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.     

Identification and spatial arrangement of high molecular weight proteins (Mr 300 000-330 000) co-assembling with microtubules from a cultured cell line (rat glioma C6)

The distribution of three high molecular weight proteins, MAP-1 (Mr 330 000), MAP-2 (Mr 300 000) and plectin (Mr 300 000) in various fractions obtained in cycles of temperature-dependent polymerization/depolymerization of microtubules from rat glioma C6 cells was studied. Using gel electrophoresis and immunoautoradiography/immunoblotting all three proteins were found to codistribute only partially with tubulin because considerable parts remained in the cold-insoluble fractions. Moreover, the proteins, particularly MAPs, were proteolytically degraded during cycling. By contrast, when microtubules were polymerized with taxol after isotonic cell lysis a considerable enrichment of MAP-1 and MAP-2 was achieved; again, plectin co-distributed only partially. In this procedure too, MAPs, especially MAP-2, were found to be highly subject to proteolysis, unless free Ca2+-ions were rigorously avoided. Proteolytic fragments generated from MAP-2 were of similar size independent of whether temperature- or taxol-dependent polymerization procedures were used, suggesting the occurrence of a MAP-2-specific protease. When the spatial arrangement of the high Mr proteins on taxol-polymerized C6 cell microtubules was directly visualized using gold-immunoelectron microscopy, a periodical, apparently helical, decoration of microtubules was found for MAP-1 and MAP-2; plectin was irregularly arrayed. A predominantly helical arrangement of both MAPs was demonstrated also for microtubules reconstituted from mammalian brain.     

Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule- associated protein (cytoplasmic dynein)

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.     

Characterization of maize microtubule-associated proteins, one of which is immunologically related to tau

Microtubule-associated proteins (MAPs) are identified as proteins that copurify with tubulin, promote tubulin assembly, and bind to microtubules in vitro. Higher plant MAPs remain mostly unknown. One example of non-tubulin carrot proteins, which bind to neural microtubules and induce bundling, has been reported so far [Cyr, R. J., & Palewitz, B. A. (1989) Planta 177, 245-260]. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in cytosolic extracts of maize cultured cells and were used as the native matrix to isolate putative plant MAPs. Several polypeptides with an apparent molecular masses between 170 and 32 kDa copolymerized with maize microtubules. These putative maize MAPs also coassembled with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of nonefficient self-assembly and induced bundling of both plant and neural microtubules. One of these proteins, of about 83 kDa, cross-reacted with affinity-purified antibodies against rat brain tau proteins, suggesting the presence of common epitope(s) between neural tau and maize proteins. This homology might concern the tubulin-binding domain, as plant and neural tubulins are highly conserved and the plant polypeptides coassembled with brain tubulin.     

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.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Widespread occurrence of polypeptides related to neurotubule-associated proteins (MAP-1 and MAP-2) in non-neuronal cells and tissues.

The occurrence and cellular localization of polypeptides related to hog brain microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2) in non-neuronal cell lines of various species and types, and in several tissues from rat was studied. When insoluble cell fractions were prepared by incubation of isotonic cell extracts with 20 microM taxol, polypeptides co-migrating with MAP-1 and MAP-2 upon gel electrophoresis were observed in virtually all cases examined. Immunoblotting of preparations from 3T6, CHO, HeLa and N2A cells, as well as pituitary, heart, testis and liver revealed immuno-reactivity with antibodies to neuronal MAP-1 for polypeptides co-migrating with MAP-1 in all cases, except for HeLa cells and liver. With similar preparations, antibodies raised to neuronal MAP-2 were barely reactive with bands of the MAP-2 size except for N2A cells and pituitary gland. In all cases of non-neuronal cells and tissues, major cross-reactive bands, however, were of mol. wt. lower than that of MAP-2, indicating, most likely, proteolytic breakdown of MAP-2 during cell fractionation. As shown by double immunofluorescence microscopy of various cultured cell lines using affinity-purified antibodies to MAPs, and monoclonal antibodies to tubulin, MAP-1-as well as MAP-2-related antigens were generally, but not exclusively, associated with typical microtubule structures of the cytoplasm, spindle, midbody and primary cilia. Antigens related to both MAPs were also localized in frozen sections of rat trachea, testis, pituitary, kidney and cardiac and skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of Microtubule Proteins in Rat Brain at Different Developmental Stages: Comparison with That Found in Neuronal Cultures

The phosphorylation of rat brain microtubule protein on intracranial injection of labeled phosphate has been analyzed. The major microtubule protein components phosphorylated in vivo in rat brain are the high-molecular-weight microtubule-associated proteins (MAPs) MAP-1A, MAP-1B, and MAP-2. A slight phospholabeling of beta-tubulin, which corresponds to the phosphorylation of a minor neuronal beta-tubulin isotype, is also observed. Whereas MAP-1B, MAP-2, and beta-tubulin are phosphorylated in the brain of 5-day-old rat pups, when most neurons of the CNS are extending processes, MAP-1A phosphorylation is observed only after neuronal maturation takes place. The phosphorylation of MAP-1A, MAP-1B, and beta-tubulin may be due mainly to casein kinase II or a related enzyme, whereas MAP-2 appears to be modified by other enzymes such as the cyclic AMP-dependent protein kinase (protein kinase A) and the calcium/phospholipid-dependent protein kinase (protein kinase C). Microtubule protein phosphorylation has also been studied in neuronal cultures. In differentiated neuroblastoma cells, only MAP-1B and beta-tubulin are phosphorylated in a manner coupled to neurite outgrowth. In primary cultures of fetal rat brain neurons, the pattern of microtubule protein phosphorylation resembles that found in vivo in rat pup brain. As phosphorylated MAP-1A and MAP-1B are present mainly on assembled microtubules, whereas the phosphorylation of MAP-2 decreases its interaction with microtubules, a role can be suggested for the phosphorylation of these proteins in the regulation of microtubule assembly and disassembly during neuronal development.     

Peptides corresponding to the second repeated sequence in MAP-2 inhibit binding of microtubule-associated proteins to microtubules

Bovine brain high molecular weight microtubule-associated proteins (MAPs) can be displaced from assembled tubules by peptides corresponding to the second of three nonidentical repeated sequences in mouse MAP-2. The octadecapeptide m2 (VTSKCGSLKNIRHRPGGG) can release MAP-1b from MAP-containing microtubules, and the extended second-sequence peptide m2' (VTSKCGSLKNIRHRPGGGRVK) displaces MAP-1a and MAP-1b as well as MAP-2a and MAP-2b. Peptides m2 and m2' stimulate tubulin polymerization in the absence of MAPs or microtubule-stabilizing agents, and m2' acts as a competitive inhibitor of radiolabeled MAP-2 binding. The dissociation constant for MAP-2 binding to taxol-stabilized tubules was 3.4 microM in the absence of m2' and 14 microM in the presence of 1.5 mM of the m2' peptide. We estimate that the inhibition constant for peptide m2' is about 0.5 mM, about 100 times lower than for the Km of MAP-2. These observations suggest that the second repeated sequence in MAP-2 may represent an important recognition site for MAP binding to microtubules and that other structural features within MAP-2 may reinforce the strength of MAP-microtubule interactions.     

Differential distribution of microtubule-associated proteins MAP-1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (clone N2A).

To study the individual location of the microtubule proteins MAP-1 and MAP-2 in neuronal tissues and cells, antisera to electrophoretically purified MAP-1 and MAP-2 components were raised in rabbits. When frozen sections through rat brain were examined by immunofluorescence microscopy the antibodies to MAP-1 strongly stained a variety of nerve cells including dendrites and myelinated axons in the cerebrum and cerebellum. Antibodies to MAP-2 showed similar staining patterns, except that myelinated axons were unstained. These results were confirmed by immunoelectron microscopy of frozen sections through cerebellum using the peroxidase technique. Thereby, the association of MAP-1 with microtubules was also clearly demonstrated. When cultured mouse neuroblastoma N2A cells were examined by immunofluorescence microscopy the antiserum to MAP-1 brightly stained filamentous structures resembling microtubules, whereas relatively weak and diffuse staining of the cytoplasm was observed with the antiserum to MAP-2. In agreement with the immunolocalization, MAP-1, but not MAP-2, was found as a prominent component of microtubules proteins polymerized in vitro by taxol from soluble N2A cell extracts. Together these results indicate that neuronal microtubules are preferentially associated with distinct high mol. wt. polypeptides. Therefore, they support the concept that different complements of associated proteins determine distinct functions of microtubules.     

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.     

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.     

In vitro polymerization of microtubules from HeLa cells

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.     

Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP- actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene- adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.     

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.     

Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells

A novel microtubule-associated protein (MAP) of M(r) 115,000 has been identified by screening of a HeLa cell cDNA expression library with an anti-serum raised against microtubule-binding proteins from HeLa cells. Monoclonal and affinity-purified polyclonal antibodies were generated for the further characterization of this MAP. It is different from the microtubule-binding proteins of similar molecular weights, characterized so far, by its nucleotide-insensitive binding to microtubules and different sedimentation behavior. Since it is predominantly expressed in cells of epithelial origin (Caco-2, HeLa, MDCK), and rare (human skin, A72) or not detectable (Vero) in fibroblastic cells, we name it E-MAP-115 (epithelial MAP of 115 kD). In HeLa cells, E-MAP-115 is preferentially associated with subdomains or subsets of perinuclear microtubules. In Caco-2 cells, labeling for E- MAP-115 increases when they polarize and form blisters. The molecular characterization of E-MAP-115 reveals that it is a novel protein with no significant homologies to other known proteins. The secondary structure predicted from its sequence indicates two domains connected by a putative hinge region rich in proline and alanine (PAPA region). E- MAP-115 has two highly charged regions with predicted alpha-helical structure, one basic with a pI of 10.9 in the NH2-terminal domain and one neutral with a pI of 7.6 immediately following the PAPA region in the acidic COOH-terminal half of the molecule. A novel microtubule- binding site has been localized to the basic alpha-helical region in the NH2-terminal domain using in vitro microtubule-binding assays and expression of mutant polypeptides in vivo. Overexpression of this domain of E-MAP-115 by transfection of fibroblasts lacking significant levels of this protein with its cDNA renders microtubules stable to nocodazole. We conclude that E-MAP-115 is a microtubule-stabilizing protein that may play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells.     

Structural homology of microtubule-associated proteins 1 and 2 demonstrated by peptide mapping and immunoreactivity

The major high molecular weight microtubule-associated polypeptides from hog brain (MAP-1 and MAP-2) were compared by one- and two-dimensional peptide mapping under varied conditions and by immunological techniques. Partial digestion of MAP-1 and MAP-2 with Staphylococcus aureus V8 protease and analysis in one dimension gave rise to very similar peptide maps independent of whether 125I-, 3H-, or 32P-labeled proteins were used. One-dimensional cleavage patterns of significant similarity were also obtained by partial digestion of MAP-1 and MAP-2 using trypsin or chymotrypsin. Furthermore, a pronounced similarity, although clear nonidentity, of MAP-1 and MAP-2 was also revealed after exhaustive digestion of 125I-labeled proteins with S. aureus V8 protease or trypsin followed by analysis of peptides in two dimensions. For immunological comparison, antisera were used that had been raised in rabbits using electrophoretically purified MAP-1 and MAP-2 components as immunogens. As determined by immunoprecipitation, the antiserum raised to MAP-1 was equally reactive with MAP-1 and MAP-2 components, whereas the antiserum to MAP-2 reacted primarily with MAP-2. Indicating the presence of common as well as unique antigenic determinants on MAP-1 and MAP-2, these results, therefore, were in agreement with the peptide mapping data. Implications of these results for biosynthetic mechanisms as well as differential distribution and functions of MAPs in cells are discussed.     

Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles

Microtubule protein purified from brain tissue by cycles of in vitro assembly-disassembly contains ATPase activity that has been postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that greater than 90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-1, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.     

Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.     

Analysis of the microtubule-binding domain of MAP-2

We examined the microtubule-binding domain of the microtubule- associated protein (MAP), MAP-2, using rabbit antibodies that specifically bind to the microtubule-binding region ("stub") and the projection portion ("arm") of MAP-2. We found that (a) microtubules decorated with arm antibody look similar to those labeled with whole unfractionated MAP antibody, though microtubules are not labeled with stub antibody; (b) incubation of depolymerized microtubule protein with stub antibody prior to assembly partially inhibits the rate of microtubule elongation, presumably because MAPs that are complexed with antibody cannot bind to microtubules and stabilize elongating polymers; (c) the rate of appearance and amounts of 36- and 40-kD microtubule- binding peptides produced by digestion with chymotrypsin are distinct for MAPs associated with microtubules vs. MAPs free in solution. The enhanced stability of the 40-kD peptide when associated with microtubules suggests that this domain of the protein is closely associated with, or partially buried in, the microtubule surface; (d) MAP-2 is a slender, elongate molecule as determined by unidirectional platinum shadowing (90 +/- 30 nm), which is in approximate agreement with previous observations. Stub antibody labels MAP-2 in the terminal one-quarter of the extended protein, indicating an intrinsic asymmetry in the molecule.