Polymorphism of tubulin assembly. In vitro formation of sheet, twisted ribbon and microtubule

Abstract

Tubulin assembly in equilibrium has been investigated by electron microscopy at different pH values in the absence or presence of Ca2+ at 37 degrees C. In the presence of 1 mM EGTA, tubulin assembled into microtubules in the pH range 6.0 to 7.0 and into sheets in the pH range 5.8 to 6.2. The sheet was composed of 8 to 22 protofilaments and frequently curled in a lateral direction. Two-block-polymers of sheet and microtubule were present in the boundary of two pH ranges (6.0-6.2). In the presence of 1 mM CaCl2, rings were formed in the pH range 6.0 to 7.0: macrotubules of diameter 75 nm and microtubules were both formed in the pH range 6.2 to 6.4, and two types of twisted ribbons in the pH range 5.8 to 6.2. One type of twisted ribbons had a pitch of around 150 nm and amplitude of around 40 nm, the other had 250 nm and 90 nm, respectively. Microtubules formed in the presence of CaCl2 were not distinguishable from those formed in the absence of CaCl2 in electron-micrographs, although they had different properties. The formation of all of these polymers was inhibited by 0.2 M KCl and was temperature dependent; they disappeared at 0 degrees C and reappeared at 37 degrees C. Interconversion between these polymers was also investigated. Globular particles were formed in the absence of magnesium ions. Addition of 50 muM colchicine induced formation of filamentous polymers. On the basis of these experimental results, the structural relation among different forms of tubulin assembly was discussed.