The role of ring aggregates and other structures in the assembly of microtubules

Abstract Beef brain tubulin isolated by cycles of polymerization and depolymerization contains two components, 6S subunit and a 25–35S boundary containing ring‐shaped aggregates of tubulin. The rings disappear during microtubule polymerization, and the incorporation of ring tubulin into microtubules has been investigated by studying the changes in the sedimentation of tubulin which occur during polymerization. The “30S” boundary was separated from the 6S boundary by sedimentation at low temperatures. The temperature was then raised by letting a small amount of air into the vacuum chamber and the changes in sedimentation rate and concentration of each component determined as the tubulin polymerized. The 30S material polymerizes preferentially as determined by its decrease in concentration at polymerizing temperatures. Simultaneously with its decrease in concentration the 30S also decreases in sedimentation rate. The decrease in concentration of the 30S correlates well with polymerization while the decrease in sedimentation rate can occur independently of polymerization. The results indicate that the rings are not transformed directly into microtubules, but break down into subunits or small aggregates and these then assemble into microtubules. The rings may serve as a “storage aggregate” of active subunits. The presence of a possible storage aggregate in a dividing cell, the eggs of the surf clam, Spisula solidissima, has been indicated by measurements of particulate tubulin changes during the cell cycle. Microtubule assembly in vitro in homogenates of these eggs indicates that the amount of tubulin which forms microtubules may be controlled by the functioning of the microtubule organizing center.