Subunit Protein Of Microtubules Research Articles

Interactions of β tubulin isotypes with glutathione in differentiated neuroblastoma cells subject to oxidative stress.

Microtubules are a major component of the neuronal cytoskeleton. Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α and β exist as families of isotypes, whose members are encoded by different genes and have different amino acid sequences. The βII and βIII isotypes are very prominent in the nervous system. Our previous work has suggested that βII may play a role in neuronal differentiation, but the role of βIII in neurons is not well understood. In the work reported here, we examined the roles of the different β-tubulin isotypes in response to glutamate/glycine treatment, and found that both βII and βIII bind to glutathione in the presence of ROS, especially βIII. In contrast, βI did not bind to glutathione. Our results suggest that βII and βIII, but especially βIII, may play an important role in the response of neuronal cells to stress. In view of the high levels of βII and βIII expressed in the nervous system it is conceivable that these tubulin isotypes may use their sulfhydryl groups to scavenge ROS and protect neuronal cells against oxidative stress.

Computational predictions of volatile anesthetic interactions with the microtubule cytoskeleton: implications for side effects of general anesthesia.

The cytoskeleton is essential to cell morphology, cargo trafficking, and cell division. As the neuronal cytoskeleton is extremely complex, it is no wonder that a startling number of neurodegenerative disorders (including but not limited to Alzheimer’s disease, Parkinson’s disease and Huntington’s disease) share the common feature of a dysfunctional neuronal cytoskeleton. Recently, concern has been raised about a possible link between anesthesia, post-operative cognitive dysfunction, and the exacerbation of neurodegenerative disorders. Experimental investigations suggest that anesthetics bind to and affect cytoskeletal microtubules, and that anesthesia-related cognitive dysfunction involves microtubule instability, hyper-phosphorylation of the microtubule-associated protein tau, and tau separation from microtubules. However, exact mechanisms are yet to be identified. In this paper the interaction of anesthetics with the microtubule subunit protein tubulin is investigated using computer-modeling methods. Homology modeling, molecular dynamics simulations and surface geometry techniques were used to determine putative binding sites for volatile anesthetics on tubulin. This was followed by free energy based docking calculations for halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the tubulin body, and C-terminal regions for specific tubulin isotypes. Locations of the putative binding sites, halothane binding energies and the relation to cytoskeleton function are reported in this paper.

The distribution of β-tubulin isotypes in cultured neurons from embryonic, newborn, and adult mouse brains

Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α- and β-tubulin exist as numerous isotypes, differing in their amino acid sequences and encoded by different genes. The differences are highly conserved in evolution, suggesting that they are functionally significant. Neurons are a potentially very useful system for elucidating this significance, because they are highly differentiated cells and rich in tubulin isotypes. We have examined the distribution of β-tubulin isotypes in mouse primary cultured cortical neurons from embryonic fetus, newborn pups and adults. Neurons from both embryonic and adult mouse brains express the βI, βII, and βIII isotypes, but apparently not βIV or βV. βI, βII, and βIII are found in both cell bodies and neurites. However, the situation is different in newborn mice. Although βI and βIII are present in these neurons in both cell bodies and neurites and βIV is absent, just like in embryonic and adult mice, two striking differences were noted in the neurons from newborn mice. First, βV is apparently present evanescently in the neurons in both cell bodies and neurites. Interestingly, the βV was expressed strongly in newborn neurons after one day of culture; expression became much weaker after 3 days, and almost disappeared after 5 days. Second, the distribution of βII is different from other isotypes. After newborn mouse neurons were cultured for 3 days, βII started to disappear partly from the cell bodies; this was much more pronounced after five days in culture. Our findings suggest that βII's major function may involve the neurites and not the cell body. They also raise the possibility that βV has a unique role in the neurons of newborn mice.

NAP and D-SAL: neuroprotection against the β amyloid peptide (1–42)

IntroductionNAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln, single amino acid letter code, NAPVSIPQ), an eight amino acid neuroprotective peptide derived from activity-dependent neuroprotective protein (ADNP), exhibits some structural similarity to activity-dependent neurotropic factor-9 (ADNF-9; Ser-Alal-Leu-Leu-Arg-Ser-Ile-Pro-Ala, SALLRSIPA). Both peptides are also active in the all D-amino acid conformation, termed D-NAP and D-SAL. Original results utilizing affinity chromatography coupled to mass spectrometry identified tubulin, the subunit protein of microtubules, as the major NAP-associating protein in brain. The NAP-tubulin association was found to be diminished in the presence of ADNF-9, D-NAP, and D-SAL, suggesting a common target of neuroprotection. The β amyloid peptide interacts with microtubules, and previous studies have demonstrated protection against β amyloid (25–35) toxicity by NAP and ADNF-9. NAP also inhibits β amyloid (25–35 and 1–40) aggregation.MethodsCerebral cortical cultures derived from newborn rats were used in neuronal survival assays to test the activity of both NAP and D-SAL against the major Alzheimer's disease toxic peptide β amyloid (1–42).ResultsNAP and D-SAL protected cerebral cortical neurons against the major Alzheimer's disease toxic peptide β amyloid (1–42). Maximal protection of both peptides was observed at concentrations of 10-15 to 10-10 mol/l.ConclusionThese findings, together with those of previous in vivo studies conducted in relevant Alzheimer's disease models, pave the path to drug development. Bioavailability studies indicated that NAP penetrates cells and crosses the blood-brain barrier after nasal or systemic administration. Phase II clinical trials of NAP are currently in progress by Allon Therapeutics Inc.

NAP, A Neuroprotective Drug Candidate in Clinical Trials, Stimulates Microtubule Assembly in the Living Cell

NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP) provides neuroprotection in vitro and in vivo against a wide variety of neurotoxic substances. To further understand the mechanism by which NAP provides broad neuroprotection it was essential to find NAP's binding partners. Previous results, using affinity chromatography coupled with mass spectrometry, identified tubulin, the subunit protein of microtubules, as the major NAP binding protein in neurons and glial cells. Here, following microtubule depolymerization in the presence of nocodazole, NAP treatment enhanced rapid microtubule assembly and stimulated neurite outgrowth. Nocodazole is an established inhibitor of axoplasmic transport and cell division that exerts its effect by depolymerizing microtubules. NAP shows selectivity in interacting with brain tubulin and does not affect dividing cells. This data demonstrates that NAP functions as a neuroprotectant, at least in part, through its interaction with tubulin with a resulting increase in microtubule assembly.

Different effects of vinblastine on the polymerization of isotypically purified tubulins from bovine brain.

Vinblastine, a highly successful antitumor drug, targets the tubulin molecule. Tubulin, the subunit protein of microtubules, consists of an alpha- and a beta-subunit, both of which consist of isotypes encoded by different genes. We have purified three isotypes of bovine brain tubulin, namely, alpha(beta)II, alpha(beta)III and alpha(beta)IV. Microtubule associated protein-2 (MAP2) and Tau-induced assembly of these isotypes were compared in the presence and absence of vinblastine. MAP2-induced assembly of unfractionated tubulin and all the isotypes except alpha(beta)II tubulin was resistant to 1 microM vinblastine. Vinblastine at low concentrations (< 10 microM) progressively inhibited the assembly of all of the isotypes but the vinblastine concentration required for inhibition of MAP2-induced microtubule assembly was minimal for alpha(beta)II. The tau-induced assembly of unfractionated tubulin and alpha(beta)III were equally sensitive to 1 microM vinblastine whereas alpha(beta)II and alpha(beta)IV were much more sensitive to vinblastine. The microtubules obtained in the presence of tau from unfractionated tubulin, alpha(beta)II and alpha(beta)IV could be easily aggregated by 20 microM vinblastine whereas such as aggregation of microtubules obtained from alpha(beta)III and tau required approximatedly 40 microM vinblastine. Our results suggest that among the tubulin isotypes, alpha(beta)II is the most sensitive to vinblastine in the presence of MAPs while alpha(beta)III is the most resistant and this intrinsic resistance of alpha(beta)III dimers persists in the polymeric form of alpha(beta)III tubulin as well. These results may be relevant to the therapeutic and toxic actions of vinblastine.

Rapid Movement of Microtubules in Axons

Cytoskeletal and cytosolic proteins are transported along axons in the slow components of axonal transport at average rates of about 0.002–0.1 μm/s. This movement is essential for axonal growth and survival, yet the mechanism is poorly understood. Many studies on slow axonal transport have focused on tubulin, the subunit protein of microtubules, but attempts to observe the movement of this protein in cultured nerve cells have been largely unsuccessful. Here, we report direct observations of the movement of microtubules in cultured nerve cells using a modified fluorescence photobleaching strategy combined with difference imaging. The movements are rapid, with average rates of 1 μm/s, but they are also infrequent and highly asynchronous. These observations indicate that microtubules are propelled along axons by fast motors. We propose that the overall rate of movement is slow because the microtubules spend only a small proportion of their time moving. The rapid, infrequent, and highly asynchronous nature of the movement may explain why the axonal transport of tubulin has eluded detection in so many other studies.

Interactions of antimitotic peptides and depsipeptides with tubulin.

Tubulin is the target for an ever increasing number of structurally unusual peptides and depsipeptides isolated from a wide range of organisms. Since tubulin is the subunit protein of microtubules, the compounds are usually potently toxic to mammalian cells. Without exception, these (depsi)peptides disrupt cellular microtubules and prevent spindle formation. This causes cells to accumulate at the G2/M phase of the cell cycle through inhibition of mitosis. In biochemical assays, the compounds inhibit microtubule assembly from tubulin and suppress microtubule dynamics at low concentrations. Most of the (depsi)peptides inhibit the binding of Catharanthus alkaloids to tubulin in a noncompetitive manner, GTP hydrolysis by tubulin, and nucleotide turnover at the exchangeable GTP site on beta-tubulin. In general, the (depsi)peptides induce the formation of tubulin oligomers of aberrant morphology. In all cases tubulin rings appear to be formed, but these rings differ in diameter, depending on the (depsi)peptide present during their formation.

Spermatogenesis by Sisyphus: proliferating stem germ cells fail to repopulate the testis after 'irreversible' injury.

2,5-Hexanedione is the toxic metabolite resulting from oxidation of the commonly used solvents n-hexane and methyl n-butyl ketone. Exposure to 2,5-hexanedione or its precursors results in a slowly progressive peripheral polyneuropathy and testicular injury. The chemical basis of the injury involves reaction of 2,5-hexanedione with protein amines, such as the epsilon-amine of lysine, to form pyrroles which further react to form protein-protein crosslinks. The target cell of injury in the testis is the supportive cell in the seminiferous epithelium, the Sertoli cell. A major function of the Sertoli cell is to nurture the dependent germ cell population by secreting seminiferous tubule fluid. 2,5-Hexanedione-induced crosslinking of the microtubule subunit protein, tubulin, leads to altered Sertoli cell microtubule-dependent transport and deficient formation of seminiferous tubule fluid, compromising germ cell viability. In an established model of testicular injury, rats are exposed to 1% 2,5-hexanedione in the drinking water for a period of 3-5 weeks. Three weeks after initiating exposure, decreased seminiferous tubule fluid secretion initiates a wave of germ apoptosis which peaks during the 5th week. The germ cell content of the injured testis continues to decline after cessation of the exposure, reaching a nadir during the 12th week. From this time onward, the testis is severely atrophic with less than 1% of seminiferous tubules in a testicular cross section containing germ cells more advanced than spermatogonia. Interestingly, this persistent state of post-injury 'irreversible' atrophy in the rat is characterized by the presence of a proliferating stem germ cell population which produces differentiating spermatogonia which then die by apoptosis. Serial cross sections of bromodeoxyuridine-labeled testis were analyzed to determine the kinetics of stem germ cell proliferation. Approximately 40% of stem cells (identified as single cells in the seminiferous epithelium) were actively proliferating with a cell cycle time of 8-14 days. Analysis of the total germ cell population present and modeling using the known cell cycle times of differentiating spermatogonia indicated a block in differentiation at the level of type A3/A4 spermatogonia. Quantitation of the frequency of apoptosis indicated that all of the germ cells died prematurely by this mechanism. Leuprolide is a gonadotropin-releasing hormone agonist which produces a profound suppression of testosterone levels with chronic administration. When delivered as a series of 3 depot injections 24 days apart, leuprolide resulted in a partial reversal of the 2,5-hexanedione-induced persistent atrophy. The reinitiation of spermatogenesis follows a lowering of the intratesticular testosterone concentration, indicating that intratesticular testosterone is at least partially responsible for the persistent atrophy. The efficacy of leuprolide-induced reversal of the persistent atrophy decreases with time after injury, suggesting that atrophic seminiferous tubules are initially capable of recovery and then enter a state of irreversible injury. Injection of ethane dimethane sulfonate at the beginning of leuprolide treatment eliminated Leydig cells during therapy and ablated the recovery of spermatogenesis, indicating that a Leydig cell-associated paracrine factor is required to restart spermatogenesis. The rat, therefore, has multiple states of testicular germ cell proliferation: normal spermatogenesis and at least two forms of persistent atrophy (leuprolide reversible and leuprolide non-reversible). Partial reversal of the persistent atrophy can be achieved by lowering intratesticular testosterone. Ongoing experiments are designed to address the role of the Leydig cell in post-injury recovery, and to further characterize the molecular events contributing to the different states of persistent atrophy.

Interactions of bovine brain tubulin with pyridostigmine bromide and N,N'-diethyl-m-toluamide.

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N'-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent Kd of about 60 microM. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.

Effect of diethylstilbestrol on the polymerization and alkylation of tubulin

Diethylstilbestrol is a drug used to treat prostate cancer. It is thought to bind to tubulin, the subunit protein of microtubules, at the colchicine-binding site. We examined its interaction with tubulin in more detail. Diethylstilbestrol inhibits microtubule assembly, and seems to do so more effectively when tubulin polymerization is catalyzed by MAP2 rather than tau. Diethylstilbestrol also inhibits the intrachain cross-linking of tubulin by N,N′-ethylenebis-(iodoacetamide) in a pattern similar to that shown by colchicine and the drugs which bind to tubulin at the colchicine-binding site. Unlike most of this category of drugs, however, diethylstilbestrol accelerates, rather than inhibits, the decay of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas. It appears, therefore, that diethylstilbestrol interacts with tubulin in a manner similar to that of the analogs of the A-ring of colchicine, whose effect on tubulin cross-linking is similar to that of diethylstilbestrol and which also enhance tubulin decay. Drug Dev. Res. 48:104–112, 1999. © 1999 Wiley-Liss, Inc.

IKP104-induced decay of tubulin: role of the A-ring binding site of colchicine.

Tubulin, the major subunit protein of microtubules, has a tendency to lose its ability to assemble or to interact with ligands in a time-dependent process known as decay. Decay involves the increase in exposure of sulfhydryl groups and hydrophobic areas. The antimitotic drug IKP104 [2-(4-fluorophenyl)-1-(2-chloro-3, 5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone] accelerates the decay of tubulin [Ludueña et al. (1995) Biochemistry 34, 15751-15759]. In the presence of colchicine, however, IKP104 stabilizes tubulin against decay. We have shown that the stability and the acceleration of the decay of tubulin are mediated respectively by the high- and low-affinity binding site(s) of IKP104 [Chaudhuri et al. (1998) J. Protein Chem. 17, 303-309]. To better understand the mechanism by which colchicine protects tubulin from IKP104-induced decay, we examined the effect of colchicine and its analogues on this process. We found that IKP104 unfolds tubulin in a process involving a specific domain where colchicine interacts, although the binding sites of these two drugs are distinctly different. 2-Methoxy-5-(2',3',4'-trimethoxyphenyl) tropolone (MTPT), the bicyclic analogue of colchicine that lacks the B-ring, can also protect tubulin from IKP104-induced decay. An A-ring analogue of colchicine, 3,4,5-trimethoxybenzaldehyde (TMB), can also stop IKP104-induced unfolding of tubulin significantly. Interestingly, the C-ring analogue of colchicine, tropolone methyl ether (TME), does not prevent this process. Our results thus suggest that neither the B-ring nor the C-ring binding regions of colchicine are involved in the IKP104-induced decay and that the A-ring binding site of colchicine on tubulin plays a crucial role in IKP104-induced decay.

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay.

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)- -(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104's acceleration of decay. We found that podophyllotoxin abolished IKP104's accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole's effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.

Preparation of a monoclonal antibody specific for the class I isotype of beta-tubulin: the beta isotypes of tubulin differ in their cellular distributions within human tissues.

Tubulin, the subunit protein of microtubules, is an alpha/beta heterodimer. In many organisms, both alpha and beta consist of various isotypes. Although the isotypes differ in their tissue distributions, the question of whether the isotypes perform different functions in vivo is unanswered. In mammals, the betaI and betaIV isotypes are quite widespread, and betaII is less so, while betaIII and betaVI have narrow distributions and betaV distribution is unknown. As a tool for localizing the isotypes, we report the preparation of a monoclonal antibody specific for betaI, to add to our previously described monoclonal antibodies specific for betaII, betaIII, and betaIV [Banerjee et al., J. Biol. Chem. 263:3029-3034, 1988; 265:1794-1799, 1990; 267:5625-5630, 1992]. In order to prepare this antibody, we have purified betaI-rich rat thymus tubulin. We have used our battery of antibodies to localize the beta isotypes in four human tissues: oviduct, skin, colon, and pancreas. We have found striking differences in their tissue distributions. There is little or no betaIII in these tissues, except for the columnar epithelial cells of the colon. BetaII is restricted to very few cells, except in the skin, where it is concentrated in the stratum granulosum. BetaI is widespread in all the epithelia. In the skin it is found in the entire stratum malpighii. In the oviduct, betaI is found largely in the nonciliated epithelial cells. In the exocrine pancreas, betaI occurs only in the centroacinar cells and not in the acinar cells; the latter do not stain with any of these antibodies. BetaIV is present at very low levels in skin and pancreas. By contrast, it is prominent in the colon and also in the oviduct, where it occurs in all the epithelial cells, especially in the ciliated cells, with the highest concentrations in the cilia themselves. These results suggest that the regulation of the expression and localization of isotypes in tissues is very complex.

Multiple Forms of Tubulin: Different Gene Products and Covalent Modifications

Tubulin, the subunit protein of microtubules, is an alpha/beta heterodimer. In many organisms, both alpha and beta exist in numerous isotypic forms encoded by different genes. In addition, both alpha and beta undergo a variety of posttranslational covalent modifications, including acetylation, phosphorylation, detyrosylation, polyglutamylation, and polyglycylation. In this review the distribution and possible functional significance of the various forms of tubulin are discussed. In analyzing the differences among tubulin isotypes encoded by different genes, some appear to have no functional significance, some increase the overall adaptability of the organism to environmental challenges, and some appear to perform specific functions including formation of particular organelles and interactions with specific proteins. Purified isotypes also display different properties in vitro. Although the significance of all the covalent modification of tubulin is not fully understood, some of them may influence the stability of modified microtubules in vivo as well as interactions with certain proteins and may help to determine the functional role of microtubules in the cell. The review also discusses isotypes of gamma-tubulin and puts various forms of tubulin in an evolutionary context.

Synthesis of Lipid Derivatives of Colchicine

AbstractThe synthesis of glycero‐lipids linked to colchicine derivatives is reported. The lipid structures are designed to perform two‐dimensional crystallization experiments with tubulin, the structural subunit protein of microtubules.

Interaction of three sponge‐derived macrocyclic lactone polyethers (spongistatin 3, halistatins 1 and 2) with tubulin

AbstractMacrocyclic lactone polyethers of marine origin include a series of compounds which bind to tubulin, the subunit protein of microtubules, and inhibit microtubule assembly and vinblastine binding to tubulin. We have previously studied two of these compounds: halichondrin B and the very similar homohalichondrin B. We have found that halichondrin B enhances the time‐dependent exposure of hydrophobic areas on the tubulin molecule without affecting the exposure of the sulfhydryl groups, while homohalichondrin B has no effect on exposure of hydrophobic areas and slightly suppresses exposure of sulfhydryl groups. The spongistatins (isolated from the marine sponge Spongia sp.) resemble the halichondrins, except for having a smaller ring; spongistatin 1 inhibits vinblastine binding to tubulin and blocks microtubule assembly. Here, we have examined the effect of the closely related spongistatin 3 on the exposure of tubulin sulfhydryl groups and hydrophobic areas. We have found that spongistatin 3 inhibits formation of the same intrachain cross‐link in tubulin as is inhibited by vinblastine. Unlike vinblastine, however, spongistatin 3 has no effect on the exposure of either sulfhydryl groups or hydrophobic areas on the tubulin molecule. In short, spongistatin 3 resembles maytansine in its effects on tubulin more than it does either halichondrin B or homohalichondrin B. We also examined the interaction of tubulin with halistatins 1 and 2, which are close structural analogues of halichondrin B and homohalichondrin B, respectively. Halistatins 1 and 2 are isolated, respectively, from the marine sponges Phakellia carteri and Axinella carteri. We find that both halistatins resemble halichondrin B in that they have no effect on the exposure of sulfhydryl groups. Our results suggest that very small structural changes in these compounds can significantly alter the pattern of their effects on the tubulin molecule. © 1995 Wiley‐Liss, Inc.

Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.

Spatial patterns from oscillating microtubules.

Microtubules are fibers of the cytoskeleton involved in the generation of cell shape and motility. They can be highly dynamic and are capable of temporal oscillations in their state of assembly. Solutions of tubulin (the subunit protein of microtubules) and guanosine triphosphate (GTP, the cofactor required for microtubule assembly and oscillations) can generate various dissipative structures. They include traveling waves of microtubule assembly and disassembly as well as polygonal networks. The results imply that cytoskeletal proteins can form dynamic spatial structures by themselves, even in the absence of cellular organizing centers. Thus the microtubule system could serve as a simple model for studying pattern formation by biomolecules in vitro.

The α-subunit of tubulin is preferentially associated with brain presynaptic membrane

Tubulin, the subunit protein of microtubules, is a heterodimer composed of two polypeptides OLand /3of -55 000 1M, each [ 1,2]. The LYand @bunits show microheterogeneity and both have been resolved into several components [3-71. We have recently shown that cytoplasmic tubulin microheterogeneity is most prominent in the brain and increases with rat brain maturation [8]. Tubulin is not only confined to the cytoplasm, as it is found to be associated with various membranes [9-131 including the presynaptic membranes [ 14,151. It was therefore of interest to determine the presence and properties of membrane bound tubulin from brain and compare it to tubulin from the cytoplasmic fraction. By the criteria of molecular weight, isoelectric point, peptide mapping and vinblastine binding we show, in the present study, that synaptosomal membranes isolated from rat cerebral cortex contain significant amounts of tubulin. Furthermore, we find that the (Yand /3-tubulins differ in their association with these membranes.