Molecular basis of physical and chemical probes for spindle assembly

Microtubules are major cytoskeletal components of the mitotic spindles in echinoderm eggs. Well preserved isolated spindles from the first cleavage of sea urchin eggs reveal that 3, 000 to 5, 000 orderly arranged microtubules are present. Considering the sensitive nature of spindle bi-refringence, it is highly probable that the spindle assembly is regulated by the labile association of tubulin molecules and the involvement of kinetic centers such as the kinetochores and MTOCs in the centrosome region.Heavy water (D2O) increases the spindle volume and birefringence in living and dividing sea urchin eggs. Through the in vitro experiments, we confirmed that the initial rate and the final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of D2O. Yields were higher as the D2O concentration increased. D2O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concent-ration in the presence of D2O. We obtained linear van't Hoff plots and calculated thermodynamic parameters which were positive and increased with the elevation of the D2O concentration. The enhancement of the polymerization of tubulin by D2O in vitro could, therefore, be the result of the strengthening of intra-and/or inter-molecular hydrophobic interactions of tubulin molecules. We believe that the increase in length and number of microtubules of the mitotic spindles in dividing cells of eukaryotes with D2O is caused by the direct involvement of D2O in the polymerization of tubulin.When fertilized sea urchin eggs were exposed to dilute solutions of alkyl-resorcinols, i.e. T-1, an isolate from the culture medium of Pseudomonas sp. , its derivatives or analogs such as Ansamycin, Curvularin or Macbecin I, spindles were more or less arrested in metaphase and became either barrel-shaped or ex-tremely enhanced. T-1 and Curvularin, which induced barrel-shaped spindles, possessed the same structural features with respect to 1, 3-benzenediol system with long lipophilic side chains. Structural modification of one of the hydroxyl groups of Curvularin provides a remarkable increase in chemical efficacy. This effect can be considered to be a freezing of the state of dynamic equilibrium or partial association/dissociation of tubulin molecules along with the microtubules by the given drugs. The barrel-shaped spindles consisted of bundles of straight microtubules of equal length, and we believe this might reflect the stabilized state of tubulin polymers caused by the local increase of molecular hydrophobicity. The anaphase sequence was usually delayed and chromosomes moved very slowly. We found that this unusual spindle shape was induced by the spread, discoidal redistribution of centrosomal MTOCs, and comparable with the irregular sea urchin spindles induced with mercaptoethanol.The finding of the TO series compounds turned our attention to 1, 4-benzo-quinones with long hydrophilic side chains, and Macbecin I was recognized to be a unique mitotic arrester. This compound could be a lead compound to search new mitotic arresters and its analogues obtained by synthesis. The unique in vivo assay system developed by us using the mitosis in dividing sea urchin eggs can be widely applied for the experimental survey of new mitotic arresters. Fur-ther massive screening effort would provide us more opportunity to find new drugs which can be used as the molecular probes to analyze the physico-chemical basis of the assembly and/or disassembly of cytoskeleton.