Brain Microtubule-associated Proteins Research Articles

Immunolocalization and molecular properties of a high molecular weight microtubule-bundling protein (syncolin) from chicken erythrocytes.

A protein of apparent molecular weight 280,000 (syncolin), which is immunoreactive with antibodies to hog brain microtubule-associated protein (MAP) 2, was purified from chicken erythrocytes. Immunofluorescence microscopy of bone marrow cells revealed the presence of syncolin in cells at all stages of erythrocyte differentiation. In early erythroblasts syncolin was diffusely distributed throughout the cytoplasm. At later stages it was found along microtubules of the marginal band, as confirmed by immunoelectron microscopy. The association of syncolin with the marginal band was dependent on the integrity of microtubules, as demonstrated by temperature-dependent de- and repolymerization or marginal band microtubules. Syncolin cosedimented in a saturable manner with microtubules assembled in vitro, and it was displaced from the polymer by salt. Brain as well as erythrocyte microtubules, reconstituted with taxol from MAP-free tubulin and purified syncolin, were aggregated into dense bundles containing up to 15 microtubules, as determined by electron microscopy. On the ultrastructural level, syncolin molecules were visualized as globular or ringlike structures, in contrast to the thin, threadlike appearance of filamentous MAPs, such as brain MAP 2. According to ultrastructural measurements and gel permeation chromatography, syncolin's molecular weight was approximately 1 x 10(6). It is suggested that syncolin's specific function is the cross-linking of microtubules in the marginal band and, by implication, the stabilization of this structure typical for nucleated (chicken) erythrocytes.

Intracellular localization of MAP2-related protein (O-map) in prophase I and metaphase II oocytes of Xenopus

An antibody directed against rat brain microtubule-associated protein 2 (MAP2) immunoprecipitated a protein of 240 kDa from a Xenopus oocyte extract. In contrast, in Xenopus brain extract, this antibody recognizes a protein of 280 kDa. The oocyte MAP2-related protein (called O-MAP) is present in both prophase I and metaphase II-blocked oocytes as demonstrated by immunoblotting experiments; it is in vivo phosphorylated. Immunocytochemical studies using the anti-rat brain MAP2 antibody demonstrated that the O-MAP colocalizes within the cortical microtubular array present in both prophase I and metaphase II oocytes. However, O-MAP is not associated with the microtubular structures which are organized during the oocyte prophase-metaphase transition, i.e., a giant cytoplasmic network and both the meiotic spindles. O-MAP therefore appears as a microtubule-associated protein oocyte specific and may play a role in the regulation of microtubule stability and the organization of the oocyte cytoskeleton.

Association between microtubules and golgi vesicles isolated from rat parotid glands

Summary We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 μg of microtubule protein bound to 500 μg of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 μg/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10–20% of the total tubulin content of the parotid cell.

Phosphorylation of microtubule-associated protein-2 in GH3 cells. Regulation by cAMP and by calcium.

The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.

Phosphorylation of microtubule-associated protein tau: identification of the site for Ca2(+)-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles.

The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth. While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role. Tau is one of the MAPs in mammalian brain. When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing. Tau can be phosphorylated by several protein kinases. Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity. Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles. By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats. Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier. The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles.

The Phosphorylation of a Putative Sperm Microtubule-Associated Protein 2 (MAP2) is Uniquely Sensitive to Regulation1

We have identified a bovine sperm phosphoprotein, pp255 (Mr = 255,000), which reacts strongly and specifically with an antibody to rat brain microtubule-associated protein 2 (MAP2). The phosphorylation state of this putative sperm MAP2 in intact bovine epididymal sperm is uniquely sensitive to regulation by intracellular pH (pHi), calcium, isobutyl-3-methylxanthine (MIX), H-8, and fluoride. Increasing pHi by approximately 0.4 units or exposure to calcium (0.1 microM with the ionophore A23187) or to the protein kinase inhibitor, H-8, decreases sperm MAP2 phosphorylation. Decreasing sperm pHi or exposure to MIX or fluoride increases MAP2 phosphorylation. Numerous other detectable sperm phosphoproteins are either unresponsive to most of these modulators or are considerably less sensitive to them. This phosphoprotein co-sediments with the particulate sperm heads during subcellular fractionation, and is not detectable in other sperm fractions. Two-dimensional electrophoresis separates sperm MAP2 into multiple species, indicative of varying degrees of phosphorylation. Sperm MAP2 is phosphorylated on serine residues, changes electrophoretic mobility slightly on one-dimensional gels with changes in phosphorylation levels, and exhibits the highest specific radioactivity of any sperm phosphoprotein observed. The phosphorylation state of sperm MAP2 can be uncoupled from sperm motility levels under several conditions. The co-localization of sperm MAP2 with the head fraction and the unique sensitivity of its phosphorylation level to modulators, which are known to regulate capacitation and the acrosome reaction, suggest that sperm MAP2 phosphorylation may be an intermediate step in the regulation of one or both of these sperm processes.

Traces of brain microtubule-associated proteins affect dynamic properties of microtubules

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin-colchicine complex in the mixture to block the repolymerization. For a total of 24 microM tubulin in a polymerization mixture, 10 microM of the sample polymerized originally under the conditions used. When 1.05 microM tubulin-colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23-2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.

Presence of a new microtubule cold-stabilizing factor in bull sperm dynein preparations

Brain tubulin polymerized with dynein isolated from bull spermatozoa forms cold-stable microtubules, in contrast with microtubules made of brain tubulin polymerized by brain microtubule-associated proteins (MAPs). The level of cold-stable microtubules depends on the concentration of dynein used. Addition of dynein to cold-unstable microtubules renders these microtubules stable to cold. Although ATP and a non-hydrolysable ATP analogue increase the formation of microtubules made of tubulin and dynein, these nucleotides have no effect on dynein cold-stabilizing properties. The data suggests that a new factor, not involving the dynein ATPase active site and present in bull sperm dynein preparations, confers cold-stability to microtubules.

Characterization and Intracellular Distribution of Microtubule‐Associated Protein 2 in Differentiating Human Neuroblastoma Cells

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.

Microtubule motors and other maps

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

The tubulin-binding sequence of brain microtubule-associated proteins, tau and MAP-2, is also involved in actin binding

The interaction of actin with a synthetic peptide which corresponds to one of the repeated tubulin-binding sites present in tau and MAP-2 (microtubule-associated protein 2) proteins has been analysed. The analysis, which uses affinity chromatography of G-actin on a column containing the synthetic peptide, and the co-sedimentation and co-localization of F-actin and the peptide (as determined by immunoelectron microscopy), indicates that the part of the amino acid sequence of tau involved in the binding of tubulin is also involved in actin binding.

Binding of exocytic vesicles from MDCK cells to microtubules in vitro

Microtubules have been implicated in the transport of vesicles carrying newly synthesized proteins from the trans-Golgi network (TGN) to the cell surface. We have established a quantitative in vitro binding assay to investigate the putative interaction between these exocytic carrier vesicles and the microtubules at the molecular level. TGN-derived exocytic carrier vesicles, labeled with C6NBD-ceramide metabolites or viral glycoproteins, were obtained from polarized filter-grown MDCK II cells by perforation of the apical membrane with a nitrocellulose filter. These exocytic vesicles were incubated with taxol-polymerized tubulin and cytosol, layered on top of a 30% sucrose cushion and subjected to centrifugation. Quantitation of vesicles co-sedimenting with microtubules was done by measuring NBD-fluorescence of viral glycoproteins in the pellet and supernatant fractions. About 25% of the label sedimented through the cushion in the presence of microtubules and cytosol. Both apically and basolaterally targetted carrier vesicles containing influenza virus HA2 or vesicular stomatitis virus G protein, respectively, associated with the microtubules. Only 2-5% NBD-fluorescence was obtained in the pellet when no cytosol or microtubules were added to the vesicles. Negative-stain electron microscopy of resuspended pellets showed distinct microtubule-vesicle complexes. Heat inactivation or treatment of cytosol with N-ethylmaleimide (NEM), or trypsinization of vesicles inhibited the binding of vesicles to microtubules. Furthermore, coating of microtubules with brain microtubule-associated proteins abolished binding. These data suggest that NEM-sensitive cytosolic proteins are required for microtubule-vesicle association, and that the vesicles are bound via trypsin-sensitive receptor proteins on their surface.

Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2.

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.

A minor microtubule-associated protein is responsible for the stimulation of vesicular stomatitis virus transcription in vitro

An activity found in bovine brain microtubule-associated proteins (MAPs) which stimulated vesicular stomatitis virus (VSV) transcription in vitro co-eluted from a gel filtration column (Sepharose CL-6B) with a minor MAP subset of an apparent Mr of 100,000 to 250,000. The activity did not appear to be closely associated with MAPs 1, MAPs 2, tau or tubulin. Anti-MAPs 1 and anti-MAPs 2 IgG did not reduce or abolish the stimulatory activity. The bovine brain MAPs stimulatory activity was similar to that found in HeLa cell extracts: both were heat-resistant, and both co-purified with the MAPs fraction of cell extracts; also amounts of each which gave maximum stimulation when used separately did not give additional stimulation when combined. Fractions from the Sepharose CL-6B column that contained the greatest amount of stimulatory activity exhibited little or no cAMP-dependent or -independent kinase activity. The MAPs stimulatory activity required the presence of both L and NS polymerase subunits.

Effects of brain microtubule‐associated proteins on microtubule dynamics and the nucleating activity of centrosomes

In this paper, we report on the effect of brain microtubule-associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated per centrosome. This number remains constant with time. This suggests that the number of centrosome-nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.

Association between microtubules and Golgi vesicles isolated from rat parotid glands

We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.

Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly-disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (greater than 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.

Modulation of a 190-kD microtubule-associated protein in pigment epithelium by VIP

Vasoactive intestinal peptide stimulates phosphorylation of six high molecular weight cytosolic proteins in the cultured retinal pigment epithelium (RPE). Of these, the 190-kD phosphoprotein is associated with the microtubules assembled by taxol/GTP and is immunologically related to the brain microtubule-associated protein 2 (mol.wt. = 280 kD). VIP is also shown here to stimulate secretion in the cultured RPE. VIP-stimulated phosphorylation of a 190-kD microtubule-associated protein is also demonstrated here in the retinal glia.

Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells.

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 220K (K = 10(3) Mr), which are immunologically related to brain MAP-1B. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to the neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-1B and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in vitro, which suggests that they may participate in microtubule assembly in vivo; the 325K protein could perform such a role during the entire cell cycle, while the 220K protein could be implicated in the formation of the mitotic spindle.

In situ localization of microtubule-associated protein mRNA in the developing and adult rat brain

We have used cDNA probes specific for three of the major brain microtubule-associated proteins (MAPS), MAP1, MAP2, and MAPS, to study the timing of appearance, relative abundance, and intracellular compartmentalization of MAP gene transcripts in developing rat brain. The MAPI probe hybridizes throughout the brain, in both grey and white matter. MAN mRNA is detected only in grey matter and appears in cerebral neurons only after they have ceased dividing and have migrated to the cortical plate. The MAPS cDNA hybridizes throughout the embryonic brain, but by P12, MAP5 mRNA distribution is restricted to relatively immature areas. MAN mRNA, found in dendrites in the developing brain, persists in some adult dendrites. MAP5 mRNA, like β-tubulin mRNA, is found only in the cell bodies of developing neurons, indicating that the protein must be transported from the soma into processes. MAP1 mRNA is found only in the proximal regions of cortical pyramidal cell dendrites in both developing and adult brain. The diverse distributions of MAP gene transcripts emphasize the importance of these proteins in generating heterogeneity of microtubule function and indicate that MAP compartmentalization within neurons is regulated in part by differential mRNA transport.