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 growth and shortening has been proposed to depend on end conformation where 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 "conformational cap" model predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tubulin protofilaments at a depolymerizing end will depend on the ratio of the rate of peeling to the rate of breakage of the longitudinal bonds between adjacent curved dimers. We have tested this model for the plus and minus ends of microtubules assembled with pure porcine tubulin off the ends of axoneme fragments in standard assembly buffer. Individual microtubule ends were imaged using video-enhanced differential interference 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+ and no Ca2+, shortening occurred at 20 (plus) and 45 (minus) microns/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 negative stain electron microscopy. Above 20 mM Mg2+ or above 5 mM Ca2+, microtubule shortening occurred at 60 (plus) and 115 (minus) microns/min or faster and "knobs" were distinctly visible at depolymerizing ends, particularly at the faster minus ends, and knob contrast remained constant during many micrometers of rapid shortening. Negative stain electron microscopy revealed that these knobs were "blossoms" of inside-out curved protofilaments, some extending for several helical turns (30 to 60 dimers in length) at constant curvature from the ends. At these high shortening velocities, the peeling of curved protofilaments was confined to within several dimers of the end of the microtubule cylinder, suggesting that dimer curling and protofilament peeling is constrained to the tip by interactions between adjacent straight protofilaments. Depolymerization is produced by conformational changes in GDP-tubulin 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 constants can control the steady-state average length of curved GDP-tubulin protofilaments at the depolymerizing end.
Tran
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Biology Department, University of North Carolina, Chapel Hill 27599, USA.
Joshi
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Salmon
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D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D018715:Microscopy, Video; D008870:Microtubules / Q000502:physiology*; D009010:Monte Carlo Method; D011487:Protein Conformation; D014404:Tubulin / Q000502:physiology*
D000998:Antiviral Agents; D014404:Tubulin; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; D008274:Magnesium; D002118:Calcium