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Molecular and cellular mechanisms underlying neural tube defects in the loop-tail mutant mouse
Michel Gravel1, Alexandra Iliescu, Cynthia Horth
1Department of Biochemistry and Complex Traits Program, McGill University, Montreal, Quebec, Canada H3G 0B1.
Abstract:
Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis) caused by mutations in the Vangl2 gene (D255E, S464N). Mammalian Vangl1 and Vangl2 are membrane proteins that play critical roles in development such as establishment of planar cell polarity (PCP) in epithelial layers and convergent extension movements during neurogenesis and cardiogenesis. Vangl proteins are thought to assemble with other PCP proteins (Dvl, Pk) to form membrane-bound PCP signaling complexes that provide polarity information to the cell. In the present study, we show that Vangl1 is expressed exclusively at the plasma membrane of transfected MDCK cells, where it is targeted to the basolateral membrane. Experiments with an inserted exofacial HA epitope indicate that the segment delimited by the predicted transmembrane domains 1 and 2 is exposed to the extracellular milieu. Comparative studies of the Lp-associated pathogenic mutation D255E indicate that the targeting of the mutant variant at the plasma membrane is greatly reduced; the mutant variant is predominantly retained intracellularly in endoplasmic reticulum (ER) vesicles colocalizing with the ER marker calreticulin. In addition, the D255E variant shows drastically reduced stability with a half-life of approximately 2 h, compared to >9 h for its wild type counterpart and is rapidly degraded in a proteasome-dependent and MG132 sensitive pathway. These findings highlight a critical role for D255 for normal folding and processing of Vangl proteins, with highly conservative substitutions not tolerated at that site. Our study provide an experimental framework for the analysis of human VANGL mutations recently identified in familial and sporadic cases of spina bifida.
Insights
Mutations in the Vangl2 gene cause severe neural tube defects in mice. The D255E mutation impairs Vangl1 protein targeting and stability, offering insights into spina bifida causes.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Mammalian Vangl1 and Vangl2 are crucial membrane proteins for embryonic development, regulating planar cell polarity (PCP) and cell movements.
- Vangl proteins are believed to form signaling complexes with other PCP proteins to guide cellular polarity.
- Mutations in Vangl2 cause severe neural tube defects, such as craniorachischisis, in Loop-tail (Lp) mice.
Purpose of the Study:
- To investigate the cellular and molecular mechanisms underlying Vangl protein function and the impact of pathogenic mutations.
- To analyze the localization, stability, and processing of wild-type and mutant Vangl1 proteins.
- To establish a framework for studying human VANGL mutations associated with spina bifida.
Main Methods:
- Transfection of MDCK cells with wild-type and mutant Vangl1 constructs.
- Immunofluorescence microscopy to determine protein localization (plasma membrane, basolateral, ER).
- Analysis of protein stability using half-life measurements and proteasomal degradation assays (MG132 treatment).
Main Results:
- Wild-type Vangl1 localizes to the plasma membrane, specifically the basolateral domain, with an extracellularly exposed N-terminus.
- The pathogenic D255E mutation significantly reduces Vangl1 plasma membrane targeting, leading to retention in endoplasmic reticulum (ER) vesicles.
- The D255E Vangl1 variant exhibits markedly reduced stability (half-life ~2 hours vs. >9 hours for wild-type) and is degraded via a proteasome-dependent pathway.
Conclusions:
- The D255 residue is critical for proper Vangl protein folding, processing, and stability.
- Intracellular retention and rapid degradation of the D255E Vangl1 mutant explain its pathogenic effects.
- This study provides a model for understanding human VANGL mutations in spina bifida and related developmental disorders.
