Structural impact of heparin binding to full-length Tau as studied by NMR spectroscopy

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.     

FTDP-17 mutations in Tau alter the regulation of microtubule dynamics: an "alternative core" model for normal and pathological Tau action

Mutations affecting either the structure or regulation of the microtubule-associated protein Tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among the most characterized activities of Tau is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here we have tested the hypothesis that Tau mutations causing neurodegeneration also alter the ability of Tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions, and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or interrepeat regions of Tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of Tau regulatory action based on an "alternative core" of microtubule binding and regulatory activities composed of two repeats and the interrepeat between them. In this model, the interrepeat serves as the primary regulator of microtubule dynamics, whereas the flanking repeats serve as tethers to properly position the interrepeat on the microtubule. Importantly, since there are multiple interrepeats on each Tau molecule, there are also multiple cores on each Tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for Tau-mediated neuronal cell death and provide a novel mechanistic model for normal and pathological Tau action.     

Microtubule bundling by tau proteins in vivo: analysis of functional domains.

Tau varies both in the N-terminal region (three types) and in the C-terminal repeated microtubule binding domain (two types), generating six isoforms through alternative splicing. To understand the differences between the isoforms and to determine which domains are important for microtubule bundling, we performed transfection studies on fibroblasts using tau isoforms and deletion mutants to quantify their ability to bundle microtubules. By comparing the isoforms, we found that a longer N-terminal region induced microtubule bundling more efficiently, but changes in the microtubule binding domain did not. Mutants lacking the proline rich region or the repeated domain did not bind to microtubules. Although all the other mutants could bind to and bundle microtubules, deletion in the N-terminal neutral region or the first half of the C-terminal tail caused a significant decrease in microtubule bundling, indicating the importance of these regions in microtubule bundling.     

The C terminus of tubulin, a versatile partner for cationic molecules: binding of Tau, polyamines, and calcium

The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.     

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 ~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 K_d) 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.     

PACSIN1, a Tau-interacting Protein,Regulates Axonal Elongation and Branching by Facilitating Microtubule Instability

Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.     

Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.     

NMR investigation of the interaction between the neuronal protein tau and the microtubules

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.     

Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly.

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.     

Surface-decoration of microtubules by human tau

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a "halo" of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on alpha-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.     

The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau

We report the complete sequence of the microtubule-associated protein MAP1B, deduced from a series of overlapping genomic and cDNA clones. The encoded protein has a predicted molecular mass of 255,534 D and contains two unusual sequences. The first is a highly basic region that includes multiple copies of a short motif of the form KKEE or KKEVI that are repeated, but not at exact intervals. The second is a set of 12 imperfect repeats, each of 15 amino acids and each spaced by two amino acids. Subcloned fragments spanning these two distinctive regions were expressed as labeled polypeptides by translation in a cell-free system in vitro. These polypeptides were tested for their ability to copurify with unlabeled brain microtubules through successive cycles of polymerization and depolymerization. The peptide corresponding to the region containing the KKEE and KKEVI motifs cycled with brain microtubules, whereas the peptide corresponding to the set of 12 imperfect repeats did not. To define the microtubule binding domain in vivo, full-length and deletion constructs encoding MAP1B were assembled and introduced into cultured cells by transfection. The expression of transfected polypeptides was monitored by indirect immunofluorescence using anti-MAP1B-specific antisera. These experiments showed that the basic region containing the KKEE and KKEVI motifs is responsible for the interaction between MAP1B and microtubules in vivo. This region bears no sequence relationship to the microtubule binding domains of kinesin, MAP2, or tau.     

A nonmotor microtubule binding site in kinesin-5 is required for filament crosslinking and sliding

Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our?results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to?maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.     

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3β

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.     

Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.     

PASK (proline-alanine-rich Ste20-related kinase) binds to tubulin and microtubules and is involved in microtubule stabilization

Proline–alanine-rich Ste20-related kinase (PASK, also referred to as SPAK) has been linked to ion transport regulation. Here, we report two novel activities of PASK: binding to tubulin and microtubules and the promotion of microtubule assembly. Tubulin binding assay showed that full-length PASK and its kinase domain bound to purified tubulin whereas the N-terminal or C-terminal non-catalytic domains of PASK did not. The full-length PASK and its kinase domain were sedimented with paclitaxel-stabilized microtubules by ultracentrifugation. These results indicate that the kinase domain of PASK can interact directly with both microtubules and soluble tubulin in vitro. Truncated PASK lacking the N-terminal non-catalytic domain promoted microtubule assembly at a subcritical concentration of purified tubulin. FLAG–PASK expressed in COS-7 cells translocated to the cytoskeleton when the cells were stimulated with hypertonic sodium chloride, and stabilized microtubules against depolymerization by nocodazole. Our findings suggest that PASK may regulate the cytoskeleton by modulating microtubule stability.     

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.     

The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.     

Generation of microtubule stability subclasses by microtubule- associated proteins: implications for the microtubule "dynamic instability" model

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.     

The interaction of TOGp with microtubules and tubulin

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.