Doublecortin reinforces microtubules to promote growth cone advance in soft environments.

Doublecortin (DCX) is a microtubule (MT)-associated protein in immature neurons. DCX is essential for early brain development,¹ and DCX mutations account for nearly a quarter of all cases of lissencephaly-spectrum brain malformations^2,3 that arise from a neuronal migration failure through the developing cortex.⁴ By analyzing pathogenic DCX missense mutations in non-neuronal cells, we show that disruption of MT binding is central to DCX pathology. In human-induced pluripotent stem cell (hiPSC)-derived cortical i³Neurons, genome edited to express DCX-mEmerald from the endogenous locus, DCX-MT interactions polarize very early during neuron morphogenesis. DCX interacts with MTs through two conserved DCX domains^5,6 that bind between protofilaments and adjacent tubulin dimers,⁷ a site that changes conformation during guanosine triphosphate (GTP) hydrolysis.⁸ Consequently and consistent with our previous results,⁵ DCX specifically binds straight growth cone MTs and is excluded from the GTP/guanosine diphosphate (GDP)-inorganic phosphate (Pi) cap recognized by end-binding proteins (EBs). Comparing MT-bound DCX fluorescence to mEmerald-tagged nanocage standards, we measure approximately one hundred DCX molecules per micrometer growth cone MT. DCX is required for i³Neuron growth cone advance in soft microenvironments that mimic the viscoelasticity of brain tissue, and using high-resolution traction force microscopy, we find that growth cones produce comparatively small and transient traction forces. Given our finding that DCX stabilizes MTs in the growth cone periphery by inhibiting MT depolymerization, we propose that DCX contributes to growth cone biomechanics and reinforces the growth cone cytoskeleton to counteract actomyosin-generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated traction may be insufficient for productive growth cone advance.