Elucidating the structural mechanism of microtubule depolymerization by kinesin-13 family proteins

Abstract

KMesin-13 family proteins are a group of motors that use energy from ATP hydrolysis to destabilize the microtubule (MT) structure rather than to move along the MT track like other kinesins. Such activity makes them MT dynamic regulators during cell division and their proper functions are important for accurate segregation of chromosomes. We aim to elucidate the structural mechanism of kinesin-13-driven MT depolymerization to gain more insight into its cellular function. In Chapter 1, we first introduced MT dynamic instability and its various regulators. We then focused on the biochemical and structural characteristics of kinesin-13 family proteins and compared them with other motile kinesins. At the end of Chapter 1 the major technique used in our research, electron microscopy (EM) and image reconstruction, was introduced.;In Chapter 2, we described in detail the methods we used and the procedures we followed. In Chapter 3, we presented our observation that kinesin-13 motor domain (MD) in its ATP-bound form, has the unusual ability to form rings/spirals around MTs. We also presented a medium resolution three-dimensional (3D) density map of this kinesin-13-ring-MT complex obtained by cryo-electron microscopy and image analysis. And an atomic model of the complex was built by docking the crystal structures into the 3D density map. Our model indicates that the ring is composed of an outer layer of curved tubulins and an inner layer of kinesin-13 MD. In each asymmetric unit of the structure, one kinesin-13 MD interacts with both the curved tubulin in the protofilament ring and the straight tubulin in the MT lattice. By providing a 3D view of the complex formed between the kinesin-13 MDs and a curved tubulin protofilament, the model reveals a snapshot of depolymerization and insights to mechanism. In addition, a new tubulin-binding site on the kinesin-13 MD was identified. Mutations at this family-conserved site selectively disrupt the formation of MT-associated ring complexes.;In Chapter 4, we discussed the significance of our findings on our overall understanding of the molecular mechanism of kinesin-13's action and its functions in vivo.