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

Abstract

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 β-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 β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-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 β-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.

Asymmetric requirement for α-tubulin over β-tubulin

“…Knocking out the α-tubulin gene TUBA1A causes severe brain malformation and is perinatal lethal, but knocking out any of several β-tubulin genes results in milder malformations or subsequent axon repair defects (Bittermann et al 2019; Latremoliere et al 2018). Missense mutations in any of these tubulin isotypes are linked to severe brain malformations in humans, demonstrating the important roles of these genes in supplying functional tubulin (Bahi-Buisson and Cavallin 1993; Romaniello et al 2018; Park et al 2021). That the full knockouts of β-tubulin genes are less severe than knocking out α-tubulin TUBA1A indicates a stronger requirement for α-tubulin gene copy number than β-tubulin gene copy number, reminiscent of our findings in the single celled yeast.…”

“…Isotypes might serve as transcriptional modules for cells and organisms to meet a tubulin demand, with tissue specific function and expression (Raff, 1984; Kemphues et al, 1982; Latremoliere et al, 2018; Dumontet et al, 1996). Humans have 8-10 α and 7-9 β-tubulin isotypes, and these are expressed at different levels according to cell type and developmental stage (Findeisen et al, 2014; Leandro- García et al, 2010; Park et al, 2021). The expansion of the number of α- and β-tubulin genes provides a variety of transcriptional modules for creating programs of tubulin expression in specific cellular contexts.…”

Emerging cellular themes in leukodystrophies

“…H-ABC is caused by mutations in the TUBB4A gene which codes for β-tubulin, a major structural protein that dimerizes with ⍺-tubulin to form heterodimers that are the building blocks of the microtubule cytoskeleton. Compared to other β-tubulin genes, TUBB4A is highly expressed by oligodendrocytes and its expression increases postnatally, consistent with a role in myelination (Zhang et al, 2014;Park et al, 2021). In oligodendrocytes, microtubules play important roles in building the structure of oligodendrocyte branches and myelin sheaths (Fu et al, 2019) as well as in transporting mRNA and other cargos (Carson et al, 1997;Herbert et al, 2017).…”

Understanding molecular mechanisms and predicting phenotypic effects of pathogenic tubulin mutations

“…For instance, TUBβ3 mutations that alter the charged surface of the microtubule prevent molecular motors from binding and thus have profound impacts on cellular transport [ 33 ]. Changes in microtubule properties could also induce a gain of function, as has been proposed for the pathogenic T178M variant in TUBβ2A and TUBβ3, which has been reported to make microtubules more stable and cause altered microtubule growth dynamics [ 34 ].…”