Microtubule Nucleation and Stabilization by Doublecortin

Abstract

Doublecortin (DCX) is a microtubule (MT)-associated protein indispensable for migrating neurons during brain development. DCX nucleates and stabilises MTs via homologous DC domains. The C-terminal DC domain (C-DC) is separated from the N-terminal counterpart (N-DC) by a 42-residue linker. Intellectual disability (smooth brain) and epilepsy-causing mutations cluster around both DC domains. Thus, both domains are thought to interact with MTs, but their exact roles remain obscure. For MT nucleation, the naturally bent tubulin dimers associating longitudinally and laterally need to straighten up to form cylindrical lattice. Stabilization of such lattice relies on counteracting the natural tendency of tubulin to curl outward. The DCX binding site on the MT lattice is at the vertex of four tubulin dimers, so DCX binding stabilizes both longitudinal and lateral lattice contacts. But it is neither clear which of the two DC domains mediates this MT-stabilizing interaction nor which is involved in MT nucleation. We found that the construct in which C-DC was replaced with another N-DC showed much lower MT-nucleating activity and – in contrast to wild-type DCX – it produced not only the physiological 13-protofilament (PF) MT architecture, but also 14-PF MTs. Near-atomic resolution cryo-EM reconstructions enabled visualisation of the structural differences between the DC domains suggesting that N-DC has higher affinity for the mature MT lattice while the newly-formed lattice is predominantly decorated with C-DC. Molecular modelling suggests that C-DC can bind between moderately curved PFs, whereas N-DC cannot. The predicted C-DC binding mode largely overlaps with patient mutations. Taken together, the data suggest that C-DC facilitates nucleation of physiological 13-PF MTs, while N-DC gradually replaces C-DC along the lattice, ensuring robust stabilisation of the straight polymer.