Microtubules assembly

Microtubule-associated proteins bind to microtubules in vivo and subserve a number of functions including the promotion of microtubule assembly and bundling, chemomechanical force generation, and the attachment of microtubules to transport vesicles and organelles (Olmsted, 1986). Tubulin purified from brain tissue by repeated polymerization-depolymerization contains up to 20% MAPs. The latter can be dissociated from tubulin by ion-exchange chromatography. The MAPs from brain can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 

The microtubule-associated proteins MAP2 and tau both have two separate functional regions (Lewis et al., 1989). One is the microtubule-binding site, which nucleates microtubule assembly and controls the rate of elongation (by slowing the rate of assembly). The second functional domain shared by MAP2 and tau is a short C-terminal a-helical sequence that can cross-link microtubules into bundles by self-interaction. This domain has some of the properties of a leucine zipper. Likely it is responsible for the organization of microtubules into dense stable parallel arrays in axons and dendrites (Lewis et al., 1989). 

Another drug is taxol, which is extracted from the bark of the Pacific yew tree, Taxus brevijolia. Unlike colchicine and the vinca alkaloids, taxol binds tightly to microtubules and stabilizes them against depolymerization by Ca. It also enhances the rate and yield of microtubule assembly, thereby decreasing the amount of soluble tubulin in the cytosol pool. Again, the overall effect of taxol is to arrest dividing cells in mitosis. Taxol is used in cancer chemotherapy. 

Jensen KG, Onfelt A, Wallin M (1991b) Effects of organotin compounds on mitosis, spindle structure, toxicity and in vitro microtubule assembly. Mutagenesis, 6(5) 409-416. 

Neurofibrillary tangles are intracellular and consist of abnormally phosphorylated tau protein which is involved in microtubule assembly. Tangles interfere with neuronal function... 

Docetaxel, another taxane, binds to tubulin to promote microtubule assembly. The pharmacokinetics of docetaxel are best described by a three-compartment model, with an a half-life of 0.08 hours, a 3 half-life of 1.6 to 1.8 hours, and a terminal half-life of 65 to 73 hours.14 Docetaxel has activity in the treatment of breast, non-small cell lung, prostate, bladder, esophageal, stomach, ovary, and head and neck cancers. Dexamethasone, 8 mg twice daily for 3 days starting the day before treatment, is used to prevent the fluid retention syndrome associated with docetaxel and possible hypersensitivity reactions. The fluid... 

Estramustine, an oral drug, also inhibits microtubule assembly and has weak estrogenic activity at the estradiol hormone receptors of the cell. Approximately 75% of a dose of estramustine is absorbed.15 The terminal half-life ranges between 20 to 24 hours, with nonrenal excretion as the major route of elimination. This drug is used primarily for the treatment of prostate cancer, but its use is limited by the side effects, which include nausea and vomiting, diarrhea, thromboembolic events, and gynecomastia. 

Delacourte A, Buee L. Normal and pathological tau proteins as factors for microtubule assembly. Int Rev Cytol 1997 171 167-224. 

The answer is d. (Hardman, p 1258. Katzung, pp 935-9.36.) Vinblastine binds to tubulin and blocks the protein from polymerizing to microtubules. The drug-tubulin complex binds to the developing microtubule, resulting in inhibition of microtubule assembly and subsequent depolymerization. 

The answer is c. (Hardman, pp 1260—1262.) Paclitaxel is a large structural molecule that contains a 15 membered taxane ring system. This anti cancer agent is an alkaloid derived from the bark of the Pacific yew tree. Its chemotherapeutic action is related to the microtubules in the cell. Paclitaxel promotes microtubule assembly from dimers and causes microtubule stabilization by preventing depolymerization. As a consequence of these actions, the microtubules form disorganized bundles, which decreases... 

DMT, TMT, DBT, TBT and DPhT chlorides exhibited in vitro spindle disturbance in V79 Chinese hamster cells of brain tubulin. The V79 cells lose stainable spindles at higher concentration. The cell mitosis activity effect at low concentration increased with the lipophilicity of the OTC, but all compounds showed a concentration dependence on microtubules. The OTC seem to act through two different cooperative mechanisms inhibition of microtubule assembly and interaction with hydrophobic sites. The latter mechanism might involve Cl/OH ion exchange28. 

Triethyllead was suggested to be one of the factors causing progressive damage of European forests144. The toxic effect of triethylleads on cells was attributable to inhibition of microtubule assembly. Triethylleads react with SH groups present in tubulin, whereupon the latter loses its capability for microtubule assembly144. 

Lindwall, G. and Cole, R.D. Phosphorylation affects the ability of tau protein to promote microtubule assembly. /. Biol. Chem. 259 5301-5305,1984. 

C5a and C5a des Arg stimulate aerobic glycolysis, hexose monophosphate shunt activity, glucose uptake and the respiratory burst of human neutrophils. All of these processes are stimulated in neutrophil suspensions incubated in the absence of cytochalasin B, but the responses are considerably enhanced if this inhibitor of microtubule assembly is present. Stimulated rates of oxidative metabolism are maximal within 2 min of addition of peptides, with half-maximal responses obtained at 30-60 nM C5a and 1-3 pM C5a des Arg. 

The role of microtubules in secretion is more clearly defined. Colchicine and vinblastine inhibit secretion, even in cytochalasin-B-treated cells, and D2O (which promotes tubulin assembly) enhances secretion in cytochalasin-treated cells. Microtubules may also be necessary for the translocation of phagocytic vesicles from the neutrophil periphery into the central region of the cytoplasm. Drugs affecting microtubule assembly may inhibit particle-induced oxidase activation or else increase oxidase activation in response to soluble agents such as fMet-Leu-Phe. 

MICROTUBULE ASSEMBLY A REVIEW OF PROGRESS. PRINCIPLES, AND PERSPECTIVES... 

For special purposes, tubulin may be prepared by vinblastine-induced paracrystal formation and subsequent centrifugal manipulation (Wilson et al, 1976). Dimethyl sulfoxide has been useful in promoting microtubule assembly (Himes, 1977), and deuterium oxide is also effective as a promoter of tubule assembly (Borisy et al, 1975). An example of the latter is provided by the isolation of microtubule proteins from neuroblastoma cells (Olmsted and Lyon, 1981). 

Biain adenosinetriphosphatase This enzymatic activity is persistendy associated with brain micFotubules even after multiple cycles of warm-induced microtubule assembly, centrifugation to separate protomer and polymer, cold-induced disassembly, and subsequent centrifugation to remove cold-stable aggregates (White et aL, 1980). The enzyme hydrolyzes boA GTP and ATP, and recent work by Tominaga and Kaziro (1982) indicates that there are two distinct ATP-ases, one that is of low M, (around 33,000) and tubulin dependent in the presence of calcium ion, and the other of larger size and associated with membrane vesicles... 

This protein or group of proteins is characterized as four to five bands on SDS—polyacrylamide gel electropherograms in the range from 58,(K)0 to 65,000, and by their ability to recycle with tubulin and stimulate microtubule assembly. [See pp. 33—37 of the review by Kirschner (1978) for an excellent overview of this protein and its properties]... 

Summary of Proposed Models for Microtubule Assembly from Depolymerized Microtubule Protein... 

---