Isolation from bovine brain of a superstable microtubule subpopulation with microtubule seeding activity

Cold-stable microtubule protein isolated from beef brain is capable of seeding microtubule assembly under conditions that prevent the initiation of self-assembly of cold-labile microtubules. We have developed a quantitative assay for the determination of seeding activity. Using this assay, we find that seeding activity is apparently due to microtubule fragments that resist -80 degrees C, a condition that causes the depolymerization of cold-stable microtubules ("cold stability" is defined as resistance to 0 degree C disassembly), but rapidly depolymerize when exposed to 3.0 mM free calcium, to micromolar Ca2+-calmodulin, or to 0.2 M NaCl at 4 degrees C. After salt treatment, seeding activity is permanently lost although microtubule cold stability is retained through further assembly cycles. Similarly, after sedimentation of microtubule seeds the supernatant protein assembles into cold-stable microtubules, which are permanently devoid of seeding activity. By contrast, seeding activity can be recovered by recycling of supernatant protein from preparations exposed to 3.0 mM calcium or to Ca2+-calmodulin prior to centrifugation, indicating the solubilization of an active component (designated "preseeds") under these conditions. Polyacrylamide gels show some differences in polypeptides between seeding and non-seeding cold-stable microtubule preparations. Approximately 35% of the microtubule population assembled from beef brain crude extract is cold stable, while approximately 2% constitutes -80 degrees C resistant seeds. The formation of seeds from seed-forming subunits (preseeds) occurs rapidly, is apparently a cooperative phenomenon, and occurs on preexisting microtubules under either assembly initiating or steady-state conditions.