“Snapshots of a 13-protofilament microtubule nucleator at secondary structure resolution”
Franck Fourniol – Department of Biological Sciences, Birkbeck, London
During brain development, immature neurons grow neurites and migrate to form the highly ordered layers of the cortex. The doublecortin (DCX) family of microtubule-associated proteins (MAPs) was found to be essential for this process. Single point mutations in DCX were found to cause severe neuronal migration disorders in humans. Previous studies have opened the way to an understanding of the relationship between the structure of DCX and its impact on microtubule dynamics, the details of which are still being explored. DCX is a 40 kDa protein with a tandem of 11 kDa globular microtubule binding domains (called DC domains) and a C-terminal Serine/Proline rich domain. A previous study based on cryo-electron microscopy (cryoEM) and helical reconstruction showed how a truncated construct of DCX, lacking the C-terminal Ser/Pro rich sequence, stabilised the microtubule lattice by bridging adjacent protofilaments with one DC domain, a unique mechanism among neuronal MAPs. Using cryoEM and state-of-the-art single particle algorithms, we have generated a 8.8 Å-resolution reconstruction of 13-protofilament microtubules co-polymerised in presence of full-length DCX, and decorated with conventional kinesin motor domain. We show that the N-terminal DC domain binds at the interface between 4 tubulin heterodimers, in a way that apparently does not interfere with kinesin binding, in good agreement with previous reports. Complementary biochemical characterisation revealed tubulin C-terminal tails are not required for the DCX-tubulin interaction.
These results provide insight into the complex interplay between DCX-like MAPs, tubulin and kinesin in neurons.