed to inherent mechanistic differences between the bound isoforms.

4R and 3R Tau Have Opposite Effects on the MT Growth Rate at Low Tau:Tubulin Ratios—The in vitro dynamic instability behavior at the plus ends of individual MTs assembled in the presence of 3RS tau or 4RS tau was analyzed during the early elongation phase of MT polymerization. Examples of the dynamic instability of MTs assembled with either no tau, 3RS tau, or 4RS tau at a tau:tubulin ratio of 1:55 are shown in Fig. 2A. Because these studies were performed during the early elongation phase of MT polymerization, we observed mostly growth events with few catastrophes or attenuations. Consistent with previous findings (20, 22), quantitative analysis of many MTs revealed that 4RS tau increased the average MT growth rate significantly (p < 0.01) from 2.8 ± 0.1 µm/min for control MTs to 3.3 ± 0.1 µm/min. Most surprisingly, the same concentration of 3RS tau had the opposite effect, significantly (p < 0.01) decreasing the average growth rate to 2.3 ± 0.1 µm/min (Fig. 2B).



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  		FIG. 2.
3R tau decreases and 4R tau increases the MT growth rate. The growing and shortening of individual MTs grown during early elongation were determined by video microscopy. A, typical life history traces of 15 µM tubulin in the absence of tau (gray solid lines) or in the presence of 0.27 µM 3RS (black solid lines) or 4RS (black dashed lines) tau. The gray trace demarcated with an asterisk exhibits a growth (~0–240 s), an attenuation (~240–370 s), a catastrophe (~370 s; the transition to shortening from growth or attenuation), and a shortening (~370–390 s). B, average MT growth rates of 15 µM tubulin grown in the absence or presence of 0.27 µM 3RS or 4RS tau (1:55 tau:tubulin dimer). Student's t test, *, p < 0.01.

 
To ensure that the unexpected decrease in MT growth rate was not unique to an idiosyncratic aspect of a given tau or tubulin preparation, the above experiments were repeated using independent preparations of all six tau isoforms and a different tubulin preparation. At a 1:55 tau:tubulin ratio, 4RS, 4RM, and 4RL each significantly (p < 0.05) increased the MT growth rate (2.6 ± 0.1, 2.7 ± 0.1, and 2.7 ± 0.1 µm/min, respectively) relative to MTs assembled in the absence of tau (2.3 ± 0.1 µm/min), whereas 3RS, 3RM, and 3RL each significantly (p < 0.05) decreased the growth rate (2.0 ± 0.1, 2.0 ± 0.1, and 1.9 ± 0.1 µm/min, respectively; Table I and Fig. 3). Consistent with earlier work, all six isoforms dramatically decreased the catastrophe frequency (Table I).


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  		TABLE I
Effects of all tau isoforms on microtubule growth and shortening

Average growth rate values are given as mean ± standard error of the mean.

 



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  		FIG. 3.
N-terminal inserts do not affect MT growth rate. Average growth rates of 15 µM tubulin grown in the presence or absence of 0.27 µM of each tau isoform (1:55 tau:tubulin dimer). Student's t test, *, p < 0.05

 
Two additional points are worthy of note. First, although the data indicate that low concentrations of 3R tau reduce the MT growth rate, the data in Table I also show that 3R tau increases the percentage of time spent growing and decreases the catastrophe frequency. Therefore, the overall effect of low concentrations of 3R tau may be promotion of net microtubule assembly, albeit growth at a slower rate. Second, the N-terminal inserts may have some subtle effect on time spent in attenuation phase. The short (4RS and 3RS), medium (4RM and 3RM), and long (4RL and 3RL) isoforms spent ~5, ~13, and ~9% of the time in attenuation phase, respectively. However, because attenuation events are relatively rare during early elongation, these apparent differences may be due to normal variation within a small sample size.

Both 4R and 3R Tau Increase the Growth Rate at High Tau:Tubulin Ratios—Given that the N-terminal inserts do not appear to dramatically affect MT dynamics (compare values in Table I), we focused our subsequent investigations on a comparison of 4RS and 3RS tau. To characterize further the differences between 4R and 3R tau, we assayed the effect of 4RS and 3RS tau on MT growth rate at four tau:tubulin molar ratios (1:55, 1:45, 1:38, and 1:20). In agreement with previous studies (19, 20, 22), increasing concentrations of 4R tau led to increases in the MT growth rate (Fig. 4 and Table II). Compared with an average MT growth rate in the absence of tau of 2.8 ± 0.1 µm/min, 4R tau:tubulin ratios of 1:55, 1:45, and 1:38 induced increasing growth rates of 3.3 ± 0.1, 3.9 ± 0.2, and 4.4 ± 0.2 µm/min, respectively. This effect, however, began to plateau between ratios of 1:38 and 1:20, with the 1:20 ratio yielding a growth rate of 4.9 ± 0.3 µm/min. In contrast, the suppression of MT growth rates by 3R tau at low tau:tubulin ratios converted to an acceleration of MT growth rates at higher tau: tubulin ratios; 3R tau:tubulin ratios of 1:55 and 1:45 yielded suppressed growth rates of 2.3 ± 0.1 and 2.2 ± 0.2 µm/min, whereas 1:20 yielded an increased growth rate 3.9 ± 0.2 µm/min. Because different tubulin preparations are derived from different bovine brains, they may vary in tubulin isotype composition and extent of post-translational modification. These differences could affect MT dynamics. Therefore, we repeated these assays with independent tau and tubulin preparations to confirm our observations; these experiments yielded similar results (Tables II and III).



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  		FIG. 4.
Effect of increasing concentrations of 3RS tau ({blacktriangleup}) and 4RS tau ({blacksquare}) on MT growth rates. 15 µM tubulin grown in the presence of 0.27 µM (1:55 tau:tubulin), 0.33 µM (1:45), 0.39 µM (1:38), or 0.75 µM (1:20) 3RS or 4RS tau. The horizontal dashed line indicates the average growth rate in the absence of tau.

 


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  		TABLE II
Dose-response of 3RS and 4RS tau on microtubule growth and shortening for tubulin preparation 1

Average growth rate values are given as mean ± standard error of the mean. Distribution of growth events is estimated by calculating the area under each curve for the best-fit Gaussian equation.

 


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  		TABLE III
Dose-response of 3RS and 4RS tau on microtubule growth and shortening for tubulin preparation 2

Average growth rate values are given as mean ± standard error of the mean. Distribution of growth events is estimated by calculating the area under each curve for the best-fit Gaussian equation.

 
MT Growth Rates Segregate into Two Distinct Subpopulations in the Absence of Tau—Examination of the distribution of growth rates revealed two distinct subpopulations both in the absence and presence of tau. The distribution of growth rates in the absence of tau and in the presence of increasing amounts of 3RS and 4RS tau are presented for two different tubulin preparations (left and right columns in Figs. 5 and 6). Although the average growth rates observed for the two different tubulin preparations in the absence of tau varied somewhat (2.8 ± 0.1 versus 2.3 ± 0.1 µm/min), as is commonly observed between independent tubulin samples, the bipolar distribution of growth rates was present with both tubulin preparations. Each tubulin growth rate distribution contained a large, relatively slow growing subpopulation (Gaussian distribution medians of 2.6 and 2.2 µm/min) and a small, faster growing subpopulation (Gaussian distribution medians of 4.4 and 3.7 µm/min, respectively).



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  		FIG. 5.
Frequency histograms of the MT growth rates of 15 µM tubulin (tub) at various 3RS tau:tubulin molar ratios for two different tubulin preparations (A–E and F–J). The tau:tubulin molar ratio is demarcated in the upper left corner of each panel. Vertical lines in each panel indicate the medians of the two Gaussian distribution peaks of 15 µM tubulin in the absence of tau.

 



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  		FIG. 6.
Frequency histograms of the MT growth rates of 15 µM tubulin (tub) at various 4RS tau:tubulin molar ratios for two different tubulin preparations (A–E and F–J). The tau:tubulin molar ratio is demarcated in the upper left corner of each panel. Vertical lines in each panel indicate the medians of the two Gaussian distribution peaks of 15 µM tubulin in the absence of tau.

 
Increasing 3RS Tau Concentrations Increases the Size of the Faster Growing Subpopulation of MTs—Histograms of individual MT growth rates for two independent 3RS tau preparations and two independent tubulin preparations at tau:tubulin ratios of 1:55, 1:45, 1:38, and 1:20 revealed that 3RS tau modulates the growth rate distribution in two distinct manners (Figs. 5 and 7 and Tables II and III). At lower 3R tau concentrations (1:55 and 1:45), the slow growing MT subpopulation was left-shifted (MTs grew slower) relative to the no-tau control MTs. Median MT growth rates for the no tau, 1:55, and 1:45 conditions were 2.6, 2.2, and 2.0 µm/min, respectively, for tubulin preparation 1 (Table II) and 2.2, 2.0, and 1.9 µm/min, respectively, for tubulin preparation 2 (Table III). At these low concentrations, however, 3R tau appeared not to affect the small, faster growing subpopulation of MTs. When the 3R tau concentration was increased to 1:38 and 1:20, the growth rate of the slower growing subpopulation was not suppressed any further; instead, individual MTs redistributed from the slower growing subpopulation to the faster growing subpopulation. Whereas <10% of the MTs were found in the faster growing subpopulation at 3R tau:tubulin ratios of 1:55 and 1:45, 16–34% of the MTs were found in the faster growing group at a 3R tau:tubulin ratio of 1:38. At an even higher 3R tau:tubulin ratio of 1:20, nearly all (80–100%) of the MTs were found to be in the faster growing group. Thus, 3R tau modulated MT growth rates in two distinct ways that we designate as subpopulation shifting and subpopulation redistribution. At 1:55 and 1:45, 3RS tau suppressed the overall growth rate by subpopulation shifting leftward. At 1:20, 3RS tau increased the overall growth rate by subpopulation redistribution to the faster growing subpopulation.



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  		FIG. 7.
Best-fit Gaussian distributions of MT growth rate frequency histograms for two different tubulin preparations at 1:55 (gray line), 1:45 (gray dashes), 1:38 (black dashes), and 1:20 (black line) tau:tubulin molar ratios. A, 3RS tau, tubulin preparation 1. B, 3RS tau, tubulin preparation 2. C, 4RS tau, tubulin preparation 1. D, 4RS tau, tubulin preparation 2. Vertical lines in each panel indicate the medians of the two Gaussian distribution peaks of 15 µM tubulin in the absence of tau.

 
Increasing 4RS Tau Concentrations Increases the Size of the Faster Growing Subpopulation of MTs and Increases the Median Growth Rates for All Subpopulations—Histograms of individual MT growth rates from two independent 4RS tau and two independent tubulin preparations at various 4RS tau:tubulin ratios revealed that 4RS tau modulated the MT growth rate quite differently from 3RS tau (Figs. 6 and 7 and Tables II and III). Increasing concentrations of 4R tau generally caused a rightward shift (MTs grew faster) of both MT subpopulations. For tubulin preparation 1 (Table II), 4R tau increased the median growth rates of the two subpopulations from 2.6 and 4.4 µm/min for the no-tau control condition to maxima of 3.6 and 6.2 µm/min for the 1:20 4R condition. For tubulin preparation 2 (Table III), 4R tau increased the median growth rates of the two subpopulations from 2.2 and 3.7 µm/min for the no-tau control condition to maxima of 3.0 µm/min (1:38 4R condition) and 4.3 µm/min (1:20 4R condition). The right shift of both subpopulations by 4R tau at all concentrations tested was in sharp contrast to the action of 3R tau, which left-shifted the slower subpopulation at low tau:tubulin ratios. A second effect of increasing 4R tau concentrations was to increase the size of the faster growing subpopulation (subpopulation redistribution). Although 3R tau only caused a redistribution to the faster growing subpopulation at high tau:tubulin ratios, 4R tau caused a redistribution at all concentrations tested. The no-tau control, 1:55, 1:45, 1:38, and 1:20 4R tau conditions yielded 10, 23, 33, 28, and 49%, respectively, of the MTs in the faster growing subpopulation for tubulin preparation 1 (Table II) and 6, 8, 68, 78, and 100% of the MTs in the faster growing subpopulation for tubulin preparation 2 (Table III). Thus, although 4RS and 3RS tau both caused subpopulation shifting and subpopulation redistribution, they did so in qualitatively and quantitatively different ways.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We found the following. 1) All 3R tau isoforms at low tau:tubulin ratios reduce the average MT growth rate relative to the growth rate of MTs in the absence of tau, whereas all 4R tau isoforms increase the average MT growth rate. Thus, the presence or absence of the exon 10-encoded sequence is important in the regulation of MT dynamics, whereas the amino end insertions have no major effect. 2) At high tau:tubulin ratios, both 4RS and 3RS tau increase the average MT growth rate. 3) Both in the presence and absence of tau, there are two distinct subpopulations of MTs, one growing at a relatively slow rate and the other growing at a relatively fast rate. 4) Increasing concentrations of both 4RS and 3RS tau result in an increase in the size of the faster growing MT subpopulation, with 4RS tau causing a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3RS tau (5). Low 3RS tau:tubulin ratios result in a left shift of the large, slow growing subpopulation, whereas both low and high 4RS tau:tubulin ratios generally result in a right shift of both subpopulations.

Multiple Tau Isoforms and the Regulation of MT Dynamics—A large body of literature (17, 18) has shown that precise regulation of MT dynamics is essential for many cellular functions, including mitosis, axonal transport, and the establishment and maintenance of cell morphology. Aberrant regulation of MT dynamics can lead to cell dysfunction and eventual cell death (39, 40). Thus, it is important to understand at a mechanistic level how MT dynamics are regulated, and furthermore, how MT regulatory proteins are themselves regulated.

Previous work has firmly demonstrated that tau is a potent regulator of MT dynamics, both in vitro and in cells. Initially, Drechsel et al. (19) observed that an adult bovine tau preparation containing multiple tau isoforms promoted the MT growth rate, inhibited the shortening rate, and decreased the catastrophe frequency in vitro at high tau:tubulin ratios (1:30 to 1:2). Trinczek et al. (22) assessed recombinant human 4RL, 4RS, and 3RS tau at high tau:tubulin ratios (1:5 to 1:1) and made similar observations, noting that both 4R isoforms were more potent than 3RS. Recent work from our laboratories has shown similar quantitative differences between 4R and 3R tau, both in vitro and in cells (20, 23). Prior to the present work, all six tau isoforms had not been compared methodically in a single study. Here we show that whereas the N-terminal inserts have no obvious effects on MT dynamics, 4R and 3R tau isoforms regulate MT growth rates in both quantitatively and qualitatively different ways.

Additionally, we show that at low tau:tubulin ratios, all 3R tau isoforms reduced the MT growth rate. Although this finding may appear to conflict with our earlier work (Table II in Ref. 20), the slight increases in growth rates observed there were small and not statistically significant; indeed, the earlier work focused on the strong differential effects of 3R and 4R tau upon shortening events. Here we show that 3R tau caused a significant reduction in MT growth rates on six different occasions (see Tables I, II, III), demonstrating that this reduction does indeed occur.

MT Growth Rates Segregate into Two Subpopulations, Potential Mechanisms—One of the unexpected findings described here is that MTs growing in the absence of tau, i.e. from pure tubulin preparations, segregate into a large, relatively slow growing subpopulation and a small, relatively fast growing subpopulation. Although distinct subpopulations of MT growth rates have not been reported previously, several groups (19, 44–47) have noted surprisingly wide distributions in growth and shortening rates of MTs assembled with purified tubulin. Gildersleeve et al. (46) demonstrated that the wide distributions are likely an inherent property of MTs. Although the two discrete subpopulations were difficult to distinguish within the wide growth rate distributions we and others observed in the absence of tau, they became more apparent with the redistribution caused by the addition of tau. All conditions were repeated using different tau and tubulin preparations, and a similar tau-dependent subpopulation redistribution was observed. To further control for experimental artifact, the sequence of growth events of individual MT life histories was analyzed to ensure that relatively fast and slow growth events did not occur in a time-dependent manner, as might be expected with depletion of a given substrate; growth rates of the relatively fast and slow growing subpopulations did not grossly correlate with time after polymerization initiation. Taken together, these data indicate that the subpopulations observed are likely an inherent property of MTs.

What molecular mechanisms could account for the presence of discrete subpopulations of growing MTs? Any mechanism must ultimately be acting through a change in the kinetics of tubulin addition at MT ends. Because structure determines function, one possibility is that MT ends can assume conformations that differ from one another in their growth rate kinetics. Indeed, MT ends are known to have at least two distinct conformational states with differing dynamics as follows: a GTP- or GDP-Pi-tubulin cap during growth events and a GDP-tubulin end during shortening events (48–54). To achieve two distinct growing subpopulations, two different GTP- or GDP-Pi-tubulin cap conformations may also exist, one that corresponds to the slower growing subpopulation and the other that corresponds to the faster growing subpopulation.

How might two such GTP- or GDP-Pi-tubulin cap end conformations form? One possibility is that the two conformations represent two distinct, relatively stable, low energy conformations of MT ends. The tubulin conformation at the end of an MT might be determined by the conformation of the adjacent MT lattice, which can exist in multiple states (55–57). Microtubules typically contain between 13 and 15 protofilaments, which must result in different lattice structures with distinct properties (55, 58, 59). Furthermore, protofilament number can vary along the length of a single MT (55), indicating that the axoneme-seeded MTs observed in this and other studies may not be limited to the protofilament number of their nucleator.

Another potential source for different conformational states of the MT end may be the tubulin composition of a given MT. Purified bovine tubulin used in most in vitro dynamics studies contains a diverse population of tubulin isotypes and post-translational modifications; these may be differentially incorporated into a given MT, potentially leading to discrete conformations and thereby to a discrete change in MT dynamics. Indeed, isotype composition has been reported to alter MT dynamics (60), with more homogenous isotype populations generally producing faster assembling MTs (61, 62). In addition, tubulin post-translational modifications have been found to affect MT structure or overall morphology (63).

Potential Mechanisms for Differential Regulation of MT Dynamics by 4R and 3R Tau, Quantitative and Qualitative Differences—Both 4R and 3R tau binding promoted a concentration-dependent conversion of slower growing MTs to faster growing MTs, although 4R tau caused this conversion at lower tau: tubulin ratios than 3R tau. Mechanistically, at high tau concentrations, tau may bind at the MT end, a common binding site for MT regulatory proteins (64), and stabilize or promote conversion to a faster growing end conformation locally. Alternatively, tau could bind to the MT lattice and induce a conformational change (such as altered protofilament number) that propagates along the length of the MT. If the conformational change reaches the end of the MT, the MT could convert from slow growing to fast growing. Consistent with this notion, both taxol and microtubule-associated proteins decrease MT protofilament number under conditions that also alter MT dynamics (58, 65, 66). Additionally, tau binding to MTs appears to induce conformational changes in the MT lattice (56, 57). Furthermore, a tau-induced change in the MT lattice could explain how tau can affect MT dynamics at extremely low tau:tubulin ratios, such as 1:175 (21). Finally, the difference in the MT-binding domains of 4R and 3R tau (26) makes it possible to induce tau isoform-specific conformational changes and hence isoform-specific regulation of MT dynamics.

Additionally, both 4R and 3R tau shifted the median growth rates of each subpopulation of MTs; however, they did so in qualitatively different ways. At low tau:tubulin ratios, 3R tau left-shifted the median growth rate of the large, slow growing subpopulation. In contrast, 4R tau generally right-shifted both subpopulations in a concentration-dependent manner. Several mechanisms could account for the graded shifts in median growth rate observed here. The binding of a tau molecule may induce a localized and propagated conformational change in the MT lattice, i.e. an allosteric wave. Depending on the proximity of tau to the MT end, this allosteric wave could alter the growth rate kinetics in a graded manner. Increasing concentrations of tau would increase the average proximity of tau to the MT end and thereby the extent of altered kinetics. Alternatively, tau may bind to the MT end and promote concentration-dependent structural changes locally, thus altering the growth rate. In either case, the graded change in kinetics could be mediated by relatively subtle molecular alterations in the GTP- or GDP-Pi-tubulin cap. Thermodynamically, one could view the two stable energy minima, corresponding to slow and fast growing MT structures, as containing numerous micro-minima that make the graded changes in growth rate possible. One alternative explanation of the 3R tau-induced reduction of MT growth rate medians is that 3R tau may be sequestering free tubulin. However, the finding that 3R tau causes this reduction at a 1:55 tau:tubulin ratio does not support this hypothesis because there is insufficient tau to reduce the soluble tubulin level. Thus, for any potential mechanism, 4R and 3R tau are likely binding at or near the MT ends, causing different changes in MT conformation and thus qualitatively different end kinetics.

Opposing and Collaborative Effects of 4R and 3R Tau, Concentration Dependence—As noted above, 4R tau caused a concentration-dependent increase in the average MT growth rate, whereas 3R tau caused a decrease in average MT growth rate at low tau:tubulin ratios and an increase at high tau:tubulin ratios. Thus, 4R and 3R tau may be acting in opposition at low tau:tubulin ratios but in collaboration at high tau:tubulin ratios. Might these differences be physiologically relevant to developing and/or mature neurons? Whereas tau levels in brain extracts provide minimal information about tau isoform expression levels in individual neuronal cells, cultured neuronal cell lines provide some insight. In undifferentiated PC12 cells, which may be analogous to neuronal precursor cells, the ratio of tau:tubulin is 1:68, whereas in differentiated PC12 cells, the ratio is 1:17 (1). These numbers suggest that the ranges of tau:tubulin ratios that we have examined, and the differential effects of 4R and 3R tau at these ratios, are likely to be biologically meaningful. During development, neurons express increasing levels of only 3RS tau (10, 14–16, 24, 25); thus, fine regulation of the intracellular 3RS concentration could enable the cell to decrease or increase MT growth rates as necessary to promote neuronal maturation. In the adult, where 3R and 4R tau are expressed equally, the opposing activities of 4R and 3R tau at some concentrations and the collaborative activities at other concentrations may be utilized by the cell to finely regulate MT dynamics under different cellular circumstances or in different cellular compartments.

Mechanistic Implications for Tau-mediated Neuronal Cell Death—Although the FTDP-17 tau mutations establish a clear link between tau dysfunction and disease, the underlying molecular mechanism(s) leading to neuronal cell death are not understood. The presence of abundant and abnormal tau aggregates in many neurodegenerative disorders, including Alzheimer disease and FTDP-17, has led to the hypothesis that these aberrant tau aggregates are cytotoxic (10, 35). However, no direct evidence supports cytotoxicity of the aggregates (37, 67). Additionally, because all of the tau present is wild type in FTDP-17 splicing mutants, in these cases, it is unclear what changes in the tau protein cause aggregation, unless the aggregation were a downstream consequence of abnormal tau activity, i.e. misregulated MT dynamics.

An alternative "MT misregulation" model suggests that alterations in tau structure-function or isoform expression ratios result in misregulation of MT dynamics, which in turn leads to cell death. This model is supported by a large body of literature demonstrating that tight regulation of MT dynamics is essential for proper cell function and viability (17, 18, 39, 40). Consistent with this model, tau molecules harboring amino acid substitutions that cause FTDP-17 possess deficits in the ability to bind and assemble MTs (68–70), as well as to regulate MT dynamics.2,3 Most relevant to this work, the MT misregulation model can readily accommodate the FTDP-17 tau RNA-splicing mutations. Given the qualitative and quantitative differences in the mechanistic capabilities of 4R and 3R tau described here and elsewhere, it is likely that alterations in the expression ratios of the tau isoforms will generate deleterious changes in the regulation of MT dynamics in neurons.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants NS35010 (to S. C. F), NS13560 (to L. W.), and CA57291 (to M. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Neuroscience Research Institute, Bldg. 571, Rm. 6129, University of California, Santa Barbara, CA 93106. Tel.: 805-893-2659; Fax: 805-893-2659; E-mail: feinstei{at}lifesci.ucsb.edu.

1 The abbreviations used are: MT, microtubule; FTDP-17, fronto-temporal dementia with parkinsonism linked to chromosome 17; 3R, 3-repeat tau; 4R, 4-repeat tau; 3RS, 3-repeat short tau; 3RM, 3-repeat medium tau; 3RL, 3-repeat long tau; 4RS, 4-repeat short tau; 4RM, 4-repeat medium tau; 4RL, 4-repeat long tau; HPLC, high pressure liquid chromatography; Pipes, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid. Back

2 J. Bunker, M. A. Jordan, L. Wilson, and S. C. Feinstein, submitted for publication. Back

3 S. F. Levy, A. C. LeBoeuf, M. R. Massie, M. A. Jordan, L. Wilson, and S. C. Feinstein, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Herb Miller for generously providing purified bovine tubulin and Dr. Jennifer Ross for assistance with data analysis.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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