TUBA1A tubulinopathy mutants disrupt neuron morphogenesis and override XMAP215/Stu2 regulation of microtubule dynamics
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Evaluation Summary:
Tubulin mutations underlie a number of neurodevelopmental diseases, but their effect on microtubule function remains largely unknown. Using a combination of approaches and model systems, the authors provide evidence that the disease-associated alpha-tubulin mutations V409A and V409I may perturb microtubule polymerization. In addition, the mutations in alpha tubulin directly impact Tumor Overexpressed Gene domain proteins from recruiting tubulins to regulate microtubule dynamics. This study demonstrates a link between regulators of microtubule dynamics and disease pathogenesis. It will be of interest to the cytoskeleton and neurobiology fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Heterozygous, missense mutations in α- or β-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 (tumor overexpressed gene) 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.
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Evaluation Summary:
Tubulin mutations underlie a number of neurodevelopmental diseases, but their effect on microtubule function remains largely unknown. Using a combination of approaches and model systems, the authors provide evidence that the disease-associated alpha-tubulin mutations V409A and V409I may perturb microtubule polymerization. In addition, the mutations in alpha tubulin directly impact Tumor Overexpressed Gene domain proteins from recruiting tubulins to regulate microtubule dynamics. This study demonstrates a link between regulators of microtubule dynamics and disease pathogenesis. It will be of interest to the cytoskeleton and neurobiology fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
Tubulin mutations underlie a number of neurodevelopmental diseases, but their effect on microtubule function remains largely unknown. Using a combination of approaches and model systems, the authors provide evidence that the disease-associated alpha-tubulin mutations V409A and V409I may perturb microtubule polymerization. In addition, the mutations in alpha tubulin directly impact Tumor Overexpressed Gene domain proteins from recruiting tubulins to regulate microtubule dynamics. This study demonstrates a link between regulators of microtubule dynamics and disease pathogenesis. It will be of interest to the cytoskeleton and neurobiology fields.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Two different mutations in a single alpha-tubulin residue, V409, are found in patients who have cortical malformations caused by abnormal neuronal migration. In this manuscript Hoff and colleagues set out to determine the effect of these disease-associated mutations on microtubules. The authors first determine that V409A and V409I disrupt the migration of cortical neurons in mouse brains and alter the neurite growth of cultured cortical neurons, consistent with the V409 mutations being associated with neuronal migration disorders in humans. The authors then delve into the effect of the mutations on tubulin and microtubules, and here the authors to turn to a yeast model (human V409 is equivalent to yeast V410). In yeast, V410 mutant tubulin results in microtubules that polymerize faster and have fewer …
Reviewer #1 (Public Review):
Two different mutations in a single alpha-tubulin residue, V409, are found in patients who have cortical malformations caused by abnormal neuronal migration. In this manuscript Hoff and colleagues set out to determine the effect of these disease-associated mutations on microtubules. The authors first determine that V409A and V409I disrupt the migration of cortical neurons in mouse brains and alter the neurite growth of cultured cortical neurons, consistent with the V409 mutations being associated with neuronal migration disorders in humans. The authors then delve into the effect of the mutations on tubulin and microtubules, and here the authors to turn to a yeast model (human V409 is equivalent to yeast V410). In yeast, V410 mutant tubulin results in microtubules that polymerize faster and have fewer catastrophes. Based on decreased affinity between the V410 mutants and the microtubule polymerase Stu2 (the yeast XMAP215 ortholog), the authors ultimately suggest a model in which the disease-associated V409 mutations enhance microtubule polymerization by altering tubulin dimer conformation.
Overall, the authors do a nice job of probing the effects of the disease-associated V409/V410 mutations on neuronal morphogenesis and microtubule behavior. However, a major concern is that is unclear how the defects in microtubule behavior uncovered in yeast relate to microtubule function in neurons and how these perturbations might then disrupt neuronal morphogenesis. The strength of the manuscript is the characterization of the tubulin mutations in yeast; the mouse cortical neuron experiments are nice to include but are predominantly descriptive and have some weaknesses (not all the conclusions drawn from the cortical neuron experiments are supported by the authors' data). Also, while consistent with their data, the authors' model that the V409/V410 mutations disrupt tubulin conformation seems somewhat speculative.
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Reviewer #2 (Public Review):
The authors study tubulinopathy causing alpha tubulin mutations which impact microtubule cytoskeleton building block – the alpha/beta tubulin – and lead to human to neuronal and developmental defects. The authors study the mechanism of Valine 409 to alanine or isoleucine in alpha tubulin. Their studies reveal a major impact of these mutant alpha tubulins on neuronal development and migration. Studies of this conserved alpha tubulin valine in budding yeast (410 in the yeast) reveal an enhancement in polymerization and depolymerization. Most critically the authors find that the mutations interfere with ability of Tumor Overexpressed Gene (TOG) domain regulators from binding these mutant tubulin. The work reveals for the first time a mechanistic link between ability of TOG domain Regulators of microtubule …
Reviewer #2 (Public Review):
The authors study tubulinopathy causing alpha tubulin mutations which impact microtubule cytoskeleton building block – the alpha/beta tubulin – and lead to human to neuronal and developmental defects. The authors study the mechanism of Valine 409 to alanine or isoleucine in alpha tubulin. Their studies reveal a major impact of these mutant alpha tubulins on neuronal development and migration. Studies of this conserved alpha tubulin valine in budding yeast (410 in the yeast) reveal an enhancement in polymerization and depolymerization. Most critically the authors find that the mutations interfere with ability of Tumor Overexpressed Gene (TOG) domain regulators from binding these mutant tubulin. The work reveals for the first time a mechanistic link between ability of TOG domain Regulators of microtubule dynamics and developmental defects caused by tublinopathy.
Microtubule polymerization dynamics are essential for cell development and cell division. The alpha/beta tubulin dimer is the conserved microtubule building block. A range of mutations, termed tubulinopathies, are found in human alpha and beta tubulin genes and result in range of defects in microtubule polymerization, their regulation or their capacity to be used as tracks for microtubule-based motor proteins. Understanding the mechanism for tublinopathies has been hampered by the lack of molecular understanding in the mechanism of these mutations within tubulins and how they impact alpha/beta tubulin capacity to polymerize into microtubules, or be regulated by conserved proteins.
The authors focus on two related tubulinopathy mutations in alpha tubulin, where valine 409 is mutated to Isoleucine (V409I) or to alanine (V409A), with the alanine mutations being the more severe. The authors study the mutants and wild type alpha tubulins in neuronal cells showing how they impact neuronal migration and neurite initiation, revealing the V409A severely impacts neuronal migration compared to V409I and wild type tubulin. This correlates with side neurite initiation, which is not well studied property even in neuronal cultures.
The authors then turn to budding yeast and introduce these mutations in the budding yeast tub1 and tub3 and show that V409I and V409A increase both the polymerization rate and depolymerization rates with alanine mutant being more severe. The authors correlate the location of a mutant with impact on Tumor Over-expressed gene (TOG) domain binding. These domains interact with alpha-beta tubulins in the curved conformation and their binding sites, are suggested to weaken upon tubulin straightening when incorporating into microtubules. The authors show that budding yeast Stu2p TOG domain arrays recruit wild type tubulin from yeast extracts in vitro, in contrast to the V409A and V409I which do not bind and become enriched with TOG domains. The authors postulate that the defects in polymerization are likely related inhibiting yeast TOG protein Stu2p binding . Stu2 plus-end tracking is significantly decreased in the V409A and less so in the V409I compared to wild type. When stu2p expression is decreased in living yeast cells, the authors observe no impact on microtubule dynamics in the mutants. In vitro studies of purified tubulins reconstitute these alpha/beta tubulin mutants in vitro showing that they somewhat impact tubulin polymerization. Although pure tubulin mutant studies were not successful in the case of the V409A , 1:1 ratio mixing with wild type tubulin lead strong enhancement of polymerization and depolymerization rates.
The authors conclude: the two mutants impact microtubule dynamics in two ways :
- Impacting the ability of TOG array proteins from binding microtubules through defects in the alpha tubulin TOG interaction interface. the mutants disable the TOG domain alpha/beta tubulin interaction interface in different degrees of severity with the alanine mutant being more severe.
- Impact on the curved conformation of alpha/beta tubulin and its ability to form a straight conformation. The authors suggest the alpha-tubulin mutations enhance the straight conformation enhancing the microtubule stabilizing state of these proteins.
Assessment:
Overall, the manuscript presents very important data to exploring the mechanism of tubulinopathy causing mutations and their impact on microtubule dynamics. The approach presented fairly thorough and the effort undertaken to understand these mutations spans multiple systems with multiple levels of analysis. However, the work lacks a structural presentation of the mutants and structural models for potential functional effects. These models are very valuable to making careful interpretation for the mechanism of the mutation. The one of the authors conclusions regarding the enhanced straightening of the tubulin mutants is likely not well supported by the data.Major concern:
The main concern is the interpretation for the V409A and V409I mutant mechanism relating to straightening the tubulin dimer. Although the authors present the location of the mutation near the alpha-beta tubulin interface, they do not present structural view of this mutant. Valine 409 in alpha tubulin resides at H11' region of C-term domain of alpha tubulin. Although this region is near the alpha/beta tubulin interface, V409 extending towards the solvent exposed surface. I compared the position of Valine 409 in both the straight and curved tubulin conformations. Thus, generally the impact of this mutant would be predicted to affect alpha/beta-tubulin binding to regulators such as TOG array proteins. This mutant is not likely to impact alpha-beta tubulin conformation. The authors make a claim that the enhancement in tubulin polymerization/depolymerization observed with V409A and V409I is related to its conformation. However there is another interpretation. The inability of the soluble pool of the V409A and V409I mutants to bind TOG proteins, increases the effective polymerizing concentration for microtubule dynamics. So the observed enhancement in microtubule polymerization maybe related to the dissociation of V409A or V409I from TOG proteins in the cytoplasm, which may substantially increase of soluble tubulin concentration available for polymerization within cells. These mutants may lead to a higher effective tubulin concentration resulting in higher polymerization rates. The in vitro polymerization experiments do not provide convincing supporting the enhanced dynamics of the mutants, in particular for V409A, due to the need to mix with wild type tubulin.
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Reviewer #3 (Public Review):
In this article Hoff et al. address the molecular, cellular and developmental consequences of two mutations in 𝛼-tubulin that in human cause malformations of cortical development (lissencephaly and pachygyria). In a first part, the authors show that overexpression of mutated 𝛼-tubulin alters neuronal positioning in the developing neocortex and affects neuronal morphology and microtubule acetylation in culture. In a second part, they endogenously insert these mutations in yeast and demonstrate altered microtubule dynamics, including faster polymerization and reduced catastrophe. They show that this effect also occurs in vitro, independently of any microtubule regulator and is therefore due to intrinsic effects on tubulin polymerization. Finally, they show that these mutations are able to rescue microtubule …
Reviewer #3 (Public Review):
In this article Hoff et al. address the molecular, cellular and developmental consequences of two mutations in 𝛼-tubulin that in human cause malformations of cortical development (lissencephaly and pachygyria). In a first part, the authors show that overexpression of mutated 𝛼-tubulin alters neuronal positioning in the developing neocortex and affects neuronal morphology and microtubule acetylation in culture. In a second part, they endogenously insert these mutations in yeast and demonstrate altered microtubule dynamics, including faster polymerization and reduced catastrophe. They show that this effect also occurs in vitro, independently of any microtubule regulator and is therefore due to intrinsic effects on tubulin polymerization. Finally, they show that these mutations are able to rescue microtubule defects in Stu2/XMAP215 loss of function, confirming that these tubulin mutants stimulate microtubule polymerization.
The conclusions of the yeast experiments are novel and well supported by the data. Some aspects of the neuronal experiments are less convincing and would require additional experiments and clarifications:
1. The link between the neuronal positioning defects observed in vivo in figure 1 and the mild branching phenotype observed in figure 2 is not clear. The authors discuss branching defects as a cause of altered multipolar to bipolar transition but this is far from established. Moreover, the authors do not show here if the defect is indeed due to a block at the multipolar stage or to a bone fide neuronal migration phenotype.
2. The neuronal positioning defects are documented in figure 1 and reveal reduced fraction of cells is the fourth quartile. The representative image is however not convincing for V9409I, and no statistical test is provided. The mild defects may suggest a delay in migration, and the same analysis one day earlier (3 days post-electroporation) would be informative.
3. Figure 2C shows that in mutant tubulin-expressing neurons, neurites resist better to cold-induced retraction. The authors first present this as an assay to probe for neurite retraction defects, which may be a cause of the neurite branching phenotype. It is however not clear whether this cold treatment is a proper assay to test this. Live imaging of neurite growth and shrinkage at 37oC would be a much more convincing assay. The alternative interpretation of this experiment, which the authors make later on in the manuscript, is more convincing: that this is a readout for increased microtubule stability. The experiment is not quantified.
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