Microtubule-associated protein 2 and the organization of cellular microtubules

Microtubule-associated proteins (MAPs) are prominent components of the neuronal cytoskeleton that can promote microtubule formation and whose expression is under strong developmental regulation. They are thought to be involved in organizing the structure of microtubule fascicles in axons and dendrites, although whether they form active cross-links between microtubules or serve as strut-like spacer elements has yet to be resolved. In the experiments reported here we explored their influence on microtubules by expressing them in non-neuronal cells using DNA transfection techniques. We confirm earlier reports that microtubule-associated proteins of the MAP2/tau class can induce bundling of microtubules. In addition we find that MAP2 causes the rearrangement of microtubules in the cytoplasm in a manner that is dependent on the length of the microtubule bundles. Short bundles are straight and run across the cytoplasm whereas long bundles form a marginal band-like array at the periphery. We suggest that the latter arrangement is produced when microtubule bundles that are too long to fit inside the diameter of the cell bend under the restraining influence of the cortical cytoskeleton. In confirmation of this, we show that when the cortical actin network is depolymerized by cytochalasin B the MAP2-containing microtubule bundles push out cylindrical extensions from the cell surface. These results suggest that the induction of stiff microtubules bundles by MAP2, coupled with a breach in the cortical actin network, can confer two of the properties characteristic of neuronal processes; their cylindrical form and the presence of fasciculated microtubules.     

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.     

Morphological transformation of liposomes caused by assembly of encapsulated tubulin and determination of shape by microtubule-associated proteins (MAPs)

To examine the role of cytoskeletons in cellular morphogenesis, we generated liposomes encapsulating tubulin, with or without microtubule-associated proteins (MAPs), and observed their transformation using dark-field microscopy. When tubulin was polymerized with MAPs in liposomes, liposomes were transformed into a "bipolar" shape with a central sphere and two tubular membrane protrusions that aligned in a straight line. On the other hand, when pure tubulin was polymerized in liposomes without MAPs, they initially transformed into a bipolar shape but subsequently re-transformed into a "monopolar" shape, i.e. a sphere with only one straight tubular portion. This re-transformation occurred in two ways: first, by shortening of one of the tubular portions due to microtubule disassembly; or second, by fluctuation of the central sphere toward one of the ends without shortening of the tube portion. MAPs prevented this re-transformation, and their role in stabilizing the shape of transformed liposomes was studied by the co-sedimentation method. The results show that MAPs, particularly MAP1 and MAP2, mediate binding between microtubules and the liposomal membrane. However, MAP2 by itself did not bind to liposomes, but was able to stabilize bipolar liposomes. This stabilization is caused not only by direct links between microtubules and liposomes, but also by prevention of Brownian motion of microtubules through an increase in friction.     

CYP2D6 mRNA expression in circulating peripheral blood mononuclear cells correlates with in vivo debrisoquine hydroxylase activity in extensive metabolizers

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 sequential affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behaviour 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.     

Assembly of Atlantic cod (Gadus morhua) brain microtubules at different temperatures: dependency of microtubule-associated proteins is relative to temperature

Isolated cod (Gadus morhua) brain microtubules were found to have a broad temperature interval for assembly. In contrast to mammalian microtubules they assembled even at as low temperatures as 14 degrees C. Evidence was found that temperature alters the dependency of microtubule-associated proteins (MAPs) for assembly. The assembly was MAPs-dependent at low, but not at higher temperatures. Assembly at +18 degrees C was inhibited by both NaCl and estramustine phosphate. These compounds are well known to inhibit the binding of MAPs to tubulin. At higher temperatures there was no MAPs dependency for assembly, despite that MAPs bound to the microtubules. Cow MAPs had the same effect as cod MAPs, suggesting that despite differences in MAP composition, the effect is not caused by the unusual composition of cod MAPs. The results therefore suggest that these differences in MAPs dependency are due to intrinsic properties of cod tubulin or tubulin-to-tubulin interactions. Small temperature-induced conformational changes of tubulin and a slight enrichment of acetylated and detyrosinated tubulin in microtubules assembled at +30 degrees C as compared to +15 degrees C, were observed. The ability to alter the assembly stimulating effect of MAPs may be important for the cell to regulate microtubule dynamics and stability. In addition, changes in tubulin conformation and composition of tubulin isoforms may reflect adaptations for microtubule assembly at low temperatures.     

A new model for microtubule-associated protein (MAP)-induced microtubule assembly. The Pro-rich region of MAP4 promotes nucleation of microtubule assembly in vitro

The microtubule-binding domains of microtubule-associated protein (MAP) 2, tau, and MAP4 are divided into three distinctive regions: the Pro-rich region, the AP sequence region and the tail region (Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Electron microscopic observation showed that the taxol-stabilized microtubules alone and those mixed with the A4T fragment (containing the AP sequence region and the tail region) had a long, wavy appearance, while those mixed with the PA4T fragment (containing the Pro-rich region, the AP sequence region, and the tail region) or the PA4 fragment (containing the Pro-rich region and the AP sequence region) were shorter and straighter. Stoichiometries of the binding between the fragments and the tubulin dimers were approximately between 1 and 2, suggesting that not all of the AP sequences in the AP sequence region bound to tubulin. Binding affinity of the PA4T fragment is only four times higher than that of the A4T fragment, while the microtubule nucleating activity of the PA4T fragment is far greater. Based on these results, we propose that the nucleation of microtubule assembly is promoted by the bridging activity of the Pro-rich region in the MAPs.     

Microtubule-associated proteins and the stimulation of tubulin assembly in vitro

Microtubules, purified by cycles of assembly and disassembly in vitro, are composed of tubulin and several microtubule-associated proteins (MAPs). When the MAPs were separated from the tubulin by phosphocellulose chromatography, the tubulin by phosphocellulose chromatography, the tubulin no longer assembled at 37 degrees C as measured by turbidity. If the MAPs and tubulin were recombined and warmed to 37 degrees C, microtubules assembled. MAPs stimulated tubulin assembly by affecting both the initiation and elongation processes. The effect on initiation was indicated by results showing an increase in initial rate and a decrease in average microtubule length as the MAP:tubulin ratio was increased. The initiation and elongation activities of the MAPs at 4 degrees C during which time the initiating activity decreased while the ability to affect the total amount of assembly remained constant. The decrease in initiating ability was correlated with the loss of the two major components of the MAP fraction, MAPs 1 and 2.     

An immunohistochemical study of neuronal microtubule-associated proteins in Hirschsprung's disease

Neuronal microtubule-associated proteins (MAPs) are important components of neurons and are believed to regulate neuronal function and development by controlling the assembly of microtubules and the interaction of microtubules with other cytoplasmic organelles. We studied the immunohistochemical localization of MAPs 1, 2, 5, and tau in the intestinal tissues of five patients with Hirschsprung's disease and in five normal controls using monoclonal antibodies. Microtubule-associated proteins 5 and tau proved to be excellent enteric neuronal markers; they were specifically located in the nerve cell bodies and processes of normal intestine as well as in the abnormal hypertrophied nerve fibers of aganglionic colon. Fine fibrillar structures in the neuroplasm were revealed in greater detail than were those obtained from studies with conventional markers, including neuron-specific enolase, S-100 protein, and neurofilament protein. A slight reduction of MAPs 5 and tau immunoreactivity was observed in the aganglionic colon compared with normal colon. Microtubule-associated proteins 1 and 2 were absent from the nerve fibers in both normal and aganglionic colon. This study suggests that immunostaining for MAPs 5 and tau may be superior to other immunohistochemical methods for diagnosing Hirschsprung's disease; however, in view of its limited retrospective nature these findings need to be corroborated by a large prospective evaluation.     

EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes

The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly has a Mr of 77,000. The 77,000 Mr polypeptide is heat- and acid-labile, and is antigenically distinct from the mammalian brain MAPs, MAP-2 and tau. Affinity-purified antiserum against the 77,000 Mr polypeptide was used to survey a variety of cells and tissues for the presence of antigenically related polypeptides. A cross-reacting polypeptide, ranging in Mr from 72,000 to 80,000, was found in microtubule preparations from a wide variety of echinoderms, including sea urchins, starfish and sand dollars. Indirect immunofluorescence showed that the polypetide was found in interphase as well as mitotic microtubule arrays. No cross-reacting material was detected in microtubules isolated from marine molluscs, mammalian brain or mouse B16 cultured cells. Because the 77,000 Mr MAP is abundant in echinoderms, we have called it EMAP for echinoderm microtubule-associated protein. Although the precise function of the EMAP is not known, our data suggest that the EMAP is involved in the attachment of ribosomes to microtubules. Large numbers of ribosomes are attached to the walls of EMAP-containing microtubules, but not EMAP-deficient microtubules. Removal of the EMAP from the microtubule by salt-extraction results in the release of ribosomes from the microtubule, indicating that the EMAP may form part or all of the long tapered stalk that connects these two organelles.     

Characterization of in vitro glycation sites of tau

Tau is a microtubule-associated protein that loses microtubule binding activity and aggregates into paired helical filaments (PHFs) in Alzheimer's disease. Nonenzymic glycation is one of the posttranslational modifications detected in PHF-tau, but not in normal tau. PHF-tau has reduced ability to bind to microtubules. To determine whether glycation of tau occurs in its microtubule binding domains, we have characterized in vitro glycation sites of the longest isoform of tau, which has four microtubule binding domains (Tau-4). The identified glycation sites are Lys-87, 132, 150, 163, 174, 225, 234, 259, 280, 281, 347, 353, and 369. We have also studied glycation of another isoform of tau, which has only three microtubule binding domains (Tau-3). This isoform is modified by glucose 15-20% more slowly than Tau-4. However, the glycation sites appear to be the same in both isoforms, except for Lys-280 and 281; these are located in the second microtubule binding domain, which is missing in Tau-3. Lys-150, 163, and 174 are located within or proximal to the sequence of tau that is involved in the microtubule nucleation activity, and Lys-259, 280, 281, 347, 353, and 369 are located in the microtubule binding domains. Glycation at these sites can affect the functional properties of tau, and advanced glycation at these sites might lead to the formation of insoluble aggregates similar to the ones seen in Alzheimer's disease.     

Characterization of the microtubule-binding domain of microtubule-associated protein 1A and its effects on microtubule dynamics

To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.     

Neuronal abnormalities in microtubule-associated protein 1B mutant mice

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.     

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.     

Parallel coding of conjunctions in visual search

The microtubule associated protein tau is the main structural component of paired helical filaments (PHFs), aberrant polymers found intracellularly in neurons of brains with the Alzheimer's disease. Glycation is one of the posttranslational modifications that has been found in tau from PHFs, but not in normal brain tau. Studies were carried out with purified tau protein subjected to chemical modifications, in order to further investigate the mechanisms of tau self-association into PHFs. Tau was subjected to modifications affecting reactive lysyl residues, e.g., carbamoylation with potassium cyanate and glycation reaction with glucose. The effects of these modifications to produce functional alterations in tau capacity to bind brain tubulin and to induce microtubule assembly were investigated. Chemically-modified tau and tau of Alzheimer's type exhibited a similar microtubule interaction behavior as analysed by overlay assays, but those were different than normal tau controls. On the other hand, studies of the microtubule assembly kinetics indicated that the reported tau modifications resulted in a loss of its capacity to promote microtubule assembly from purified tubulin preparations. The data on the differences in the electrophoretic profiles, Western blots and the overlay patterns, along with those on the microtubule polymerisation of normal brain tau as compared with both modified and Alzheimer's tau, suggest changes in the functional behavior of this protein as a result of its structural modifications. These studies were complemented with an immunogold analysis at the electron microscope level, which indicated that the modified tau did not incorporate into assembled microtubules. These findings, combined with the results on tau chemical modifications suggest that the reactive lysine residues within functional domains on tau, e.g., those of the repetitive binding motifs, were affected by these modifications. Furthermore, these observations provide new clues to understand the anomalous interactions of tau in Alzheimer's disease.     

Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue

The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 microM EMAP to 21 microM tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.     

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (SP-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and SP-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.     

The microtubule-associated protein tau cross-links to two distinct sites on each alpha and beta tubulin monomer via separate domains

The interaction between tubulin subunits and microtubule-associated proteins (MAPs) such as tau is fundamental for microtubule structure and function. Previous work has suggested that the "microtubule binding domain" of tau (composed of three or four imperfect 18-amino acid repeats, separated by 13- or 14-amino acid inter-repeat regions) can bind to the C-terminal ends of both alpha and beta tubulin monomers. Here, using covalent cross-linking strategies, we demonstrate that there are two distinct tau cross-linking sites (designated as "C-terminal" and "internal") on each alpha and beta tubulin monomer. The C-terminal tau cross-linking site is located within the 12 C-terminal amino acids of both alpha and beta tubulin, while the internal tau cross-linking site is located within the C-terminal one-third of alpha and beta tubulin but not within the last 12 amino acids. In addition, we show that tau cross-links to the C-terminal site via its repeat 1 and/or the R1-R2 inter-repeat. The cross-linking of tau to the internal site is mediated by some subset of its other repeat units. Integrating these and earlier data with the 3.7 A resolution model of the alphabeta tubulin dimer recently presented by E. Nogales et al. [(1998), Nature 391, 199-203], we propose a new model for the tau-microtubule interaction.