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 specific binding of the microtubule-associated protein 2 (MAP2) to the outer membrane of rat brain mitochondria.

Purified mitochondria from rat brain contain microtubule-associated proteins (MAPs) bound to the outer membrane. Studies of binding in vitro performed with microtubules and with purified microtubule proteins showed that mitochondria preferentially interact with the high-molecular-mass MAPs (and not with Tau protein). Incubation of intact mitochondria with Taxol-stabilized microtubules resulted in the selective trapping of both MAPs 1 and 2 on mitochondria, indicating that an interaction between the two organelles occurred through a site on the arm-like projection of MAPs. Two MAP-binding sites were located on intact mitochondria. The lower-affinity MAP2-binding site (Kd = 2 x 10(-7) M) was preserved and enriched in the outer-membrane fraction, whereas the higher-affinity site (Kd = 1 x 10(-9) M) was destroyed after removing the outer membrane with digitonin. Detergent fractionation of mitochondrial outer membranes saturated with MAP2 bound in vitro showed that MAPs are associated with membrane fragments which contain the pore-forming protein (porin). MAP2 also partially prevents the solubilization of porin from outer membrane, indicating a MAP-induced change in the membrane environment of porin. These observations demonstrate the presence of specific MAP-binding sites on the outer membrane, suggesting an association between porin and the membrane domain involved in the cross-linkage between microtubules and mitochondria.     

Use of a heat-stable microtubule-associated protein class-specific antibody to investigate the mechanism of microtubule binding.

Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule-binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat-stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val-Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co-sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding.     

Specificity and biological significance of microtubule-associated protein-DNA interactions in chick

The interactions between chick brain microtubule associated proteins (MAPs) and chick DNA have been examined using DNA-cellulose chromatography, cross-blotting, and nitrocellulose filter-binding. Comparison of nitrocellulose filter-binding and cross-blotting results show that while MAPs and a minor, M_r 48 000, protein show significant binding at 50 mM NaCl, only the latter continues to bind a significant amount of DNA at 150 mM NaCl, suggesting an ionic basis for the MAP-DNA interactions. MAP-DNA interactions also show weak preference for AT-rich fractions, and are sensitive to S1 nuclease digestion. We suggest that the MAPs bind preferentially to single-stranded DNA. The binding may involve an interaction between the DNA phosphates and the highly cationic tubulin-binding domain of the MAPs. Repetitive fractions of the chick genome prepared both by hydroxyapatite chromatography and by S1 nuclease digestion show binding to a number of minor proteins present in preparations of microtubule proteins, as well as to the MAPs. We conclude that the MAPs probably do not bind specifically to repetitive DNA, in contrast to earlier reports using mouse DNA. MAP-DNA interactions are therefore unlikely to be involved in the attachment of microtubules to mitotic chromosomes.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics

We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule- associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.     

Towards an understanding of microtubule function and cell organization: an overview.

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.     

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequencial affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behavior of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.Abbreviations MAPs microtubule-associated proteins - C-terminal carboxyl-terminal - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - DTT dithiotreitol - BSA bovine serum albumin     

Plant microtubule cytoskeleton complexity: microtubule arrays as fractals

Biological systems are by nature complex and this complexity has been shown to be important in maintaining homeostasis. The plant microtubule cytoskeleton is a highly complex system, with contributing factors through interactions with microtubule-associated proteins (MAPs), expression of multiple tubulin isoforms, and post-translational modification of tubulin and MAPs. Some of this complexity is specific to microtubules, such as a redundancy in factors that regulate microtubule depolymerization. Plant microtubules form partial helical fractals that play a key role in development. It is suggested that, under certain cellular conditions, other categories of microtubule fractals may form including isotropic fractals, triangular fractals, and branched fractals. Helical fractal proteins including coiled-coil and armadillo/beta-catenin repeat proteins and the actin cytoskeleton are important here too. Either alone, or in combination, these fractals may drive much of plant development.     

A common mechanism for cytoplasmic dynein-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes

During infection, adenovirus-associated virus (AAV) undergoes microtubule-dependent retrograde transport as part of a program of vectorial transport of viral genome to the nucleus. A microtubule binding assay was used to evaluate the hypothesis that cytoplasmic dynein mediates AAV interaction with microtubules. Binding of AAV serotype 2 (AAV2) was enhanced in a nucleotide-dependent manner by the presence of total cellular microtubule-associated proteins (MAPs) but not cytoplasmic dynein-depleted MAPs. Excess AAV2 capsid protein prevented microtubule binding by AAV serotypes 2, 5, and rh.10, as well as adenovirus serotype 5, indicating that similar binding sites are used by these viruses.     

TOR signaling regulates microtubule structure and function

The functional diversity and structural heterogeneity of microtubules are largely determined by microtubule-associated proteins (MAPs) [1] [2]. Bik1p (bilateral karyogamy defect protein) is one of the MAPs required for microtubule assembly, stability and function in cell processes such as karyogamy and nuclear migration and positioning in the yeast Saccharomyces cerevisiae [3]. The macrocyclic immunosuppressive antibiotic rapamycin, complexed with its binding protein FKBP12, binds to and inhibits the target of rapamycin protein (TOR) in yeast [4] [5]. We report here that TOR physically interacts with Bik1p, the yeast homolog of human CLIP-170/Restin [6] [7]. Inhibition of TOR by rapamycin significantly affects microtubule assembly, elongation and stability. This function of TOR is independent of new protein synthesis. Rapamycin also causes defects in spindle orientation, nuclear movement and positioning, karyogamy and chromosomal stability, defects also found in the bikDelta mutant. Our data suggest a role for TOR signaling in regulating microtubule stability and function, possibly through Bik1p.     

The number of repeat sequences in microtubule-associated protein 4 affects the microtubule surface properties

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a Repeat region with tandemly organized repeat sequences. In this study, we focused on the variations of the Repeat region, and searched for MAP4 isoforms with diverse Repeat region organizations. We successfully isolated four types of MAP4 cDNAs, which differed from each other in both the number and the arrangement of the repeat sequences, from a single source (bovine adrenal gland). To examine the functional differences among the isoforms, we prepared the microtubule-binding domain polypeptides of three of the four isoforms, and examined their activities. The isoform fragments showed similar degrees of microtubule assembly promoting activity and microtubule binding affinity. This result suggested that the Repeat region variation is not important for the control of microtubule dynamics, which is believed to be the main function of MAPs. On the other hand, the microtubule bundle-forming activity differed among the isoform fragments. The bundle formation was augmented by increasing the number of repeat sequences in the fragments. Based on these results, we propose the hypothesis that the role of the MAP4 isoforms is to regulate the surface charge of microtubules.     

Radial microtubule organization by histone H1 on nuclei of cultured tobacco BY-2 cells

In acentriolar higher plant cells, the surface of the nucleus acts as a microtubule-organizing center, substituting for the centrosome. However, the protein factors responsible for this microtubule organization are unknown. The nuclear surfaces of cultured tobacco BY-2 cells possess particles that generate microtubules. We attempted to isolate the proteins in these particles to determine their role in microtubule organization. When incubated with plant or mammalian tubulin, some, but not all, of the isolated nuclei generated abundant microtubules radially from their surfaces. The substance to induce the formation of radial microtubules was confirmed by SDS-PAGE to be a protein with apparent molecular mass of 38 kDa. Partial analysis of the amino acid sequences of the peptide fragments suggested it was a histone H1-related protein. Cloning and cDNA sequence analysis confirmed this and revealed that when the recombinant protein was incubated with tubulin, it could organize microtubules as well as the 38-kDa protein. Histone H1 and tubulin formed complexes immediately, even on ice, and then clusters of these structures were formed. These clusters generated radial microtubules. This microtubule-organizing property was confined to histone H1; all other core histones failed to act as organizers. On immunoblot analysis, rabbit antibodies raised against the 38-kDa protein cross-reacted with histone H1 proteins from tobacco BY-2 cells. These antibodies virtually abolished the ability of the nucleus to organize radial microtubules. Indirect immunofluorescence showed that the antigen was distributed at the nuclear plasm and particularly at nuclear periphery independently from DNA.     

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Inhibition of microtubule assembly by poly(L-glutamic acid) and the site of its action

Poly(L-glutamic acid) (PGA) suppresses the polymerization of porcine brain microtubule proteins and induces the depolymerization in vitro in a concentration-dependent manner. The extent of inhibition increases with increasing molecular weight of the PGA tested. A 50% inhibition of the protein polymerization was observed at a PGA (molecular weight = 60,000) to microtubule protein ratio of 0.04 (w/w), and complete inhibition was obtained at a ratio of 0.07. Such an inhibition on the polymerization by PGA is greatly decreased when Mg2+ is present at a higher concentration. The addition of PGA raises the critical concentration of microtubule proteins necessary for assembly. During incubation with PGA, microtubule proteins retain the ability to assemble, i.e., substoichiometric amounts of taxol considerably relieve the inhibition of assembly by PGA. PGA interacts with microtubule-associated proteins (MAPs) preferentially, because the amount of MAPs binding to PGA-Sepharose 4B is much larger than that of tubulin. Tau proteins were observed only in adsorbed fractions, while MAP-2 was present in both unbound and adsorbed fractions.     

Rapid phosphorylation of microtubule-associated proteins through distinct mitogenic pathways

Mitogenic stimulation of sparse quiescent Swiss 3T3 cells with serum induces a transient reorganization of microtubules which may be necessary for generation or transduction of the mitogenic signal(s). Recently, several studies have shown that microtubule-associated proteins (MAPs) modulate microtubule-mediated functions in vitro and in vivo. We have analyzed, by two-dimensional electrophoresis, the molecular changes in MAPs associated with microtubules in situ following cell activation. By as early as 15 min after addition of serum, several of the MAPs present in quiescent cells are lost from the assembled microtubule fraction while one additional MAP becomes evident. This new MAP is a phosphoprotein whose appearance is independent of protein synthesis. Four additional MAPs also become phosphorylated, and this phosphorylation is accompanied by a partial redistribution of MAPs into the unassembled soluble fraction. Stimulation of cells with purified platelet-derived growth factor or phorbol tumor promoter, a direct activator of protein kinase C, also induces phosphorylation of the same MAPs and DNA synthesis. These results demonstrate that activation of the protein kinase C pathway is sufficient to promote the phosphorylation of MAPs and mitogenesis. However, epidermal growth factor, which does not activate protein kinase C, also stimulates phosphorylation of MAPs and DNA replication. Furthermore, down-regulation of the protein kinase C pathway does not prevent these responses. We conclude that phosphorylation of MAPs and mitogenesis can proceed through protein kinase C-dependent and -independent pathways in 3T3 cells.     

Regulation of the microtubule steady state in vitro by ATP.

ATP increases microtubule steady state assembly and disassembly rates in vitro in a concentration-dependent manner. Bovine brain microtubules, composed of 75% tubulin and 25% high molecular weight microtubule-associated proteins (MAPs), were purified by three cycles of assembly and disassembly in the absence of ATP. When assembled to steady state, these microtubules add dimers at one end and lose them at the other in a unidirectional assembly-disassembly process. In the presence of 1.0 mM ATP the unidirectional flow of tubulin from one end of the microtubules to the other increases as much as 20 fold, as revealed by loss of 3H-GTP from uniformly labeled microtubules under GTP chase conditions and by the rate of disassembly following addition of 50 microM podophyllotoxin. UTP, CTP and 5' adenylylimidodiphosphate (AMP-PNP) cannot substitute for ATP in producing this effect. Furthermore, the increase in steady state flow rate persists afer ATP is removed. Thus microtubules assembled in ATP and centrifuged through sucrose cushions to separate them from nucleotides continue to exhibit increased rates in the next assembly cycle in the absence of ATP. It is possible that an ATP-dependent microtubule protein kinase is responsible for the observed increase in tubulin flow rate. A kinase activity associated with brain MAPs has been reported to be cAMP-dependent (Sloboda et al., 1975). We have found an adenylate cyclase activity associated with these microtubules. Whether the adenylate cyclase is a contaminant or due to a specific microtubules-associated protein, and whether its activity is functionally linked to the increased rate of assembly and disassembly in the presence of ATP, remain to be determined.     

Tau protein binds to microtubules through a flexible array of distributed weak sites

Tau protein plays a role in the extension and maintenance of neuronal processes through a direct association with microtubules. To characterize the nature of this association, we have synthesized a collection of tau protein fragments and studied their binding properties. The relatively weak affinity of tau protein for microtubules (approximately 10(-7) M) is concentrated in a large region containing three or four 18 amino acid repeated binding elements. These are separated by apparently flexible but less conserved linker sequences of 13-14 amino acids that do not bind. Within the repeats, the binding energy for microtubules is delocalized and derives from a series of weak interactions contributed by small groups of amino acids. These unusual characteristics suggest tau protein can assume multiple conformations and can pivot and perhaps migrate on the surface of the microtubule. The flexible structure of the tau protein binding interaction may allow it to be easily displaced from the microtubule lattice and may have important consequences for its function.     

The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure-function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH(2)-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability.