The microtubule-associated protein Doublecortin restricts neurite branching through tubulin modifications

Microtubules exist in expanded and compacted states, as defined by the lattice spacing of α & β-tubulin dimers. Changes in lattice spacing has been linked to factors such as GTP-hydrolysis, the binding of microtubule-associated proteins (MAPs), the tubulin code, and microtubule bending. These diverse factors exert opposing molecular driving forces on the microtubule lattice that push lattice spacing towards expanded or compacted states. Doublecortin is a neuronal MAP that regulates microtubule structure in neurons and binds compacted lattice in model cells. Mutations in Doublecortin cause lissencephaly and subcortical band heterotopia by impairing neuronal migration. Using a combination of reconstitution assays, and cell-based systems, we studied DCX’s properties to understand how it contributes to neuronal branching. 

First, we used CRISPR/Cas9 to knock-out the Doublecortin gene in induced pluripotent stem cells and differentiate the cells into cortical neurons. DCX-KO neurons showed reduced velocities of nuclear movements and an increased number of neurites early in neuronal development, consistent with previous findings. EB comet dynamics were unchanged in DCX-KO neurons; however, we observed a significant reduction in α-tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching are rescued by expression of Doublecortin or of TTLL11, an α-tubulin glutamylase. Using U2OS cells as an orthogonal model system, we show that DCX and TTLL11 act synergistically to promote polyglutamylation. 

Second, we observed the behavior of DCX when in presence of paclitaxel, a lattice spacing expander. Using an in vitro reconstitution approach, we showed that paclitaxel, and others stabilizing drugs expand microtubules. In cells, high concentrations of paclitaxel caused DCX to relocalize to compacted lattices found at concave bends. When the concentration of DCX is increased, however, we find that DCX re-compacts the previously expanded microtubules in vitro. Consistently, high expression levels of DCX prevented its relocalization in paclitaxel-treated cells. When the competition between paclitaxel and DCX is ″balanced″, we observed a complex phenotype: DCX simultaneously localized to both long, straight clusters and concave bends, while other regions on the microtubule network remained DCX-free. 

Finally, we propose that Doublecortin acts as a positive regulator of α-tubulin polyglutamylation, restricting neurite branching by cooperatively modifying tubulin conformations. Our results indicate an unexpected role for Doublecortin in the homeostasis of the tubulin code. 

Muriel Sébastien est invitée par Isabelle Marty.