The molecular biology of tubulinopathies: Understanding the impact of variants on tubulin structure and microtubule regulation

Abstract

Heterozygous, missense mutations in both α- and β-tubulin genes have been linked to an array of neurodevelopment disorders, commonly referred to as "tubulinopathies." To date, tubulinopathy mutations have been identified in three β-tubulin isotypes and one α-tubulin isotype. These mutations occur throughout the different genetic domains and protein structures of these tubulin isotypes, and the field is working to address how this molecular-level diversity results in different cellular and tissue-level pathologies. Studies from many groups have focused on elucidating the consequences of individual mutations; however, the field lacks comprehensive models for the molecular etiology of different types of tubulinopathies, presenting a major gap in diagnosis and treatment. This review highlights recent advances in understanding tubulin structural dynamics, the roles microtubule-associated proteins (MAPs) play in microtubule regulation, and how these are inextricably linked. We emphasize the value of investigating interactions between tubulin structures, microtubules, and MAPs to understand and predict the impact of tubulinopathy mutations at the cell and tissue levels. Microtubule regulation is multifaceted and provides a complex set of controls for generating a functional cytoskeleton at the right place and right time during neurodevelopment. Understanding how tubulinopathy mutations disrupt distinct subsets of those controls, and how that ultimately disrupts neurodevelopment, will be important for establishing mechanistic themes among tubulinopathies that may lead to insights in other neurodevelopment disorders and normal neurodevelopment.

Keywords: cytoskeleton; microtubule; neurodevelopment; protein structure; tubulinopathy.

FIGURE 1

Microtubule dynamics are regulated at multiple control points. (A) α-tubulin is human TUBA1A in magenta and β-tubulin is human TUBB3 is in cyan (PDB structure: 5JCO). Regions that participate in longitudinal and lateral interactions are described along the sides of the structure. α- and β-tubulin each bind one GTP molecule. The non-exchangeable “N-site” on α-tubulin sits at the intradimer surface and the GTP is not hydrolyzed. The exchangeable “E-site” on β-tubulin is at the interdimer interface and the GTP molecule is hydrolyzed upon incorporation of the heterodimer into the microtubule lattice. (B) Lateral interactions occur between neighboring protofilaments and are illustrated by the dotted lines. Longitudinal interactions occur between at the interdimer interface between two stacked heterodimers. (C) Microtubule-associated proteins (MAPs) and motors can have specific preferences for associating with different segments of microtubules. For example, XMAP215 (gray) associates at microtubule plus ends and kinesins (purple) walk along the lattice toward the plus end. Tubulin undergoes a series of conformational changes. Free, curved heterodimer subsequently straightens as it is assembled into microtubule lattice.

FIGURE 2

Depiction of the development of the cerebral cortex. (A) Neural progenitor cells (blue) undergo symmetric and asymmetric divisions before differentiating into specific cell populations, such as neurons (orange). (B) Neurons radially migrate along glial cells from the ventricular zone (VZ) to the cortical plate (CP). Neurons are bipolar in the VZ before switching to a multipolar state in the subventricular zone (SVZ). To continue radial migration, neurons then revert back to a bipolar state through the intermediate zone (IZ) until they reach the developing CP. (C) Upon reaching the CP, neurons undergo further morphogenesis, including maturation of the axon and extension of multiple dendrites.

FIGURE 3

Mapping TUBA1A tubulinopathy mutations. (A) Mapping individual mutants described in section “Investigating specific residues affected by mutations in patients; can they tell us more about the etiology of tubulinopathies?” on TUBA1A (PDB structure: 5JCO). α-tubulin is human TUBA1A in magenta and β-tubulin is human TUBB3 in cyan. Spheres represent TUBA1A residues S140 (green), R264 (blue), R402 (orange), and V409 (gray). Four views of the structure are shown, each rotated 90° along the longitudinal axis. The 0° angle represents the outer surface of the microtubule that is considered the MAP binding region. 90° and 270° angles represent lateral interfaces with adjacent protofilaments. 180° represents the luminal side of the microtubule. (B) Known TUBA1A mutations to date mapped on primary sequence according to predominant cortical malformation. If no clinical imaging data is available to us, the mutation is highlighted in red. Functional regions described in section “Mapping the tubulinopathy mutations” are labeled in various colors above the secondary structures.

FIGURE 4

Quantifying TUBA1A mutations by type of amino acid side chain change. TUBA1A mutations were sorted by the type of amino acid change that occurs in patients (loss of charge or charge swap, gain of charge, gain or loss of hydrophobicity, or no change in charge or hydrophobicity). TUBA1A mutations were also sorted by the primary cortical malformation resulting from each mutation (lissencephaly, microlissencephaly, pachygyria, or polymicrogyria). For each cortical malformation, the number of mutations that qualify as one (or multiple) of these types of amino acid changes were reported.

FIGURE 5

Quantifying TUBA1A mutations by secondary structure. (A) TUBA1A mutations were sorted by the secondary structural element (helix, loop, or sheet) in which they reside. Observed and expected number of mutations were compared using a Chi-squared test [5.99 critical value when α = 0.05; X² (df = 2, N = 119) = 1.202; p = 0.55]. The expected number of mutations in each secondary structure was calculated by determining the percentage of amino acids that reside in each structure, then multiplying that by 119 (the number of TUBA1A missense mutations known to date). This value represents the number of mutations that would be expected to appear in each structure if all 119 mutations were randomly distributed. (B) TUBA1A mutations were sorted by the primary cortical malformation resulting from each mutation (lissencephaly, microlissencephaly, pachygyria, or polymicrogyria). For each cortical malformation, the number of mutations in each secondary structural element was reported. A Fisher’s exact test was run for each cortical malformation category to determine if mutations were enriched in one secondary structure over the others. Asterisk (*) on bar indicates p-value < 0.05 (polymicrogyria helices p = 0.03).

FIGURE 6

Quantifying TUBA1A mutations by functional region. (A) TUBA1A mutations were sorted by the functional domains (longitudinal, lateral, MAP binding, GTP binding, lumen, intradimer, or other) in which they reside. Observed and expected number of mutations were compared using a Chi-squared test [12.59 critical value when α = 0.05; X² (df = 6, N = 119) = 5.177; p = 0.5]2). The expected number of mutations in each functional domain was calculated by determining the percentage of amino acids that reside in each domain, then multiplying that by 119 (the number of TUBA1A missense mutations known to date). This value represents the number of mutations that would be expected to appear in each domain if all 119 mutations were randomly distributed. (B) TUBA1A mutations were sorted by the primary cortical malformation resulting from each mutation (lissencephaly, microlissencephaly, pachygyria, or polymicrogyria). For each cortical malformation, the number of mutations in each functional domain was reported. A Fisher’s exact test was run for each cortical malformation category to determine if mutations were enriched in one functional domain over the others. Asterisk (*) on bar indicates p-value < 0.05 (pachygyria MAP binding p < 0.01).