HOW TUBULIN SUBUNITS ARE LOST FROM THE SHORTENING ENDS OF MICROTUBULES

Microtubules exhibit dynamic instability, switching between persistent states of growth and shortening at their ends. The switch between gro wth and shortening has been proposed to depend on end conformation whe re growing ends have ''straight'' tubulin protofilaments stabilized by a terminal cap of GTP-tubulin, while shortening ends have lost their GTP-tubulin cap, allowing terminal GDP-tubulin dimers to curve inside- out and peel rapidly away from the microtubule lattice. This ''conform ational cap'' model predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tu bulin protofilaments at a depolymerizing end will depend on the ratio of the rate of peeling to the rate of breakage of the longitudinal bon ds between adjacent curved dimers. We have tested this model for the p lus and minus ends of microtubules assembled with pure porcine tubulin off the ends of axoneme fragments in standard assembly buffer. Indivi dual microtubule ends were imaged using video-enhanced differential in terference contrast light microscopy. The rate of rapid shortening was systematically increased by isothermal dilution into assembly buffer containing various concentrations of Mg2+ or Ca2+ ions. At 1 mM Mg2+ a nd no Ca2+, shortening occurred at 20 (plus) and 45 (minus) mu m/min. The ends appeared similar in contrast to growing ends and the core of the microtubule and the ends appeared blunt or slightly frayed by nega tive stain electron microscopy. Above 20 mM Mg2+ or above 5 mM Ca2+, m icrotubule shortening occurred at 60 (plus) and 115 (minus) mu m/min o r faster and ''knobs'' were distinctly visible at depolymerizing ends, particularly at the faster minus ends, and knob contrast remained con stant during many micrometers of rapid shortening. Negative stain elec tron microscopy revealed that these knobs were ''blossoms'' of inside- out curved protofilaments, some extending for several helical turns (3 0 to 60 dimers in length) at constant curvature from the ends. At thes e high shortening velocities, the peeling of curved protofilaments was confined to within several dimers of the end of the microtubule cylin der, suggesting that dimer curling and protofilament peeling is constr ained to the tip by interactions between adjacent straight protofilame nts. Depolymerization is produced by conformational changes in GDP-tub ulin since microtubules assembled with a slowly hydrolizable analog of GTP, GMPCPP, are stable even at 20 mM Mg2+ or 5 mM Ca2+. Monte Carlo simulations show that the ratio of the peeling to breakage rate consta nts can control the steady-state average length of curved GDP-tubulin protofilaments at the depolymerizing end. (C) 1997 Academic Press.