The investigation of the microtubule binding of human doublecortin-like kinase and microtubule dynamics, by cryo-electron microscopy and cryo-electron tomography

The microtubule (MT) cytoskeleton forms a complex and dynamic network which facilitates important processes such as intracellular signalling, vesicular transport, cell motility and cell division. The cell exerts fine control over the MT cytoskeleton through diverse regulatory mechanisms, one facet of which is through the activities of MT associated proteins (MAPs), which bind MTs and modify their behaviour. Doublecortin (DCX) and doublecortin-like kinase (DCLK) are members of the DCX family of MAPs and contain tandem MT binding domains, NDC and CDC, which allow them to nucleate, bind and stabilize MTs. DCX is involved in the proper migration of cortical neurons during neurogenesis, and dysfunction of DCX and its relatives are implicated in a range of disorders associated with improper neuronal function. DCLK shows remarkable similarity in sequence and fold to its family archetype DCX but surprisingly, we have found that it appears to bind MTs by a different mechanism. Cryo-EM reconstructions of MT-bound DCLK at different timepoints post-nucleation, at near atomic resolution, show that the CDC domain predominates on the mature MT lattice, where NDC predominates in DCX. This raises interesting questions around the nature of the DC domain tandems in DCLK and the other members of the DCX family. In addition, I investigated the link between MT dynamics and + end tip morphology, by cryo-electron tomography. While more work is required here, the preliminary results suggest that there may indeed be different distributions of +end tip morphologies which occur in populations of MTs with different dynamic characteristics