NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.