Tau mutants bind tubulin heterodimers with enhanced affinity Shana Elbaum-Garfinklea,1,2, Garrett Cobba,1, Jocelyn T. Comptona,3, Xiao-Han Lib, and Elizabeth Rhoadesa,c,4 Departments of aMolecular Biophysics and Biochemistry, bChemistry, and cPhysics, Yale University, New Haven, CT 06511 Edited by Marc W. Kirschner, Harvard Medical School, Boston, MA, and approved March 20, 2014 (received for review August 23, 2013)

microtubule-associated protein single molecule FRET

| intrinsically disordered proteins |

T

au is a microtubule (MT)-associated protein that plays a critical role in the pathology of several neurodegenerative disorders including Alzheimer’s disease, frontotemporal dementias, and traumatic brain injury (1). Normally, tau is found primarily in the axons of neurons where it regulates the dynamic instability of MTs (2, 3) and plays an important role in axonal transport (4, 5). Both in vitro and in vivo measurements find that tau increases the rate of MT polymerization, as well as decreasing rates of catastrophe (2, 6, 7). In disease, tau is found as aggregated, filamentous deposits that are the defining feature of a diverse class of neurodegenerative diseases, called tauopathies (1, 8). Pathological mutations to tau are thought to alter the native interactions of tau with MTs, in addition to increasing the propensity of tau to aggregate (9–11). Although the precise cause or mechanism by which tau contributes to toxicity in disease is unknown, both a loss of native function and a gain of toxic function are implicated (1, 12, 13). Tau consists of a C-terminal microtubule binding domain (MTBR) composed of imperfect repeats (R1–R4; Fig. 1A) (14), a flanking proline-rich region that enhances MT binding and assembly (3), and an N-terminal projection domain with putative roles in MT spacing (15) and membrane anchoring (16). Alternative splicing results in the expression of six isoforms of tau in the adult human brain, with zero, one, or two N-terminal inserts and three or four MT binding repeat units. The repeat units contain an 18-residue imperfect repeat sequence, which terminates with a highly conserved proline-glycine-glycine-glycine (PGGG), and are linked by a 13–14 residue interrepeat sequence (Fig. 1C). Early biochemical studies depicted the conserved regions as binding weakly to MTs, with the interrepeats acting as spacers between www.pnas.org/cgi/doi/10.1073/pnas.1315983111

them (14, 17). More recently, it was shown that the interrepeats are also directly involved in binding and polymerization (18, 19), with the N-terminal proline-rich region playing a regulatory role (20). Tau binds MTs with a biphasic pattern indicative of two distinct binding sites on the MT lattice with unequal affinities (21, 22). Binding to MTs is negatively regulated by phosphorylation on sites in the MTBR and adjacent regions (23). Tau derived from aggregates in the brain is hyperphosphorylated (24), suggesting a role for aberrant phosphorylation in disease. Attempts to obtain resolution structural information of tau have been hindered by the fact that it is intrinsically disordered under physiological conditions (25) and remains largely disordered even on binding to MTs. Contrasting models derived from cryo-EM suggest tau binds either along the outer protofilament ridge (26) or to the inner surface (27) of MTs. The MTBR carries a net positive charge, and binding of tau to the acidic carboxy termini of α and β tubulin has been observed both in the context of MTs (28) and for the isolated peptides corresponding to these tubulin sequences (29, 30). In addition, a secondary binding site has been mapped to an independent region within the C-terminal third of tubulin (30). Cleavage of the C-terminal tail of tubulin is sufficient to increase polymerization rates (30), and it has been suggested that tau may promote MT polymerization through a similar charge neutralization mechanism. The MTBR also plays a central role in tau pathology in that it forms the core of the aggregates found in disease (31) and contains the minimum sequence necessary to recapitulate relevant features of aggregation in vitro (32). Moreover, the majority Significance The microtubule binding protein tau is implicated in the pathology of a number of devastating neurological disorders. Both loss of function arising from altered interactions with microtubules and gain of toxic function tied to tau aggregation are thought to have a role in disease etiology. Here, we demonstrate that point mutants of tau implicated in disease enhance the affinity between tau and tubulin dimers. Our results lend insight into the mechanism of tau-mediated microtubule polymerization. Importantly, our findings highlight the significance of the interaction between tau and free tubulin, introducing it as a new focus for research on the role of tau in neurodegeneration. Author contributions: S.E.-G., G.C., J.T.C., and E.R. designed research; S.E.-G., G.C., J.T.C., and X.-H.L. performed research; S.E.-G., G.C., J.T.C., X.-H.L., and E.R. analyzed data; and S.E.-G., G.C., and E.R. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

S.E. and G.C. contributed equally to this work.

2

Present address: Department of Chemical and Bioengineering, Princeton University, Princeton, NJ 08544.

3

Present address: Columbia University College of Physicians and Surgeons, New York, NY 10032.

4

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1315983111/-/DCSupplemental.

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Tau is a microtubule binding protein that forms pathological aggregates in the brain in Alzheimer’s disease and other tauopathies. Disease etiology is thought to arise from loss of native interactions between tau and microtubules, as well as from gain of toxicity tied to tau aggregation, although neither mechanism is well understood. Here we investigate the link between function and disease using disease-associated and disease-motivated mutants of tau. We find that mutations to highly conserved proline residues in repeats 2 and 3 of the microtubule binding domain have differential effects on tau binding to tubulin and the capacity of tau to enhance tubulin polymerization. Notably, mutations to these residues result in an increased affinity for tubulin dimers while having a negligible effect on binding to stabilized microtubules. We measure conformational changes in tau on binding to tubulin that provide a structural framework for the observed altered affinity and function. Additionally, we find that these mutations do not necessarily enhance aggregation, which could have important implications for tau therapeutic strategies that focus solely on searching for tau aggregation inhibitors. We propose a model that describes tau binding to tubulin dimers and a mechanism by which disease-relevant alterations to tau impact its function. Together, these results draw attention to the interaction between tau and free tubulin as playing an important role in mechanisms of tau pathology.

Fig. 1. Schematic of tau constructs and an MTBR repeat. The functional domains of tau are indicated on the longest full-length isoform with alternatively spliced regions marked by dashed lines (A). The interrepeat regions that link the conserved repeat sequences are indicated by cross-hatching. On the fragments used in this study (B), K16 (residues 198–372) and K18 (residues 244–372), the residues mutated to cysteine for attachment of fluorophores (residues 244, 322, and 354), and proline to leucine/serine mutation sites (301 and 332) are indicated. A schematic of a repeat within the MTBR (C) illustrates interrepeat and repeats sequences, with the conserved residues shown.

of mutations to tau implicated in tauopathies are either point mutations within the MTBR or mutations that interfere with alternative splicing, altering the normal ∼1:1 ratio of 3R:4R tau (8, 9). Notable among the point mutants is P301L (tauP301L) (Fig. 1B), implicated in frontotemporal dementia with Parkinsonism17, which provided one of the first genetic links between tau and neurodegenerative disease (33). The P301L variant has emerged as a particularly reliable model for tau-based neurodegenerative disease, having successfully reproduced aspects of pathology in animal models of Alzheimer’s disease and other tauopathies (34). Although significant work has focused on tau interactions with MTs, very little is known about the mechanistic details of taumediated MT polymerization, including interactions of tau with tubulin dimers during the assembly process. Although many alterations to tau implicated in disease have been shown to affect MT polymerization both in vitro and in vivo, virtually nothing is known about the mechanism by which these changes occur. TauP301L exhibits impaired tubulin polymerization (35), as well as increased aggregation propensity relative to WT protein (tauWT) (32) and thus serves as a model for both loss of function and gain of toxic function aspects of disease. This mutation is in the highly conserved PGGG sequence of R2 (Fig. 1C). To broaden our understanding of the impact of this point mutation on pathology, we designed a tau variant with the analogous proline to leucine substitution in the PGGG sequence of R3, tauP332L, as well as a double mutant, tauP301L/P332L. We use single molecule fluorescence to investigate structural and functional aspects of the interaction between tau and tubulin. In combination with ensemble polymerization and aggregation assays, this work provides insight into the relationship between functional and dysfunctional roles of tau.

importantly, confirms the absence of tubulin assembly under our buffer conditions (SI Text). The K16 fragment of tau was used for all assays involving tubulin or MTs (Fig. 1B). It is composed of the MTBR and the adjacent proline-rich region and was chosen because it is the minimal 4R fragment of tau that recapitulates functional interactions of tau with MTs. Binding measurements of WT K16 tau and proline point variants to tubulin were carried out under nonassembly conditions, namely very low concentrations of tau and in buffer (20 mM Tris and 50 mM NaCl, pH 7.4), lacking assembly promoters (SI Text). Surprisingly, we found that the disease and designed variants of tau bound to tubulin dimers with a significantly higher affinity than the WT protein (Fig. 2A and Table S1): tauWT (∼2.5 μM) < tauP301L (∼1.0 μM) < tauP332L (∼100 nM) < tauP301L/P332L (∼60 nM). The ∼25- and ∼40-fold enhancements in affinity observed for tauP332L and tauP301L/P332L, respectively, compared with the ∼2.5-fold increase for tauP301L is particularly striking as it suggests that the effect of mutating proline to leucine is not uniform for all PGGG sequences. To test the specificity of the proline to leucine substitution, we also created K16 variants with proline to serine substitutions: tauP301S and tauP332S. Both variants, which are naturally occurring tauopathy-inducing mutations (36, 37), bound to tubulin more tightly than tauWT and followed the trend in affinity observed above: tauWT < tauP301S < tauP332S (Table S1). We also assayed the binding affinity of all P to L variants for taxol stabilized MTs (SI Text). In contrast to the measurements involving soluble tubulin, only minor differences in affinity relative to tauWT were observed (Fig. S1 and Table S1). These results were not completely unexpected. Although variants of tau linked to disease, namely point mutations or phosphorylation, are often broadly described as having compromised binding to MTs, the majority of studies find more significant effects on the dynamics of polymerization (6, 35, 38–40) rather than on the affinity of the interaction. In fact, previous measurements quantifying binding of tauP301L to MTs have reported a range of results including a decrease (35), no significant difference (41), and even a slight increase (42) in binding affinity relative to tauWT. Increased Affinity for Tubulin Has a Variable Effect on MT Polymerization Kinetics. To explore the impact of enhanced tubulin binding on tau

function, we characterized the ability of the four proline to leucine K16 variants to polymerize tubulin in vitro. We find that tauP301L and tau P301L/P332L are less effective at promoting MT polymerization than tauWT, with the polymerization time greater than three

Results Tau Point Mutants Bind Tubulin More Tightly than WT Tau. Because MT function is dependent on the dynamic interchange of free and bound tubulin subunits, we hypothesized that defects in polymerization might arise from an altered interaction between tau and soluble tubulin heterodimers. To examine this interaction, fluorescence correlation spectroscopy (FCS) was used to quantify binding of tau to soluble tubulin. FCS quantifies the fluctuations in fluorescence intensity arising from the diffusion of fluorescent molecules through an experimental observation volume. The characteristic diffusion time obtained by fitting the temporal autocorrelation function reflects the size of the fluorescent molecules; smaller molecules diffuse more rapidly than larger ones. Therefore, as we have applied it here, it is a powerful approach to detect association or dissociation of molecular complexes, and 6312 | www.pnas.org/cgi/doi/10.1073/pnas.1315983111

Fig. 2. Tubulin binding (A) and MT polymerization (B). (A) Binding plots show the fraction of tau bound as a function of tubulin concentration; tauP301L, tauP332L, and tauP301L/P332L show an increased affinity for tubulin dimers relative to tauWT, whereas all four variants bind MTs with comparable affinities (Table S1). (B) The kinetic traces of tau-induced tubulin polymerization illustrate that both tauP301L and tauP301L/P332L have a compromised ability to polymerize tubulin relative to tauWT and tauP332L. The table reports T50 obtained from Eq. S4; errors are SD, n ≥ 3.

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times longer for these two variants (Fig. 2B). In contrast, MT polymerization by tauP332L, which exhibited significantly tighter binding to tubulin than tauWT, is comparable to that of tauWT. This observation supports the idea that the PGGG sequences in different repeat regions have differential roles in regulating MT assembly. When considered together with the binding affinity measurements, our data suggest that binding to tubulin dimers and the polymerization activity of tau may be mediated by different, and potentially independent, repeat regions within the MTBR. Specifically, the moderate increase in tubulin binding affinity arising from the proline to leucine mutation in the PGGG sequence of R2 (P301L) is correlated with decreased capacity to polymerize tubulin, whereas the significant increase in binding affinity arising from the same mutation in R3 (P332L) has no observable impact. Cold depolymerization of the final MT polymerization products results in a greater amount of residual signal for tauP301L and tau P301L/P332L than for tauWT or tauP332L (Fig. S2). The presence of cold-stable species may indicate greater production of off-pathway tubulin-tau structures, such as the rings that have been observed at high tau:tubulin concentrations (43), or the formation of tau oligomers or aggregates. Gel electrophoresis reveals that pelleted, cold-stable species consist of both tau and tubulin (Fig. S2). Tau Binds the Tubulin Heterodimer in an Extended Conformation.

NMR studies indicate a propensity for β-turn structure in the PGGG sequences in solution, which is lost on mutation of the proline to a leucine (44). To assess whether tubulin binding affinities may arise from the differences in the intrinsic structural propensities of these regions, we used single molecule FRET (smFRET) to characterize the conformation of tubulin-bound tau. Each tau K16 construct was labeled with a donor and an acceptor fluorophore at residues 244 and 354, positions that span the MTBR, and smFRET was measured in the absence and presence of tubulin. Our results show that binding to tubulin causes a decrease in the mean FRET efficiency (ETeff) consistent with an extended MTBR (Fig. 3 A and B). Moreover, the conformational states of the various constructs are correlated with their binding affinities and functional capacity. To illustrate, both tauP332L and tauP301l/P332L, which bind to tubulin dimers with a significantly higher affinity than tauWT and tauP301L, have more extended tubulin-bound structures (lower mean ETeff; Fig. 3C). Interestingly, tauP301L and tauP301l/P332L variants that are deficient in their ability to polymerize MTs both have reproducibly more compact states (higher mean ETeff) in solution compared with the WT and P332L mutants (Fig. 3C). Intramolecular smFRET is a particularly powerful method for characterizing conformational changes in proteins, even in the absence of canonical secondary and tertiary structure (45). Appropriate modeling allows for the conversion of the measured ETeff values to distances and the extraction of more specific information about the conformation of the protein. The measured ETeff ∼ 0.4 for tau in solution can be converted to a root mean square (RMS) distance of 66 Å using a Gaussian chain, Elbaum-Garfinkle et al.

Functional Loss Does Not Implicate Toxic Gain. In vitro, diseaseassociated mutants are generally more aggregation prone than WT (42, 46). We used the K18 fragment to characterize tau aggregation (Fig. 1B) because it demonstrates an increased propensity to aggregate relative to full-length tau and allows for comparison with our own previous aggregation studies (47), as well as to others. We find that tauP332L and tauP301L/P332L also display altered aggregation kinetics (Fig. 4), but in an unexpected manner. Because of its high solubility, tau aggregation in vitro is commonly induced by polyanions and was assayed here by two different approaches. In the first assay, the kinetics of aggregation induced by heparin was followed by monitoring the increase in fluorescence of thioflavin T (ThT), a β-sheet–sensitive dye that reports on amyloid formation. In the second assay, a critical protein:lipid (P:L) ratio was determined by FCS for lipid-induced aggregation (47) (SI Text and Fig. S3). Binding of heparin to tau causes an increase in β-sheet structure in the MTBR (44), as well as distinct conformational changes throughout the protein (45), whereas binding of tau to anionic lipid vesicles results in an increase in the α-helical content of the MTBR (48). The use of contrasting assays allows us to ascertain whether the point variants of tau are sensitive to the mechanism of induction of aggregation. Both assays revealed that tauP301L/P332L is less aggregation-prone than either tauWT or the single point variants (Fig. 4). By ThT fluorescence, introduction of the P301L mutation decreases the aggregation half-time by ∼50%, with tauP332L having an intermediate effect, whereas tauP301L/P332L has a half-time of approximately twice that of tauWT (Fig. 4A). Notably, the trend observed in the aggregation kinetics (tauP301L > tauP332L > tauWT > tauP301L/P332L) is mirrored in the measurements of the P:L ratio for lipid-induced aggregation (Fig. 4B). Although the presence of only the P301L or P332L mutations decreases the amount of protein required for aggregation relative to WT, the P301L/P332L variant requires a more than fivefold increase (Fig. 4B). The qualitative agreement between these two assays suggests that the dramatic attenuation of aggregation propensity brought about by the double

Fig. 4. Tau aggregation. The aggregation of all four variants as assayed by ThT fluorescence (A) or FCS (B). Both assays show that tauP301L and tauP332L aggregate more readily than tauWT, whereas the aggregation of tauP301L/P332L is inhibited. Error bars are SD, n ≥ 3.

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Fig. 3. Single molecule FRET of tau. Histograms of the ETeff in the absence (A) and presence (B) of 10 μM tubulin dimers illustrating that tau undergoes a distinct conformational change on binding to tubulin. A comparison of the mean ETeff values of all four variants (C) shows reproducible differences between them. Error bars are SD, n = 3–15 (average, 7).

a reasonable model for a disordered protein (45). The lack of structural information makes choosing an appropriate model for tubulin-bound tau more challenging; the mean ETeff ∼ 0.33 for tubulin-bound tauWT is equivalent to an RMS distance of between 57 and 74 Å, depending on whether the Förster equation or a polymer model is used for the conversion (SI Text). As the mean ETeff for tubulin-bound tau is shifted to a lower value than that of tau in solution, reflecting an expansion of this domain on binding tubulin, we can reasonably put a lower bound of 66 Å on this range to narrow it to 66–74 Å. These values are consistent with the dimensions of the tubulin dimer, ∼80 Å, and suggest that the MTBR is extended in its tubulin-bound conformation and most likely interacts with both subunits of the tubulin heterodimer.

proline to leucine mutation is independent of its interaction with a particular inducer. Moreover, the P301L/P332L results demonstrate that modifications to tau that impair function and enhance aggregation are not necessarily coupled. Discussion and Conclusions The mechanism by which tau stabilizes MTs is poorly understood. Characterizing the molecular details can provide critical insight into how tau mutants and other variants result in loss of function linked to disease. Here we used FCS and smFRET to probe the interaction of tau with tubulin heterodimers to investigate the effect of disease-relevant and designed point mutants on tubulin polymerization. Our results show that tau mutations have a significant impact on tubulin binding affinity (Fig. 2A) in contrast to the negligible impact on binding to stabilized MTs (Fig. S1) and provide insight into this largely overlooked regime of tau function and pathology. The vast majority of published work has focused on interactions between tau and MTs, and relatively little is known about the nature of interactions with tubulin heterodimers. One reason for this may be that much of the published work has used experimental approaches that obscure detection of this interaction. Immunofluorescence imaging of fibroblast cells transfected with tau found it colocalized with MTs (49) with no evidence of cytoplasmic tau. However, such images similarly do not detect free tubulin, shown to make up 50–60% of total cellular tubulin (50), suggesting that detection of soluble protein may be compromised by the high signal from the dense MT polymer. Although live cell imaging approaches of fluorescently labeled tau are also likely to be dominated by the more readily detected MT-associated signal, it is worth noting that a significant fraction of cytoplasmic tau was observed in a more recent live cell study of Xenopus neuronal cells expressing tau GFP (51). Analysis of cellular tau has also relied on quantification based on the extraction of soluble and insoluble proteins, and these studies have found tau to be predominantly associated with the insoluble MTcontaining fraction (52). Given the propensity of tau to self-associate, it is possible that the extraction conditions result in precipitation of tau as an aggregate such that it appears as an insoluble protein. Some preliminary work in our laboratory provides evidence in support of this hypothesis (SI Text). Investigations into the interaction between tau and soluble tubulin may also be thwarted by the challenge of measuring the binding reaction in isolation. The protein concentration and timescales required for many types of measurements have resulted in tau-induced polymerization of tubulin even in the absence of additional assembly promoters (53). FCS allows for low tau concentrations (20 nM in our measurements), enabling the quantification of tubulin heterodimer binding under conditions where tubulin assembly is disfavored. Structural differences in tau may underlie the increase in affinity of tauP301L, tauP332L, and tauP301L/P332L relative to tauWT. Our smFRET measurements (Fig. 3) suggest that the tau variants used in this study have slightly altered solution structures and also bind to tubulin dimers with a slightly larger “footprint” than tauWT. NMR models predict that tau becomes partially α-helical on binding to tubulin (48, 54). In WT tau, the proline at the beginning of the PGGG sequence may serve to terminate α-helical structure, whereas the presence of a leucine could allow for it to be slightly extended (Fig. S4). Extended helical structure is compatible with the slight decrease in ETeff observed for the tubulin-bound forms of tauP301L, tauP332L, and tauP301L/332L relative to tauWT. It may also be that these mutants are more sensitive to conformational differences between the tubulin heterodimer and polymerized tubulin in MTs. Interestingly, both phosphorylated (55) and pseudophosphorylated (56) tau have been proposed to bind MTs in a more extended structure than WT tau. The striking increase in affinity resulting from the proline to leucine mutation at residue 332 raises the question as to why this 6314 | www.pnas.org/cgi/doi/10.1073/pnas.1315983111

particular residue has such a dramatic impact on tau binding to tubulin. It has been proposed that the first two conserved repeat sequences and their connecting interrepeat form the core MT binding domain of tau (R1–R2 in four repeat isoforms and R1– R3 in three repeat isoforms) (19). Mutations to the primary core that disrupt MT interactions are thought to result in enhanced engagement of alternative core units in the MTBR (39). Our results suggest the possibility that a different region of tau dominates interactions with tubulin dimers. We propose a model whereby the conserved repeat sequence of R3, potentially as part of a core unit with R4 and their connecting interrepeat, serves as the primary mediator of interactions between tau and tubulin dimers (Fig. 5). Our strongest evidence for this comes from the double mutant, tauP301L/P332L, which mirrors the dramatic increase in tubulin affinity demonstrated for tauP332L, yet exhibits impairment of tubulin polymerization consistent with tauP301L. These characteristics of the double mutant suggest that P301L (R2) and P332L (R3) are effectively dominant with regard to tubulin polymerization and tubulin binding, respectively. One interesting outcome of this model is that it may allow for simultaneous binding of tau to tubulin dimers and MTs, at least for tau variants with four repeat domains. In three repeat tau isoforms, R3 may be engaged in both tubulin and MT binding, which could explain why a number of disease-related variants that impact 3R tau function do not similarly effect 4R tau (19). The affinities we measure here have important implications for our current understanding of tau function. TauWT displays comparable affinities for tubulin dimers and MTs, with tau likely binding both tubulin heterodimers and the MT lattice under physiological conditions. However, all of the tau variants tested in this study, including the disease-implicated mutants P301L, P301S, and P332S, exhibit tighter binding to soluble tubulin dimers (Table S1). The observed increase in affinity highlights the potential significance of altered tau-tubulin interactions to tau pathology. We find that even the relatively minor increased affinity in tauP301L is associated with altered polymerization kinetics (Fig. 2B), which suggests that reversible interactions between the R2 region of the MTBR and tubulin is relevant to the mechanism of polymerization. For example, increased affinity for free tubulin relative to MTs reduces the number of tau molecules available for binding and stabilizing MTs, or conversely, sequesters the pool of free tubulin available for polymerization. Preferential binding to free tubulin could interfere with efficient recruitment and associated structural rearrangement of tubulin within the polymer. Alternatively, increased binding to free tubulin could also prevent tau from interacting with other cellular partners, as evidence for tau binding to signaling molecules, cytoskeletal elements, and lipids suggests alternative functions for tau in the cell (57).

Fig. 5. Proposed model of tau binding to tubulin heterodimer. Tau K16 is in yellow with conserved repeat regions marked by gray boxes. A tubulin heterodimer (Protein Data Bank ID code 1FFX) is in blue, with proposed tau binding site from ref. 28 in red. The distances indicated by dotted lines are derived from single molecule FRET measurements as described in the text.

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Methods Preparation and Labeling of Proteins. Tau K16 was used for all experiments in this study except for aggregation, which used tau K18 (Fig. 1B). Sitespecific labeling was achieved by introducing cysteines at desired locations to enable maleimide attachment of dyes Alexa Fluor 488 and 594 (Life Technologies). All constructs were purified as described previously (47). See SI Text for details. FCS and smFRET Measurements. FCS and smFRET measurements were made on a laboratory-built instrument based on an inverted Olympus IX-71 microscope (Olympus) that has been described previously (47, 59). Samples were placed in eight-well chambered coverglasses (Nunc) passivated by polylysine-conjugated polyethylene glycol treatment to prevent protein adsorption to the chamber surfaces. smFRET measurements used ∼30 pM tau in the absence or presence of 10 μM tubulin and included 1 mM DTT. FCS binding measurements used ∼20 nM tau and a range of tubulin concentrations. All measurements were taken at 20 °C in 20 mM Tris buffer and 50 mM NaCl, pH 7.4, unless otherwise noted. See SI Text for details. ACKNOWLEDGMENTS. We thank Lynne Regan for the pET-HT vector used for expression of tau, Lester Binder for the tau plasmid used as a template, and John Correira for the gift of tubulin used in preliminary studies. This work was supported by National Science Foundation Grant MCB 0919853 (to E.R.), funds from the Beverly and Raymond Sackler Institute (to J.T.C. and E.R.), and National Institutes of Health Institutional Training Grants 5T32GM007223 (to S.E.-G.) and T32GM067543 (to G.C.).

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Last, we note that the model frequently evoked to describe tau aggregation in disease is one where mutations or hyperphosphorylation results in reduced interactions with MTs and consequent increase in the cytosolic concentration of tau (1, 58). If the increase affinity of modified tau for tubulin, as we have seen here, is a general feature, then this interaction may have a major role in the aggregation pathway. Going forward, it will be of significant interest to determine how generally the effects observed here are found in other variants of tau implicated in disease. Consideration of tau in the context of both function and dysfunction is necessary for a thorough understanding of how it contributes to pathology in neurodegenerative disease. Using disease and designed mutants, we demonstrated that mutations to the PGGG sequences in R2 and R3 results in an increased affinity for free tubulin relative to MTs. For the R2 mutation, this correlates with a loss of function through impaired tau mediated tubulin polymerization. We show that the mutations induce small conformational changes to tau both in solution and bound to tubulin that may contribute to altered tubulin affinity. Furthermore, we find that decreased MT polymerization does not correspond to aggregation propensity. Together these results highlight the importance of tau interactions with free tubulin and introduce it as a new focus for research aimed at elucidating the mechanisms of tau function and pathology.

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