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Doublecortin mutations cluster in evolutionarily conserved functional domains
1Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Mutations in the X-linked gene doublecortin ( DCX ) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.
Insights
Mutations in the doublecortin (DCX) gene disrupt brain development by affecting microtubule interactions. Specific mutations within the conserved Doublecortin (DC) domain impair tubulin binding and microtubule polymerization, leading to developmental disorders.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Mutations in the X-linked doublecortin (DCX) gene cause lissencephaly in males and subcortical laminar heterotopia in females.
- DCX protein is known to interact with and stabilize microtubules, crucial for neuronal development.
- Previous studies identified various mutations, including missense, nonsense, and splice site mutations, throughout the DCX gene.
Purpose of the Study:
- To analyze the evolutionarily conserved Doublecortin (DC) domain within DCX and related proteins.
- To investigate the role of the tandem repeats within the DC domain in microtubule binding and polymerization.
- To determine the functional consequences of specific DCX mutations on the microtubule cytoskeleton.
Main Methods:
- Sequence analysis of DCX and DCX-like proteins across different organisms to identify conserved domains.
- In vitro and in vivo expression of DCX and DCLK repeats to assess tubulin and microtubule interactions.
- Overexpression of seven patient-derived DCX mutations in COS7 cells to observe effects on the microtubule cytoskeleton.
Main Results:
- A conserved Doublecortin (DC) domain, consisting of two tandem 80 amino acid repeats, was identified, primarily in the N-terminus.
- The first repeat of the DC domain binds tubulin and enhances microtubule polymerization.
- Several DCX mutations disrupt the microtubule cytoskeleton, with the Y125H mutation showing the most severe effect both in cells and in vitro.
Conclusions:
- The tandem repeats within the Doublecortin (DC) domain are critical for microtubule binding and polymerization.
- Missense mutations within these conserved repeats disrupt DCX function, impacting brain development.
- Understanding DCX-microtubule interactions is essential for comprehending normal and abnormal brain development.
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