The Molecular Basis Of Microtubule Dynamics And Its Regulation

Microtubule dynamic instability plays important roles in many cell biological processes,including cell division,migration,and polarization.The key of microtubule dynamics is the polymerization kinetics and special structure of microtubule growing ends.In general,microutubule dynamics is regulated by microtubule-associated proteins(MAPs),especially plus-end-tracking proteins(+TIPs).This doctoral thesis focuses on the kinetics of microtubule ends and consists of two parts:(1)a “structure-kinetics” model to describe the relationship of structure and kinetics at microtubule ends;(2)the mechanism underlying the high end-binding affinity of EB1(end binding protein1).In the first part,we analyzed the structure and kinetics at microtubule ends in various tubulin concentrations.We found a correlation between microtubule end structure and kinetics at microtubule ends.During microtubule polymerization,the rates of on-and offreactions both increase when microtubule ends become more tapered.Based on these results,we established a “structure-kinetics” model.Furthermore,using this model,we analyzed the working mechanism of EB1 and ch TOG.We found that both of EB1 and ch TOG regulate microtubule dynamics by altering microtubule end structure.In the second part,we investigated the mechanism of how EB1 regulates microtubule dynamics.Using single-molecule imaging,we showed that the end-binding kinetics of EB1 changes along with the polymerizing and hydrolysis rate of tubulin dimers.The results of monomeric EB1 mutants suggest that two EB1 monomers in the dimer contribute to the end-binding affinity.Introducing phosphomimicking mutations into the linker domain disrupts the end-binding affinity and confers a more curved conformation to EB1 without compromising dimerization,suggesting that the overall architecture of EB1 is important for the end-binding affinity.In summary,this doctoral thesis focuses on structure and biochemical kinetics of microtubule ends,investigated the structure-kinetics characteristics of microtubule end and the working mechanism of EB1.Our work provides further mechanistic inisghts into the understanding of microtubule dynamics.