High Molecular Weight Microtubule-associated Proteins Research Articles

Immunocytochemical localization of actin and microtubule-associated protein MAP2 in dendritic spines.

To determine whether dendritic spines contain actin, we evaluated the immunocytochemical localization of actin in the hippocampal formation and cerebral cortex of the rat. Monoclonal hybridoma antibodies were prepared against adult quail breast muscle actin. The culture supernatant of two cell lines (QAB1 and QAB2) was examined. Both antibodies bound only actin in crude brain homogenates, and neither exhibited species specificity. Electron microscopic analyses of sections reacted with QAB1 revealed staining of postsynaptic densities and dendritic microtubules but little staining of the cytoplasmic compartment of spines. However, sections reacted with QAB2 exhibited staining at the cytoplasmic compartment of spines as well as the sites stained by QAB1. We also evaluated the immunocytochemical distribution of beta-tubulin and high molecular weight microtubule-associated protein (MAP2) utilizing monoclonal antibodies. MAP2 was found in the dendritic spine as well as in the parent dendrite. However, beta-tubulin was found only in the postsynaptic density and in the microtubules of the parent dendrite. The combined results indicate that actin is present in the spine along with MAP2 and that there is a difference in the actin (or the state of actin) in the spine in comparison with other neuronal compartments.

Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic-neuron-like phenotype. Comparison of the phosphoprotein patterns of the cells by SDS PAGE after various times of NGF treatment revealed a high molecular weight (Mr greater than or approximately 300,000) band whose relative intensity progressively increased beyond 2 d of NGF exposure. This effect was blocked by inhibitors of RNA synthesis and did not require neurite outgrowth or substrate attachment. The enhancement by NGF occurred in serum-free medium and was not produced by exposure to epidermal growth factor, insulin, dibutyryl cAMP, or dexamethasone. Several different types of experiments indicated that this phosphoprotein corresponds to a high molecular weight (HMW) microtubule-associated protein (MAP). These included cross-reactivity with antiserum against brain HMW MAPs, co-cycling with microtubules and co-assembly with tubulin in the presence of taxol. The affected species also co-migrated in SDS PAGE gels with brain MAP1 and, unlike MAP2, precipitated upon boiling. Studies with [35S]-methionine-labeled PC12 cells indicated that at least a significant proportion of this effect of NGF was due to increased levels of protein rather than to mere enhancement of phosphorylation. On the basis of the apparent effects of MAPs on the formation and stabilization of microtubules and of the importance of microtubules in production and maintenance of neurites, it is proposed that induction of a HMW MAP may be one of the steps in the mechanism whereby NGF promotes neurite outgrowth. Furthermore, these findings may lead to an understanding of the role of MAP1 in the nervous system.

Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging.

Mammalian neurofilaments prepared from brain and spinal cord by either of two methods partially inhibit the in vitro assembly of microtubules. This inhibition is shown to be due to the association of a complex of high molecular weight microtubule-associated proteins (MAP1 and MAP2) and tubulin with the neurofilament. Further analysis of the association reveals a saturable binding of purified brain MAPs to purified neurofilaments with a Kd of 10(-7) M. Purified astroglial filaments neither inhibit microtubule assembly nor show significant binding of MAPs. It is proposed that the MAPs might function as one element in a network of intraorganellar links in the cytoplasm.

The interaction of actin filaments with microtubules and microtubule-associated proteins.

Purified actin and microtubule proteins polymerized together form a gel, while mixtures of actin with tubulin polymers lacking microtubule-associated proteins (MAPs) have low viscosities close to the sum of the viscosities of the constituents. Mixtures of actin and MAPs also have high viscosities. Our interpretation of these observations was that there is interaction of actin filaments and microtubules which is mediated by MAPs (Griffith, L. M., and Pollard, T. D. (1978) J. Cell Biol. 78, 958-965). We report here further evidence for this interaction. 1) Actin filaments and microtubules can form gels at physiological ionic strength providing the anion is glutamate rather than chloride. Both glutamate and chloride inhibit actin-MAPs interaction, but this is compensated for in glutamate where the microtubules are longer than in chloride. 2) The low shear viscosity of mixtures of isolated MAPs and actin filaments is enhanced by acidic pH and inhibited by high ionic strength. 3) MAPs can be fractionated to yield four different fractions with actin cross-linking activity: a subset of high molecular weight MAPs, purified "MAP-2" and two different fractions of tau polypeptides. 4) We have reconstituted a gel from actin, purified tubulin, and whole MAPs, but have not yet been successful with actin, purified tubulin, and any single purified MAP.

The cytoskeletal system of nucleated erythrocytes. I. Composition and function of major elements.

We have studied the dogfish erythrocyte cytoskeletal system, which consists of a marginal band of microtubules (MB) and trans-marginal band material (TBM). The TBM appeared in whole mounts as a rough irregular network and in thin sections as a surface-delimiting layer completely enclosing nucleus and MB. In cells incubated at 0 degrees C for 30 min or more, the MB disappeared but the TBM remained. MB reassembly occurred with rewarming, and was inhibited by colchicine. Flattened elliptical erythrocyte morphology was retained even when MBs were absent. Total solubilization of MB and TBM at low pH, or dissolution of whole anucleate cytoskeletons, yielded components comigrating with actin, spectrin, and tubulin standards during gel electrophoresis. Mass-isolated MBs, exhibiting ribbonlike construction apparently maintained by cross-bridges, contained four polypeptides in the tubulin region of the gel. Only these four bands were noticeably increased in the soluble phase obtained from cells with 0 degrees C-disassembled MBs. The best isolated MB preparations contained tubulin but no components comigrating with high molecular weight microtubule-associated proteins, spectrin, or actin. Actin and spectrin therefore appear to be major TBM constituents, with tubulin localized in the MB. The results are interpreted in terms of an actin- and spectrin-containing subsurface cytoskeletal layer (TBM), related to that of mammalian erythrocytes, which maintains cell shape in the absence of MBs. Observations on abnormal pointed erythrocytes containing similarly pointed MBs indicate further that the MB can deform the TBM from within so as to alter cell shape. MBs may function in this manner during normal cellular morphogenesis and during blood flow in vivo.

Possible role of microtubules and associated proteases in organophosphorus ester-induced delayed neurotoxicity

Organophosphorus delayed neurotoxicants (phenyl saligenin cyclic phosphate and diisopropyl phosphorofluoridate) altered cyclic AMP (cAMP)-dependent phosphorylation and several other processes in brain homogenates and cytoplasmic microtubules. Phenyl saligenin cyclic phosphate slightly stimulated in vitro cAMP-dependent phosphorylation in brain homogenates of three species (rat, mouse and rabbit) that have been reported to be insensitive to delayed neurotoxicity, whereas it slightly decreased this phosphorylation in brain homogenates of three sensitive species (chicken, cow and pig) and in brain microtubules of chicken and pig. The microtubule-associated processes that were moderately inhibited by phenyl saligenin cyclic phosphate in sensitive species were: in vitro [ 3H]cAMP binding to protein kinase, in vitro assembly when tubulin rings were absent, and cAMP-dependent phosphorylation of microtubule-associated proteins (MAPs) both in vitro and on intracerebral administration of 32P i. The endogenous proteases that degrade the high molecular weight MAPs were strongly inhibited in vitro by phenyl saligenin cyclic phosphate and diisopropyl phosphorofluoridate. In contrast, treatment of chickens with diisopropyl phosphorofluoridate remarkably decreased the in vitro stability of their brain cytoplasmic high molecular weight MAPs, perhaps by enhancing the MAPs-degrading protease activity. These findings indicate that the MAPs-protease system is a possible target for organophosphorus delayed neurotoxicants.

Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins.

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.

Initial phase of dendrite growth: evidence for the involvement of high molecular weight microtubule-associated proteins (HMWP) before the appearance of tubulin.

It has recently been shown that high molecular weight microtubule-associated proteins (HMWP) in the brain are present in dendrites and are absent from axons (Matus et al., 1981, Proc. Natl. Acad. Sci. U. S. A. 78:3010-3014). In this study we followed the appearance of both HMWP and tubulin in the neonatal rat cerebellum by immunoperoxidase staining, concentrating particularly on comparing Purkinje cell dendrites with adjacent granule cell axons. In the axons both immunohistochemically demonstrable tubulin and structurally distinct microtubules are present at all stages of development. By contrast the Purkinje cell dendrites contain better neither tubulin nor microtubules at early stages of their growth. However, immunoperoxidase staining showed that these developing dendrites are rich in HMWP which are particularly concentrated in the dendritic distal regions. HMWP are also present as patches beneath the surface membrane of the cell body before the emergence of dendrites. Based on this data and the well-documented ability of HMWP to promote microtubule assembly, we propose the hypothesis that during the initial phase of Purkinje neuron differentiation HMWP form part of a specialized cytoskeletal structure which acts as a specifier for the development of dendrites as opposed to axons.

39] Purification and assay of microtubule-associated proteins (MAPs)

Publisher Summary This chapter presents procedure for purification and assay of microtubule-associated proteins (MAPS). The procedures begin with microtubules assembled in vitro from either brain or tissue culture cells. The solution of microtubule proteins is usually carried through at least two to three complete assembly-disassembly cycles prior to beginning one of the following MAP isolation procedures. The strategies employed to isolate MAPs from the total microtubule protein preparation are based on several criteria: (1) net charge distribution affecting their behavior on ion exchange resins, (2) overall molecular size and axial ratio, which affect their elution from molecular sieve columns, and (3) varying stability to the effects of elevated temperatures (100°). One or more combinations of these properties can be used as the basis for the isolation procedures. Both the high molecular weight MAPs and Tau from brain and the MAPs from tissue culture cells have been assayed by their ability to promote the assembly of tubulin in vitro. Generally, these assays are carried out by determining whether the putative MAP will enhance the initial rate and total amount of assembly of a given concentration of PC or DEAE-purified brain tubulin.

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain.

Human erythrocyte ankyrin, the membrane attachment protein for spectrin, has been detected by radioimmunoassay in a variety of cells and tissues. This report identifies polypeptides crossreacting with ankyrin in brain and HeLa cells and demonstrates that one function of these ankyrin analogues involves association with microtubules. Ankyrin immunoreactivity was localized by indirect immunofluorescence in a colchicine- and detergent-sensitive cytoplasmic meshwork in interphase cells. There also was specific nuclear staining, localized in a bright spots, which was displaced entirely by ankyrin or by high molecular weight microtubule-associated proteins (MAPs) from brain. In dividing cells, the punctate nuclear staining and the meshwork disappeared. Fluorescence was localized at the spindle pole during metaphase and was redistributed to the cleavage furrow in later stages of mitosis. An immunoreactive Mr 370,000 polypeptide comigrating with MAP1 was identified in brain extracts and copolymerized with microtubules through repeated cycles of polymerization and depolymerization. Finally, erythrocyte ankyrin associated with microtubules prepared from pure tubulin, and this binding was displaced by brain MAPs.

Effect of tau on the vinblastine-induced aggregation of tubulin.

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.

High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain.

The distributions of tubulin and high molecular weight microtubule-associated proteins (HMWPs) in brain were determined by immunoperoxidase histochemistry with specific antisera. Tubulin was found in microtubules of both neurons and glia and both axons and dendrites. HMWPs were found only in neurons where, in all cases examined, they were associated with dendritic microtubules but not those in axons. Both tubulin and HMWPs were also found in postsynaptic densities. These results indicate that brain microtubules vary in chemical composition. The preferential association of HMWPs with dendritic microtubules suggests that they may play a role in distinguishing between dendritic and axonal export routes from the cell body.

Purification of brain tubulin by affinity chromatography on immobilized lactoperoxidase.

Brain tubulin binds to lactoperoxidase coupled to Affigel 10 through a 10 A succinylated aminoalkyl spacer and can be eluted by an ionic strength gradient. The tubulin can be obtained about 90% electrophoretically pure in 2 to 3 h without glycerol or GTP. It retains its ability to bind colchicine. Compared to tubulin purified by the assembly-disassembly procedure, affinity-purified tubulin has a higher critical concentration for polymerization and the purified protein appears to be free of high molecular weight microtubule-associated proteins. Tubulin binding to the lactoperoxidase affinity column protects the colchicine binding site against decay at 4 degrees C, whereas the interaction of tubulin with soluble lactoperoxidase does not.

Microtubule-associated proteins from cultured HeLa cells. Analysis of molecular properties and effects on microtubule polymerization.

We have attempted a biochemical characterization of the microtubule-associated proteins (MAPs) of cultured HeLa cells. The HeLa MAPs consist of a group of three polypeptides of 200,000 to 220,000 molecular weight (the 210K MAP) and a protein of 125,000 molecular weight (the 125K MAP). The solution properties of the HeLa MAPs were examined using molecular sieve chromatography and sucrose gradient sedimentation. With both analytical procedures, the 125K and 210K MAPs behaved independently of one another. The effects of each of the MAPs on microtubule polymerization were also studied. Both the 125K and 210K MAPs stimulated the polymerization of pure tubulin. The effect of high levels of MAPs on microtubule polymerization was also examined. Increasing the concentration of MAPs at a constant tubulin concentration increased both the rate and extent of microtubule polymerization. The 125K and 210K MAPs showed independent behavior with respect to binding to microtubules. The 210K MAP saturated its binding sites at a level of 14.0% (210K MAP:tubulin in polymer, w/w), while the 125K MAP showed no saturation even at a level of 18.3%. These results demonstrate that the 125K and 210K MAPs are distinct molecular species, differing in their solution properties and their binding to microtubules. The 210K MAP showed some similarities and some differences when compared to the porcine brain high molecular weight MAP. The HeLa MAPs, although showing some properties similar to brain MAPs, are nevertheless distinctive in several respects and may best be considered as separate though possibly related species.

High molecular weight polypeptides (270,000-340,000) from cultured cells are related to hog brain microtubule-associated proteins but copurify with intermediate filaments.

High molecular weight polypeptides (HMWPs) of 270,000 to 340,000 were found to be major components of intermediate filaments prepared by Triton X-100 extraction after spreading of rat glioma C6, HeLa, Chinese hamster ovary, and simian virus 40-transformed Chinese hamster lung cells. C6 HMWPs were shown to resemble high molecular weight microtubule-associated proteins from hog brain by four criteria: (i) comigration in electrophoresis on high-resolution sodium dodecyl sulfate/polyacrylamide gels, (ii) one-dimensional peptide mapping, (iii) phosphorylation in vitro with [gamma-32P]ATP, and (iv) ability to promote microtubule assembly in vitro. HMWPs were also found to be major components of one-time polymerized C6 microtubule preparations, which contained a sizable amount of intermediate filaments. The predominant part of HMWPs present in these microtubule preparations was found not to copurify with microtubules in cycles of temperature-dependent assembly/disassembly but to remain with the cold-insoluble intermediate filaments. These results provide an explanation for the low yields that have hampered attempts to purify microtubule-associated porteins, in particular HMWPs, from cultured cells in the past. Moreover, they suggest that HMWPs might have a dual role in the cell, serving not only as regulators of microtubule assembly but also as linker components between microtubules and intermediate filaments.

Hybrid microtubules from mixtures of human fibroblast homogenates and chick brain microtubules: Absence of high molecular weight associated proteins

Microtubules have been assembled from a mixture of chick-brain microtubule protein and total soluble protein of cultured human fibroblasts. In this system microtubules were assembled which did not have any high molecular weight (HMW) microtubule-associated protein (MAP). Since the fibroblasts were human in origin, the hybrid microtubules were compared to microtubules assembled from human brain, which do include HMW-MAPs. To determine whether HMW-MAP is unique to brain, microtubules were assembled from mixtures of soluble proteins from non-neural mouse organs and chick brain microtubules. These hybrid microtubules contain similar HMW-MAPs to those of the chick brain alone. The absence of HMW-MAPs from the hybrid microtubules does not appear to be due to proteolysis. SDS-gel electrophoresis of all fractions prepared during the process of assembly of the hybrid microtubules reveals that the HMW-MAPs of the mixture are sedimented away from disassembled microtubules during the first centrifugation. The exclusion of the HMW-MAPs from the hybrid microtubules suggests that the assembly process in these mixtures, and in fibroblasts, may be qualitatively different from that found in extracts from brain and other organs.

Regulation of the microtubule steady state in vitro by ATP

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

Polymerization of tubulin in the presence of colchicine or podophyllotoxin: Formation of a ribbon structure induced by guanylyl-5′-methylene diphosphonate

GTP-dependent in vitro polymerization of rat brain microtubular protein is inhibited to 50% by substoichiometric concentrations of the antimitotic drugs colchicine (0.12 mol/mol of tubulin) and podophyllotoxin (0.14 mol/mol of tubulin). Substitution of pp(CH 2)pG † † Abbreviations used: pp(CH 2)pG, guanylyl-5′-methylene diphosphonate; MES, [2-( N-morpholino)]-ethane sulfonic acid; SDS, sodium dodecyl sulfate; MAP, high molecular weight microtubule-associated proteins in the nomenclature of Dentler et al. (1975); tau, microtubule-associated polypeptides in the nomenclature of Weingarten et al. (1975). for GTP, however, results in an extensive microtubular protein polymerization at such concentrations. In the presence of pp(CH 2)pG, suprastoichiometric concentrations of podophyllotoxin (19 mol/mol of tubulin) are required to inhibit the polymerization process by 50%. Colchicine is very ineffective since 3 × 10 5 moles/mole of tubulin are required to give a 50% inhibition. Electron microscopical analysis shows that the polymers formed by microtubular protein in the presence of suprastoichiometric concentrations of drugs are not the normal short microtubules typical of pp(CH 2)pG-driven polymerization, but are ribbons with three or four protofilaments. The colchicine content of the harvested ribbons has been measured directly and found to be approximately 0.8 moles colchicine/mole of tubulin. Treatment of microtubular protein with substoichiometric concentrations of drugs results in an increase in the number of protofilaments forming the ribbons. Many of the ribbons can close into morphologically normal microtubules when microtubular protein is treated with only 0.05 moles of either colchicine or podophyllotoxin per mole of tubulin.

Isolation of bovine brain microtubule protein without glycerol: Polymerization kinetics change during purification cycles

Microtubule protein was isolated from bovine brain without glycerol by three cycles of polymerization, utilizing conditions for maximum assembly. Maximum polymerization occurred over a narrow GTP concentration range in crude extracts, with peak polymerization at 2.5 m m GTP. Peak polymerization occurred at 1.5 m m GTP and 0.5 m m MgCl 2 after three polymerization cycles. In a typical purification, 72 mg of microtubule protein was obtained per 100 g (wet weight) of cerebral cortex. Of this, a minimum of 52% was tubulin and 24% was high molecular weight-microtubule associated proteins (HMW-MAPs). Microtubule polymerization kinetics changed during purification cycles. In initial cycles, the total quantity of microtubules decreased significantly after peak levels of assembly were attained. In the third cycle, the decrease was minimal and only apparent at high protein concentrations. Peak viscosity correlated with tubulin and HMW-MAPs, but the magnitude of the decrease was independent, suggesting that some other component, eliminated during purification, was responsible for the decrease.