Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different beta-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in beta-tubulins and located in the exchangeable GTP-binding pocket of beta-tubulin. To determine the impact of T178M on beta-tubulin function we created an analogous mutation in the beta-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of beta-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin's GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Automated summary

Mutations in beta-tubulin genes TUBB2A or TUBB3, specifically the T178M missense mutation, were found in two patients with brain malformations characteristic of tubulinopathies. The T178M mutation results in kinetically stabilized microtubules with decreased intrinsic assembly activity and fewer transitions to disassembly. This disruption of GTPase-dependent conformational changes in tubulin provides a mechanistic explanation for the kinetic stabilization observed.