Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima.

Abstract

Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima . At 22°C or 37°C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly . Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36°angle relative to the long axis of the polymer and were composed of a and a tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000 . Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of >95% a and ,Q tubulins . After three cycles of polymerization at 37°C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22°C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml . The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5 .8 for a tubulin and 5.6 for # tubulin . In addition, one dimensional peptide maps of oocyte and spindle a and ,Q tubulins were very similar, if not identical . These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping . These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins .In 1972, Weisenberg (68, 69) described a particulate and sedimentable pool of tubulin that was assayed indirectly by colchicine binding in homogenates of unfertilized surf clam oocytes. The sedimentable tubulin was present in unfertilized oocytes in a granular and filamentous matrix suggested to be a storage form of tubulin. The level of sedimentable tubulin decreased to a minimum level at 5 min after parthenogenetic activation (time of nuclear envelope breakdown) and rose to a maximum value at meiotic metaphase. In addition to sedimentable tubulin, Burnside et al. (10) and Rosenfeld (70) showed that homogenates of activated Spisula solidissima oocytes at 28°C would assemble into microtubules, forming aster-like structures associated with centrioles . No microtubule assembly or aster formation was observed when homogenates of unfertilized oocytes were warmed, although a crude preparation of organizing centers from activated oocytes induced aster formation in homogenates of unactivated oocytes. Unfortunately, the complexity of the fractions used did not all...

Taxol-induced assembly and characterization of microtubule proteins from developing brine shrimp (Artemia)

“…It mav be that Artemia contain a large pool of unassembled iubulin which, as occurs in Spisula oocytes (52), forms microtubules only when chromosome movement is necessary. Such a situation would be considered unlikely under conventional wisdom since it implies the formation of fully functional.…”

The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation

“…Spisula centrosomes were purified from A4' extract by centrifugation through a sucrose step gradient (6, 10). Three-cycled Spisula tubulin (3X tubulin) was isolated from activated extracts by 3 cycles of MT polymerization and depolymerization (6, 10-12). Centrosomes were incubated with 3X tubulin (0.55 mg/mL) containing dilutions of HMN-176 (2.5, 0.25, and 0.025 μM) or DMSO alone for 20 minutes at 24°C to assemble asters.…”

“…3X and 5X (glutamate-purified) Spisula MT protein was isolated as previously described (6, 12, 15). Spisula tubulin was adjusted to a final concentration of 0.55 mg/mL, supplemented with either HMN-176 (2.5, 0.25, or 0.025 μM), 2.5 μM colchicine, or DMSO, incubated on ice for 10 minutes, and then warmed to room temperature to induce polymerization.…”

Unidirectional microtubule assembly in cell‐free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH

“…diplotene of the first meiotic prophase [Allen, 19531. Microtubules of the first meiotic spindle form only after the oocyte has been fertilized or parthenogenetically activated and the cytoplasmic pH rises to physiological levels [Allen, 1953;Ii and Rebhun, 1979;Dube, 1988;Kuryama et al, 1986;Longo and Anderson, 19701. Microtubule assembly in these cell-free extracts is regulated by small changes in pH, in a manner that was not predicted by the assembly properties of purified Spisula tubulin or microtubule protein [Suprenant and Rebhun, 1984;Suprenant, 19891. Microtubule assembly in extracts is favored at alkaline pH and proceeds unidirectionally onto the plus-ends of isolated axonemes.…”