EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.
Bottom Line: By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state.The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization.This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.
Affiliation: Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.Show MeSH
Mentions: The enhanced interprotofilament contacts recognized by EBs could have a potentially crucial role in stabilizing the lattice of dynamic microtubules. If so, we would predict that such contacts are lost before microtubule depolymerization, thereby reducing EB affinity at microtubule ends prior to catastrophe. We tested this hypothesis by recording dynamic microtubules in vitro in the presence of full-length dimeric Mal3-GFP by TIRF microscopy. We measured the Mal3-GFP comet intensity as a read-out for the presence of the enhanced interprotofilament contacts and asked whether the Mal3-GFP comets disappear before catastrophe. This was indeed the case (Figures 7A and 7B). The average comet intensity began to decay several seconds before catastrophe and was strongly reduced at the moment of catastrophe (Figure 7C). This observation suggests two possible interpretations: either the loss of Mal3 or a conformational transformation within the microtubule lattice leading to the loss of most of the EB binding region causes a catastrophe. It is unlikely that the loss of Mal3 from the microtubule end triggers catastrophe because in vitro the addition of EBs is known to increase the catastrophe frequency under the conditions used here (Bieling et al., 2007; Komarova et al., 2009). Thus, we can define the extended high-affinity EB binding region as a stabilizing zone at growing microtubule ends. The loss of this zone appears to trigger depolymerization. Surprisingly, EBs were found to decrease the lifetime of their own high-affinity binding sites and hence the size of the protective zone at microtubule ends (Maurer et al., 2011). Within the framework of the model of the extended protective structural cap, this provides a direct explanation for the catastrophe-promoting effect of EBs in vitro.
Affiliation: Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.