Tubulinopathy mutations in TUBA1A that disrupt neuronal morphogenesis and migration override XMAP215/Stu2 regulation of microtubule dynamics
- University of Colorado Anschutz Medical Campus, United States
- University of Colorado School of Medicine, United States
Abstract
Heterozygous, missense mutations in a- or b-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided.
Article and author information
Author details
Funding
National Institutes of Health (Graduate Student Fellowship,T32GM136444)
- Katelyn J Hoff
National Institutes of Health (R35GM136253)
- Jeffrey K Moore
National Institutes of Health (UL1TR002535)
- Santos J Franco
Boettcher Foundation (Boettcher Webb-Waring Biomedical Research Program Award)
- Santos J Franco
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animals were housed and maintained according to protocols (#00019) approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus (PHS Animal Assurance of Compliance #D16-00171). All surgeries were performed under inhaled vaporized isoflurane anesthesia with pre- and post-operative analgesic (meloxicam). Every effort was made to minimize pain and suffering.
Copyright
© 2022, Hoff et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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