Tubulin-colchicine complexes differentially poison opposite microtubule ends

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Abstract
The kinetics of radiolabeled CTP tubulin dimer addition to preformed [bovine brain] microtubule copolymers, containing large numbers of tubulin-colchicine complexes (TC), were examined at apparent equilibrium. Radiolabeled dimer addition to copolymers occurs predominantly by a treadmilling reaction, analogous to that described for unpoisoned microtubules, and some labeled dimer uptake also occurs by equilibrium exchange. TC decrease the steady-state treadmilling reaction in a concentration-dependent manner. Since microtubule copolymers exhibited a treadmilling reaction, it was possible to differentially radiolabel opposite copolymer ends with [3H]- and [14C]guanine nucleotides and thus to measure the effects of TC on dimer loss from opposite copolymer ends upon copolymer dilution. Dimer loss from both copolymer ends was inhibited in a concentration-dependent manner, but dimer loss from copolymer net assembly (A) ends (defined under steady-state conditions) was inhibited to a far greater extent than that from the opposite, net disassembly (D) copolymer ends. TC therefore exhibited a graded, polar poisoning, with copolymer A-end association and dissociation rate constants being far more susceptible to TC inhibition than those at the opposite copolymer D ends. The potential significance of this TC effect for regulating microtuble spatial orientation in vivo is discussed.