Microtubule Lumen Research Articles

Molecular Basis for Age-Dependent Microtubule Acetylation

Microtubules are decorated with chemically diverse and evolutionarily conserved posttranslational modifications that mark them for specialized functions in cells. Other than viruses and clathrin cages, microtubules are the only known hollow polymers in eukaryotic cells. Acetylation on alpha-tubulin Lys40 stands out as the only known tubulin posttranslational modification located in the microtubule lumen. It marks stable, long-lived microtubules and is required for polarity establishment and directional migration. Tubulin acetyltransferase (TAT) presents a puzzle: its activity is stimulated by microtubules, yet the luminal location of Lys40 hinders its access. TAT was proposed to access its substrate by diffusing from microtubule ends, engaging the Lys40 loop from the exterior of the microtubule or gaining access through openings or defects in the microtubule. To elucidate substrate access by TAT and uncover the molecular basis for the in vivo correlation between acetylation and microtubule age, we combined X-ray crystallography, electron microscopy, biochemical and single-molecule fluorescence analyses, and modeling. We report the first crystal structure of TAT in complex with a bisubstrate analog that together with structure-based functional analyses constrains the active enzyme to the microtubule lumen and reveal the structural determinants for Lys40 loop recognition. Unexpectedly, acetylation by TAT proceeds stochastically along the microtubule without a preference for ends despite the luminal location of the acetylation site. Single-molecule fluorescence imaging reveals that TAT efficiently and stochastically scans the microtubule, while modeling and kinetic analyses demonstrate that TAT catalytic activity, rather than diffusion through the lumen, is rate-limiting. Consistent with this, our 1.35A X-ray complex crystal structure reveals that the TAT active site is suboptimal for supporting the proton transfer required for acetylation. Thus, by virtue of its preference for the microtubule and its low catalytic rate, TAT alone can function as an enzymatic timer for microtubule lifetimes.

Microtubule chemical complexity: mechanism of tubulin modification enzymes

Tubulin is subject to an abundant and diverse set of post-translational modifications that include phosphorylation, acetylation, poly-glutamylation, poly-glycylation and tyrosination. The highest density and variety of post-translational modifications are found in especially complex microtubule arrays like those of neurons or cilia. Not surprisingly, tubulin modification enzymes have been linked to human diseases including cancers and neurodegenerative disorders. I will present recent data from my lab on the mechanism of action of two tubulin modification enzymes that illustrate two divergent paradigms of tubulin recognition. Tubulin tyrosine ligase (TTL) adds a C-terminal Tyr to the exposed C-terminus of alpha-tubulin as part of a tyrosination/detyrosination cycle present in most eukaryotic cells. We solved the first crystal structure of tubulin tyrosine ligase that revealed how the TTL scaffold supported the expansion of the repertory of tubulin post-translational modification enzymes of the TTL like family that recognize either alpha- or beta-tubulin C-terminal tails. In addition to modifying tubulin, TTL also prevents tubulin from incorporating into microtubules by recognizing a tubulin dimer interface that would otherwise be involved in microtubule lattice interactions. I will also present recent work from my group on the structure and mechanism of action of tubulin acetyltransferase (TAT). TAT acetylates Lys-40 on alpha-tubulin in the microtubule lumen. We solved the 2.7Å structure of TAT bound to its ac-coA substrate as well as the 2.45Å structure of a catalytic inactive TAT mutant that reveals a domain swapped dimer in which the functionally essential N-terminus shows evidence of unprecedented structural plasticity. Implications for catalysis and microtubule stimulation of TAT activity will be discussed.

Molecular Basis for Age-Dependent Microtubule Acetylation by Tubulin Acetyltransferase

Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here, we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule interprotofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.

Structural and Functional Characterization of the α-Tubulin Acetyltransferase MEC-17

Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine 40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch that is critical for enzymatic activity is identified, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in Caenorhabditis elegans shows that enzymatic activity is dispensable for touch sensitivity.

Crystal Structures of Tubulin Acetyltransferase Reveal a Conserved Catalytic Core and the Plasticity of the Essential N Terminus

Tubulin acetyltransferase (TAT) acetylates Lys-40 of α-tubulin in the microtubule lumen. TAT is inefficient, and its activity is enhanced when tubulin is incorporated in microtubules. Acetylation is associated with stable microtubules and regulates the binding of microtubule motors and associated proteins. TAT is important in neuronal polarity and mechanosensation, and decreased tubulin acetylation levels are associated with axonal transport defects and neurodegeneration. We present the first structure of TAT in complex with acetyl-CoA (Ac-CoA) at 2.7 Å resolution. The structure reveals a conserved stable catalytic core shared with other GCN5 superfamily acetyltransferases consisting of a central β-sheet flanked by α-helices and a C-terminal β-hairpin unique to TAT. Structure-guided mutagenesis establishes the molecular determinants for Ac-CoA and tubulin substrate recognition. The wild-type TAT construct is a monomer in solution. We identify a metastable interface between the conserved core and N-terminal domain that modulates the oligomerization of TAT in solution and is essential for activity. The 2.45 Å resolution structure of an inactive TAT construct with an active site point mutation near this interface reveals a domain-swapped dimer in which the functionally essential N terminus shows evidence of marked structural plasticity. The sequence segment corresponding to this structurally plastic region in TAT has been implicated in substrate recognition in other GCN5 superfamily acetyltransferases. Our structures provide a rational platform for the mechanistic dissection of TAT activity and the design of TAT inhibitors with therapeutic potential in neuronal regeneration.

Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

Microtubules: MEC-17 Moonlights in the Lumen

New research characterizes a tubulin acetyltransferase that acts inside the microtubule lumen and has two separable activities that greatly affect microtubule architecture and functionality.

In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: folate conjugated noscapine (Targetin)

Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280-2286, 1998). Guided by molecular modelling calculations and structure-activity relationships we conjugated at C9 of noscapine, a folate group-a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score -11.295kcal/mol) at the interface between α- and β-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (ΔG (bind)) computed based on molecular mechanics energy minimization was -221.01kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant (K (d) value) of 149±3.0μM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC(50) in the range of 15-40μM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC(50) in the range of 0.3-1.5μM).

Tubulin acetyltransferase discovered: Ciliary role in the ancestral eukaryote expanded to neurons in metazoans

The emergence of α- and β-tubulins from an FtsZ-like prokaryotic homolog represented a defining event in eukaryogenesis. It permitted, together with actin and molecular motors, the establishment of a cytoskeleton able to support dynamic events such as cell division and trafficking of vesicles and proteins. It also served as the foundation for the microtubule-based cilium, an organelle with motility and sensory functions that replaced the less versatile prokaryotic flagellum (1). As it turns out, a mechanism to stabilize the ciliary axoneme, involving acetylation of α-tubulin, evolved in the ancestral eukaryote (2–4). This reversible modification situated inside the microtubule lumen still operates in extant ciliated protists and is exploited by metazoans to support ancillary microtubule functions, particularly in neurons (5). The concurrent discovery of an α-tubulin acetyltransferase by two separate groups, published in PNAS (3) and in Nature (4), now paves the way for a detailed understanding of the cellular and physiological roles conferred by tubulin acetylation.

The major α-tubulin K40 acetyltransferase αTAT1 promotes rapid ciliogenesis and efficient mechanosensation

Long-lived microtubules found in ciliary axonemes, neuronal processes, and migrating cells are marked by α-tubulin acetylation on lysine 40, a modification that takes place inside the microtubule lumen. The physiological importance of microtubule acetylation remains elusive. Here, we identify a BBSome-associated protein that we name αTAT1, with a highly specific α-tubulin K40 acetyltransferase activity and a catalytic preference for microtubules over free tubulin. In mammalian cells, the catalytic activity of αTAT1 is necessary and sufficient for α-tubulin K40 acetylation. Remarkably, αTAT1 is universally and exclusively conserved in ciliated organisms, and is required for the acetylation of axonemal microtubules and for the normal kinetics of primary cilium assembly. In Caenorhabditis elegans, microtubule acetylation is most prominent in touch receptor neurons (TRNs) and MEC-17, a homolog of αTAT1, and its paralog αTAT-2 are required for α-tubulin acetylation and for two distinct types of touch sensation. Furthermore, in animals lacking MEC-17, αTAT-2, and the sole C. elegans K40α-tubulin MEC-12, touch sensation can be restored by expression of an acetyl-mimic MEC-12[K40Q]. We conclude that αTAT1 is the major and possibly the sole α-tubulin K40 acetyltransferase in mammals and nematodes, and that tubulin acetylation plays a conserved role in several microtubule-based processes.

Cyclostreptin and Microtubules: Is a Low‐Affinity Binding Site Required?

Cyclostreptin (CS) is a recently discovered natural product with cytotoxic activity caused by microtubule stabilization. It is the only known microtubule-stabilizing agent (MSA) that covalently binds to tubulin. It also exhibits the fast-binding kinetics seen for other MSAs. Through careful peptide digestion and mass spectrometry analysis, Buey et al. found that two amino acids are labeled by CS: Asn228, near the known taxane-binding site, and Thr220, in the type I microtubule pore. This led Buey et al. to propose Thr220 resides at the site previously predicted to be a way station or low-affinity site. By using molecular dynamics simulations and structural considerations of the microtubule pore and tubulin dimer, we conclude that postulation of a low-affinity site is unnecessary to explain the available experimental data. An alternative explanation views the microtubule pore as a structural entity that presents a substantial kinetic barrier to ligand passage to the known taxane-binding site-an entry point to the microtubule lumen that becomes completely blocked if cyclostreptin is bound at Thr220. Simulations of the free dimer also suggest a common mechanism of microtubule stabilization for taxane site MSAs through their conformational effect on the M-loop. Such an effect explains the low tubulin polymerization caused by cyclostreptin in vitro despite its covalent attachment.

Axoneme β-Tubulin Sequence Determines Attachment of Outer Dynein Arms

Axonemes of motile eukaryotic cilia and flagella have a conserved structure of nine doublet microtubules surrounding a central pair of microtubules. Outer and inner dynein arms on the doublets mediate axoneme motility [1]. Outer dynein arms (ODAs) attach to the doublets at specific interfaces [2-5]. However, the molecular contacts of ODA-associated proteins with tubulins of the doublet microtubules are not known. We report here that attachment of ODAs requires glycine 56 in the beta-tubulin internal variable region (IVR). We show that in Drosophila spermatogenesis, a single amino acid change at this position results in sperm axonemes markedly deficient in ODAs. Moreover, we found that axonemal beta-tubulins throughout the phylogeny have invariant glycine 56 and a strongly conserved IVR, whereas nonaxonemal beta-tubulins vary widely in IVR sequences. Our data reveal a deeply conserved physical requirement for assembly of the macromolecular architecture of the motile axoneme. Amino acid 56 projects into the microtubule lumen [6]. Imaging studies of axonemes indicate that several proteins may interact with the doublet-microtubule lumen [3, 4, 7, 8]. This region of beta-tubulin may determine the conformation necessary for correct attachment of ODAs, or there may be sequence-specific interaction between beta-tubulin and a protein involved in ODA attachment or stabilization.

Luminal particles within cellular microtubules

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.

Macromolecular Accessibility of Fluorescent Taxoids Bound at a Paclitaxel Binding Site in the Microtubule Surface

The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.

Discodermolide interferes with the binding of tau protein to microtubules

We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on β-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

Fast Kinetics of Taxol Binding to Microtubules: EFFECTS OF SOLUTION VARIABLES AND MICROTUBULE-ASSOCIATED PROTEINS

The kinetics of Taxol association to and dissociation from stabilized microtubules has been measured by competition with the reference fluorescent derivative Flutax-1 (Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) J. Biol. Chem. 275, 26265-26276). The association rate constant at 37 degrees C is k(+) = (3.6 +/- 0.1) x 10(6) m(-1) s(-1). The reaction profile is similar to that of the first step of Flutax-1 binding, which probably corresponds to the binding of the Taxol moiety. The rate constant of the initial binding of Flutax-1 is inversely proportional to the viscosity of the solution, which is compatible with a diffusion-controlled reaction. Microtubule-associated proteins bound to the microtubule outer surface slow down the binding of Flutax-1 and Flutax-2 10-fold. The binding site is fully accessible to Flutax-2 in native cytoskeletons of PtK2 cells; the observed kinetic rates of Flutax-2 microtubule staining and de-staining are similar to the reaction rates with microtubule associated proteins-containing microtubules. The kinetic data prove that taxoids bind directly from the bulk solution to an exposed microtubule site. Several hypotheses have been analyzed to potentially reconcile these data with the location of a Taxol-binding site at the model microtubule lumen, including dynamic opening of the microtubule wall and transport from an initial Taxol-binding site at the microtubule pores.

High-Resolution Model of the Microtubule

A high-resolution model of the microtubule has been obtained by docking the crystal structure of tubulin into a 20 Å map of the microtubule. The excellent fit indicates the similarity of the tubulin conformation in both polymers and defines the orientation of the tubulin structure within the microtubule. Long C-terminal helices form the crest on the outside of the protofilament, while long loops define the microtubule lumen. The exchangeable nucleotide in β-tubulin is exposed at the plus end of the microtubule, while the proposed catalytic residue in α-tubulin is exposed at the minus end. Extensive longitudinal interfaces between monomers have polar and hydrophobic components. At the lateral contacts, a nucleotide-sensitive helix interacts with a loop that contributes to the binding site of taxol in β-tubulin.

Changes in Microtubule Protofilament Number Induced by Taxol Binding to an Easily Accessible Site: INTERNAL MICROTUBULE DYNAMICS

We have investigated the accessibility of the Taxol-binding site and the effects of Taxol binding on the structures of assembled microtubules. Taxol and docetaxel readily bind to and dissociate from microtubules, reaching 95% ligand exchange equilibrium in less than 3 min under our solution conditions (microtubules were previously assembled from GTP-tubulin, GTP-tubulin and microtubule-associated proteins, or GDP-tubulin and taxoid). Microtubules assembled from purified tubulin with Taxol are known to have typically one protofilament less than with the analogue docetaxel and control microtubules. Surprisingly, Taxol binding and exchange induce changes in the structure of preformed microtubules in a relatively short time scale. Cryoelectron microscopy shows changes toward the protofilament number distribution characteristic of Taxol or docetaxel, with a half-time of approximately 0.5 min, employing GDP-tubulin-taxoid microtubules. Correspondingly, synchrotron x-ray solution scattering shows a reduction in the mean microtubule diameter upon Taxol binding to microtubules assembled from GTP-tubulin in glycerol-containing buffer, with a structural relaxation half-time of approximately 1 min. These results imply that microtubules can exchange protofilaments upon Taxol binding, due to internal dynamics along the microtubule wall. The simplest interpretation of the relatively fast taxoid exchange observed and labeling of cellular microtubules with fluorescent taxoids, is that the Taxol-binding site is at the outer microtubule surface. On the contrary, if Taxol binds at the microtubule lumen in agreement with the electron crystallographic structure of tubulin dimers, our results suggest that the inside of microtubules is easily accessible from the outer solution. Large pores or moving lattice defects in microtubules might facilitate the binding of taxoids, as well as of possible endogenous cellular ligands of the inner microtubule wall.

Reply to Hameroff from Emmet Spier and Adrian Thomas

We thank Stuart Hameroff for detailing the intellectual history of the Orch OR model. However, re-rooting its foundations in a form of monism begs more questions than it answers. Even ignoring the problematic conclusion that—then—all forms of matter (rocks, air, grass, protozoa etc.) have minds, we still find it hard to understand how his mind-component of matter influences the substance-component of matter. Either its influence is local—in which case `learned behaviour' and `intensity of deterministic drives' would be neural juggernauts affected by rain drops—or global, in which case our previous, single-Platonic-mind criticism still stands.Neither are we convinced that the mechanistic side of the brain is a computer, even less a quantum computer. A computer manipulates memory elements. Merely identifing the memory elements (qubits) as `microtubule quantum superpositions' completely ignores the fact that machinery must exist for these bits to be fetched, processed and re-stored. This is the job of the central processing unit (CPU) and is wholly ignored in models of the brain as a computer. We also note that there is nothing particularly useful about being a quantum computer, unless factorizing large numbers[1xPolynomial-time algorithms for prime factorization and discrete logarithms on a quantum computer. Shor, P. SIAM J. Comp. 1997; 26: 1484–1509CrossrefSee all References][1](their one establised novel capacity) is a fundamental process of mind.Hameroff agrees with Bray's conclusions that protein molecules are the basic operational elements in living cells, but goes on to suggest that quantum mechanics might play some role too. Bray[2xProtein molecules as computational elements in living cells. Bray, D. Nature. 1995; 376: 307–312Crossref | PubMedSee all References][2]has clearly demonstrated that chemistry is sufficient to explain the complex behaviour of single-celled organisms such as E. coli. Furthermore, mutations that alter the chemistry of E. coli's proteins change its behaviour in ways that are predictable from the effect of the mutations on protein chemistry. Conventional chemical mechanisms can explain how single cells process such information—invoking the Orch OR model is neither necessary nor justified.We also note that Hameroff has suggested that the emergence of consciousness in primitive metazoans (worms) might explain the Cambrian explosion. However, recent fossil finds have reduced the Cambrian anomaly. Papers[3xThree-dimensional preservation of algae and animal embryos in a neoproterozoic phosphorite. Xiao, S., Zhang, Y., and Knoll, A.H. Nature. 1998; 391: 553–558Crossref | Scopus (446)See all References, 4xPrecambrian sponges with cellular structures. Li, C-W., Chen, J-Y., and Hua, T-E. Science. 1998; 279: 879–882Crossref | PubMed | Scopus (309)See all References]describe 570 million-year-old (Precambrian) fossils of metazoan embryos; such fossil finds establish that metazoans originated long before the Cambrian explosion[5xThe breath of life: did increased oxygen levels trigger the Cambrian Explosion?. Thomas, A.L.R. Trends Ecol. Evol. 1997; 128: 44–45Abstract | Full Text PDFSee all References][5], but these Precambrian animals did not fossilize.Crucial to the Orch OR model is the idea that `brain microtubules are quite stable' and that they can maintain some thermal isolation from the rest of the body. This is a misconception. Microtubules in the axons of neurons (but not in the dendrites or cell body) are indeed held in a stable configuration; but even in axons the microtubules turn over at high rates and being shorter than the axon their open ends are exposed to the cell contents[6xSee all References, 7xMicrotubule transport from the cell body into the axons of growing neurons. Slaughter, T., Wang, J., and Black, M.M. J. Neurosci. 1997; 17: 5807–5819PubMedSee all References]. The fluid-filled lumen of microtubules can act as a transport route—for example, during bacterial sex, DNA is transported down the centre of a microtubule-like protein[6xSee all References][6]. It seems unlikely, when molecules as large as DNA, let alone water, can flow through microtubules, that they could possibly maintain the thermal isolation that is an essential requirement for the Orch OR model.Indeed, `how many roads must a man walk down before you call him a man?'—possibly, according to the Orch OR model, `the answer [might well be] blowing in the wind'[8xSee all References][8].

The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain.

Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3 +/- 0.11 and 0.8 +/- 0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.