Cold depolymerization of microtubules to double rings: geometric stabilization of assemblies

Abstract

The kinetic pathway of microtubule depolymerization at 0.degree.C has been examined. Microtubules made of MAP-containing and MAP-free tubulins were depolymerized at 0.degree.C in the presence of [3H]GDP or [3H]GTP or of trace amounts of 125I dimeric tubulin. The products of depolymerization were separated on a column, their structures were identified by electron microscopy, and the time course of incorporation of 3H or 125I labels in the different components of the system was determined. Two predominant assembly states of tubulin found in the nonmicrotubule state were .alpha.-.beta. dimers and double rings. Kinetic data indicate that ring formation from disassembling microtubules does not occur by direct coiling of protofilaments as previously thought, but disassembling GDP subunits are in very rapid equilibrium with curved oligomers that are kinetic intermediates in the isodesmic assembly of GDP-tubulin. The formation of oligomers and rings from dimers, at concentrations as low as 10 .mu.M, is much faster than nucleotide exchange on .alpha.-.beta.-tubulin. Disassembly of double rings, in contrast, is slower than nucleotide exchange on .alpha.-.beta.-tubulin, by 1 order of magnitude in the absence of MAPs and 2 orders of magnitude in the presence of MAPs. These results support the model proposed previously to explain spontaneous oscillations in microtubule assembly. They are consistent with the existence of an equilibrium between two conformations of tubulin, "straight", i.e., microtubule forming, and "curved", i.e., ring forming, under the allosteric control of bound nucleotide. The straight conformation requires the presence of two ionizable hydroxyls on the .gamma.-phosphate in GTP or GDP-Pi.