Isolation of rat liver microtubule-associated proteins. Evidence for a family of microtubule-associated proteins with molecular mass of around 200,000 which distribute widely among mammalian cells

Heat-stable microtubule-associated proteins (MAPs) were isolated from rat liver crude extract. The most prominent species showed a molecular mass close to that of bovine adrenal 190-kDa MAP (Kotani, S., Murofushi, H., Maekawa, S., Sato, C., and Sakai, H. (1986) Eur. J. Biochem. 156, 23-29), termed rat 190-kDa MAP. Immunological studies with antiserum against the rat 190-kDa MAP showed that this MAP exists in a variety of rat cells and tissues. The characteristics of the rat 190-kDa MAP, including molecular mass, heat stability, and distribution pattern, were very similar to those of bovine adrenal 190-kDa MAP. However, one-dimensional peptide mapping revealed considerable difference, and there is little mutual immunological cross-reactivity. We also identified in mouse neuroblastoma Neuro 2a cells a MAP of around 200 kDa which is considered to be MAP4 (Parysek, L. M., Asnes, C.F., and Olmsted, J.B. (1984) J. Cell Biol. 99, 1309-1315). MAP4 was slightly immunoreactive to both anti-(rat 190-kDa MAP) antiserum and anti-(bovine 190-kDa MAP) antiserum. Taking these results together, we conclude that mammalian tissues ubiquitously contain heat-stable MAPs of 200 kDa and that these 200-kDa MAPs should be considered as species-specific homologues.     

Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.     

A common amino acid sequence in 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly

Previously we reported that chymotryptic fragments of bovine adrenal 190-kDa microtubule-associated proteins (27-kDa fragment) and bovine brain tau (14-kDa fragment) contained microtubule-binding domain (Aizawa, H., Murofushi, H., Kotani, Hisanaga, S., Hirokawa, N., and Sakai, H. (1987) J. Biol. Chem. 262, 3782-3787; Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1988) J. Biol. Chem. 263, 7703-7707). In order to study the structure of microtubule-binding domain of the two microtubule-associated proteins, we analyzed the amino acid sequence of the 27-kDa fragment and compared the sequence with that of the 14-kDa fragment. This revealed that 190-kDa microtubule-associated protein and tau contained at least one common sequence of 20 amino acid residues in their microtubule-binding domains. A synthetic polypeptide corresponding to the common sequence (Lys-Asn-Val-Arg-Ser-Lys-Val-Gly-Ser-Thr-Glu-Asn-Ile-Lys- His-Gln-Pro-Gly-Gly-Gly-Arg-Ala-Lys) was bound to microtubules competitively with the 190-kDa MAP. The apparent dissociation constant (KD) for the binding of the polypeptide to microtubules was estimated to be 1.8 x 10(-4) M, and the maximum binding reached 1.2 mol of the synthetic polypeptide/mol of tubulin dimer. This synthetic polypeptide increased the rate and extent of tubulin polymerization and decreased the critical concentration of tubulin for polymerization. The polypeptide-induced tubulin polymers were morphologically normal microtubules and were disassembled by cold treatment. The common sequence (termed assembly-promoting sequence) was thus identified as the active site of 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly. The reconstitution system of microtubules with this synthetic polypeptide with assembly-promoting sequence may be useful to elucidate detailed molecular mechanism of the promotion of microtubule assembly by microtubule-associated proteins.     

Purification and characterization of a 190-kD microtubule-associated protein from bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with molecular weight of 190,000, termed 190-kD MAP, was purified from bovine adrenal cortex. This MAP showed the same level of ability to promote tubulin polymerization as did MAP2 and tau from mammalian brains. Relatively high amounts of 190-kD MAP could bind to microtubules reconstituted in the presence of taxol. At maximum 1 mol of 190-kD MAP could bind to 2.3 mol of tubulin. 190-kD MAP was phosphorylated by a cAMP-dependent protein kinase prepared from sea urchin spermatozoa and by protein kinase(s) present in the microtubule protein fraction prepared from mammalian brains. The maximal numbers of incorporated phosphate were approximately 0.2 and approximately 0.4 mol per mole of 190-kD MAP, respectively. These values were lower than that of MAP2, which could be heavily phosphorylated by the endogenous protein kinase(s) up to 5 mol per mole of MAP2 under the same assay condition. 190-kD MAP had no effects on the low-shear viscosity of actin and did not induce an increase in turbidity of the actin solution. It was also revealed that 190-kD MAP does not cosediment with actin filaments. These data clearly show that, distinct from MAP2 and tau, this MAP does not interact with actin. Electron microscopic observation of the rotary-shadowed images of 190-kD MAP showed the molecular shape to be a long, thin, flexible rod with a contour length of approximately 100 nm. Quick-freeze, deep-etch replicas of the microtubules reconstituted from 190-kD MAP and brain tubulin revealed many cross-bridges connecting microtubules with each other.     

Purification and characterization of a new, ubiquitously distributed class of microtubule-associated protein with molecular mass 250 kDa.

A heat-stable microtubule-associated protein (MAP) with relative molecular mass 250 000, termed 250-kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250-kDa MAP was able to bind to taxol-stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western-blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250-kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250-kDa MAP colocalized with free tubulin. Under electron microscopy, 250-kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.     

Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.     

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.     

A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences.

cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.     

Green algal microtubule-associated protein with a molecular weight of 90 kDa which bundles microtubules

Summary Cytoplasmic streaming in the freshwater, coencytic green alga,Dichotomosiphon tuberosus, is regulated by light. Conspicuous changes are observed in the number of microtubules cross-linked together in bundles when the cytoplasmic streaming is modulated by light. In an attempt to identify the cross-linker, we stainedD. tuberosus cells with antibodies specific for several different microtubules-associated proteins (MAPs) from vertebrates. Antibodies raised against bovine adrenal 190 kDa MAP stained the algal cells, and the pattern of staining was quite similar to that obtained with tubulin-specific antibodies. Examination by immunoelectron microscopy revealed that the antibodies specific for the 190 kDa microtubule-associated protein (MAP) were located along the microtubules. Western blotting demonstrated that the antibodies crossreacted with a peptide fromD. tuberosus with a molecular weight of about 90 kDa. This peptide was heat-stable, a property shared by the bovine 190 kDa MAP. Moreover, this 90 kDa peptide, crossreacted with antibodies raised against a synthetic peptide, identical to the tubulin-binding domain found in the 190 kDa MAP and in a tau protein. Partially purified 90 kDa protein fromD. tuberosus has the ability to bundle microtubules when mixed with a tubulin fraction fromD. tuberosus, in the presence of taxol. These results suggest that the 90 kDa protein fromD. tuberosus is a MAP that bundles microtubules.Abbreviations APMSF (p-amidinophenyl) methanesulfonyl fluoride - BSA bovine serum albumin - CBB Coomassie Brilliant Blue R - DEAE diethylaminoethyl - DMSO dimethyl sulfoxide - DOC deoxycholic acid - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MAP microtubule-associated protein - MES 2[N-morpholino] ethanesulfonic acid - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - TLCK N-p-tosyl-lysine chloromethyl ketone     

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.     

Poliovirus infection results in structural alteration of a microtubule-associated protein.

Poliovirus infection results in profound changes in cellular metabolism and architecture. To identify alterations in cellular proteins following poliovirus infection which might account for these changes, monoclonal antibodies were prepared by screening for differences in antigen pattern in infected and uninfected cell lysates. Further characterization of the antigen of one such antibody (25 C C1) is described in this report. The 25 C C1 antigen is a cytoskeleton-associated protein which decreases in size 4 to 5 h postinfection. It copurifies with some of the protein synthesis initiation factors but not with eucaryotic initiation factor (eIF)-4F, the p220 subunit of which is cleaved following infection (D. Etchison, S. C. Milburn, I. Edery, N. Sonenberg, and J. W. B. Hershey, J. Biol. Chem. 257:14806-14810, 1982). Unlike alteration of p220, alteration of the 25 C C1 antigen is not due to a protease which can be detected by cell lysate mixing experiments. Alteration of the antigen occurs during purification, suggesting progressive proteolysis, but the alteration is more extensive in preparations from infected cells than in those from uninfected cells. A recombinant phage expressing the antigenic determinant was isolated from a human fibroblast cDNA library, and the sequence of the cDNA insert was found to be entirely contained within the established sequence of microtubule-associated protein (MAP) 4 (R. R. West, K. M. Tenbarge, and J. B. Olmsted, J. Biol. Chem. 266:21886-21896, 1991). The antigen distribution, as detected by indirect immunofluorescence, was similar to, but more diffuse than, the distribution of tubulin. The antibody recognized the largest abundant HeLa cell MAP, which copurified with tubulin after three cycles of polymerization-depolymerization, thus confirming the identity of the antigen as MAP 4. These results indicate that poliovirus infection of HeLa cells affects the structural integrity of a cytoskeletal protein, MAP 4.     

Genetic analysis of a Drosophila microtubule-associated protein.

The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of this group of nonmotor MAPs. We have used a classical genetic approach to try to identify the role of the 205K MAP in Drosophila by isolating mutations in the 205K MAP gene. An F2 lethal screen was used to acquire deficiencies of 100EF, the chromosomal location of the 205K MAP gene. Drosophila bearing a homozygous deficiency for the 205K MAP region are fully viable and show no obvious phenotype. A recently developed polymerase chain reaction screen was also used to recover five P-element insertions upstream from the 205K MAP gene. Western blot analysis has shown that these insertions result in hypomorphic mutations of the 205K MAP gene. As was seen with animals that have no 205K MAP, these mutations appear to have no phenotype. These data unambiguously demonstrate that the 205K MAP gene is inessential for development. These results also suggest that there may exist protein(s) with redundant function that can substitute for 205K MAP.     

The 72-kDa Microtubule-Associated Protein from Porcine Brain

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.     

The novel microtubule-associated protein MAP3 contributes to the in vitro assembly of brain microtubules

MAP3 is a novel microtubule-associated protein found in brain and a variety of other tissues (Huber, G., Alaimo-Beuret, D., and Matus, A. (1985) J. Cell Biol. 100, 496-507). In this study, monoclonal antibodies were used to assess its influence on the polymerization of brain tubulin. When added to unpolymerized brain microtubules, anti-MAP3 IgG produced a dose-related inhibition of subsequent assembly. Under the same circumstances, nonimmune mouse IgG did not influence either the rate or the extent of tubulin polymerization. We also used immobilized antibodies to deplete brain MAPs selectively in either MAP3 or MAP1. MAP3-depleted MAPs showed a reproducible decrease in activity compared to control preparations that had been exposed to immobilized nonimmune IgG. MAP1-depleted MAPs did not differ significantly in performance from the nonimmune treated controls. We conclude that MAP3 contributes to the net assembly of brain microtubules observed in vitro. This may be particularly relevant in neonatal animals where brain MAP3 is more abundant than in the adult.     

A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs)

The effect of the antimitotic drug taxol on the association of MAPs (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of Taxol at 37 degrees C. at 0 degrees C, and at 37 degrees C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter two conditions. At 37 degrees C and at 0 degrees C, complete assembly of both tubulin and the MAPs was observed in the presence of Taxol. However, at elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAPs. The MAPs could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAPs of high purity from small amounts of biological material. The MAPs could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAPs were prepared from bovine cerebral cortex (gray matter) and from HeLa cells. MAP 1, MAP2, and the tau MAPs, as well as species of Mr = 28,000 and 30,000 (LMW, or low molecular weight, MAPs) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 mol wt HeLa MAPs were isolated from HeLa cells. Microtubules were also prepared for the first time from white matter. All of the MAPs identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the two preparations was a fivefold lower level of MAP 2 relative to tubulin in white matter than in gray. The high molecular weigh MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. These results indicate that MAP 1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.     

CLAMP, a novel microtubule-associated protein with EB-type calponin homology

Microtubules (MTs) are polymers of alpha and beta tubulin dimers that mediate many cellular functions, including the establishment and maintenance of cell shape. The dynamic properties of MTs may be influenced by tubulin isotype, posttranslational modifications of tubulin, and interaction with microtubule-associated proteins (MAPs). End-binding (EB) family proteins affect MT dynamics by stabilizing MTs, and are the only MAPs reported that bind MTs via a calponin-homology (CH) domain (J Biol Chem 278 (2003) 49721-49731; J Cell Biol 149 (2000) 761-766). Here, we describe a novel 27 kDa protein identified from an inner ear organ of Corti library. Structural homology modeling demonstrates a CH domain in this protein similar to EB proteins. Northern and Western blottings confirmed expression of this gene in other tissues, including brain, lung, and testis. In the organ of Corti, this protein localized throughout distinctively large and well-ordered MT bundles that support the elongated body of mechanically stiff pillar cells of the auditory sensory epithelium. When ectopically expressed in Cos-7 cells, this protein localized along cytoplasmic MTs, promoted MT bundling, and efficiently stabilized MTs against depolymerization in response to high concentration of nocodazole and cold temperature. We propose that this protein, designated CLAMP, is a novel MAP and represents a new member of the CH domain protein family.     

Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross- reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross- reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.     

A microtubule-associated protein in maize is expressed during phytochrome-induced cell elongation

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P₁₀₀) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP τ: the P₁₀₀ protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P₁₀₀ protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P₁₀₀, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.