Microtubule polymerization- depolymerization

The influences of herbicides on cell division fall into two classes, ie, dismption of the mitotic sequence and inhibition of mitotic entry from interphase (G, S, G2). If ceU-cycle analyses indicate increases in abnormal mitotic figures, combined with decreases in one or more of the normal mitotic stages, the effect is upon mitosis. Mitotic effects usually involve the microtubules of the spindle apparatus in the form of spindle depolymerization, blocked tubulin synthesis, or inhibited microtubule polymerization (163). Alkaloids such as colchicine [64-86-8J,viahla.stiae [865-21-4] and vincristine [57-22-7] dismpt microtubule function (164). Colchicine prevents microtubule formation and promotes disassembly of those already present. Vinblastine and vincristine also bind to free tubulin molecules, precipitating crystalline tubulin in the cytoplasm. The capacities of these dmgs to interfere with mitotic spindles, blocking cell division, makes them useful in cancer treatment. 

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). 

Fluorescein-labeled proteins are also used to measure the translational mobility of proteins and lipids by the Fluorescence Recovery After Photo-bleaching technique [54-59]. The uniformly labeled fluorescent sample is flashed with an intense light source to bleach a spot, thus producing a concentration gradient. The rate of recovery of fluorescence in that bleached area is measured and used to calculate the diffusion coefficient of the probe dye into the bleached zone. Such diffusion coefficient measurements have been used to determine the association constants of proteins in cells [60], to measure the exchange of tubulin between the cytoplasm and the microtubules [61,62], to study the polymerization-depolymerization process of actin [63-65] and to monitor the changes that occur upon cell maturation [66,67]. 

Because microtubules are required for the separation of chromosomes along the spindle in mitosis, drugs that interfere with microtubule polymerization and depolymerization can be used to selectively attack cells that are in mitosis. This is one of the strategies used to treat cancer, and drugs such as vinblastine and vincristine, which disrupt spindle microtubules, or taxol, which stabilizes microtubules, are used as anticancer drugs. 

Much attention has been paid to taxol s unique mode of action, thanks to the pioneering efforts of Susan Horwitz [83, 84]. Mitotic spindle poisons , including colchicine, the vinca alkdoids, podophyllotoxin, and maytansine, bind to soluble tubulin and inhibit microtubule polymerization. Horwitz showed that, unlike any knovra antimitotic, taxol promotes microtubule assembly and stabilizes these structures to depolymerization by CaCl2 or cold [83, 84]. Typical of other ascomycetes and deuteromycetes tested, most of the fungi in our study do not appear to be compromised by taxol below 10 m [85]. Are yew-associated endophytes equally insensitive to spindle poisons like colchicine ... 

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. 

Microtubules have a key role in mitosis and cell-proliferation. They are dynamic assemblies of heterodimers of a and f3 tubullin. In the cell-reproduction cascade tubulin polymerizes fast and subsequently depolymerizes. Tubulin dimers are unusual guanyl nucleotide binding (G) proteins, which bind GTP reversibly at a site in the (3-tubulin. GTP irreversibly hydrolyzes to GDP during polymerization. 

The cause of the cell cycle specificity of the bisindole alkaloids may be associated with the ability of these compounds to interact with the protein tubulin and thereby inhibit the polymerization (and depolymerization) of microtubules (16,17). In this respect the cellular pharmacology of vinca alkaloids is similar to that of other cytotoxic natural products such as colchicine or podophyllotoxin. On closer inspection, however, Wilson determined that the specific binding site on tubulin occupied by vinblastine or vincristine is chemically distinct from the site occupied by the other natural products (18). Subsequent experiments have determined that the maytansinoids, a class of ansa-macrocycles structurally distinct from the bisindoles, may bind to tubulin at an adjacent (or overlapping) site (19). A partial correlation of the antimitotic activity of these compounds with their tubulin binding properties has been made, but discrepancies in cellular uptake probably preclude any quantitative relationship of these effects (20). 

The generality of the end-wise depolymerization kinetic model is indicated by the comparison of the observed and predicted time-courses of cold-induced microtubule disassembly (Fig. 3). See Self-Assembly Protein Polymerization... 

The above theory can be extended to deal with other more complex cases. For example, the two ends of a biopolymer need not behave identically, and, as noted earher, MTs are helical polymers of asymmetric protomer units. Thus, two sets of on- and off-constants might be necessary. In other cases, such as in the polymerization of tubulin in the presence of tubulin-colchicine complex (Sternlicht et one may need to consider copolymerization. The kinetics of microtubule depolymerization are the reverse of elongation, and are gener-... 

DYNAMIC INSTABILITY DEPOLYMERIZATION, END-WISE MICROTUBULE TREADMILLING POLYMERIZATION RANDOM SCISSION KINETICS DEPOLYMERIZATION, END-WISE Deracemization,... 

Each tubulin dimer binds one molecule of GTP strongly in the a subunit and a second molecule of GTP or GDP more loosely in the P subunit. In this respect, tubulin resembles actin, whose subunits are about the same size. However, there is little sequence similarity. Labile microtubules of cytoplasm can be formed or disassembled very rapidly. GTP is essential for the fast growth of these microtubules and is hydrolyzed to GDP in the process.320 However, nonhydro-lyzable analogs of GTP, such as the one containing the linkage P-CH2-P between the terminal and central phosphorus atoms of the GTP, also support polymerization.321 Since microtubules have a distinct polarity, the two ends have different tubulin surfaces exposed, and polymerization and depolymerization can occur at different rates at the two ends. As a consequence, microtubules often grow at one end and disassemble... 

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