Particulate Tubulin in Interphase and Metaphase Extracts of Oocytes of Spisula

In 1972 Weisenberg reported that surf clam oocytes contained a particulate and sedimentable pool of tubulin that could be isolated in buffers containing hexylene glycol. This "interphase particulate tubulin" (IPT) copurified with 10-20 {mu}m granular spheres, which were identified as the "tubulin-containing structures" (TCS). Approximately one TCS per oocyte was isolated and the TCS disappeared after nuclear envelope breakdown. Weisenberg postulated that the TCS was comprised of a stored form of tubulin or a microtubule-assembly intermediate. To characterize this intriguing form of stored tubulin, IPT was isolated in hexylene glycol-containing buffers as described by Weisenberg (1972, J. Cell Biol. 54, 266-278) and the amount of sedimentable tubulin was quantitated by immunoblotting during the first meiotic cell cycle. Approximately 10% of the total tubulin in Spisula oocytes sediments at g forces that are too small to pellet tubulin dimers or even single microtubules. Granular spheres, approximately 15 {mu}m in diameter, are present in the sedimentable tubulin fractions. During the first cell cycle, the granular spheres disappear while the sedimentable tubulin levels gradually decrease. Although the disappearance of the spheres corresponds with the loss of sedimentable tubulin, the spheres do not contain tubulin. An initial centrifugation of the oocyte homogenates at 650g leaves most of the tubulin in the supernatant and the granular spheres in the pellet. The tubulin-containing fractions are composed of membranes and an amorphous unidentified material associated with short microtubules. Sedimentable tubulin is not detected in homogenates prepared at 0{deg}C or in the absence of hexylene glycol, conditions that favor microtubule disassembly. It is likely that sedimentable tubulin is composed of hexylene glycol-induced polymers and not unique particulate structures that sequester tubulin. Finally, the granular spheres that contaminate the tubulin preparations are identified as nucleoli. They are morphologically identical to the nucleoli of the intact oocyte and they fluoresce brightly when stained with the Hoechst DNA dye 22358.