Mutation within the human ALS2 gene, which encodes the protein Alsin, causes a number of recessive motor neuron diseases. The ALS2 gene encodes a 180kDa protein, which has been shown to localize to early endosomes. The Alsin protein comprises three predicted guanine exchange factor (GEF) domains, the best characterised of which is the VPS9 domain for Rab5 GTPase, which is involved in the endocytosis membrane trafficking pathway, particularly in the docking and fusion of early endosomes. Furthermore, Alsin contains a Rho-GEF domain which specifically interacts with Rac-1 GTPase in the PI3K/AKT signal transduction pathway. This pathway has been implicated in numerous biological processes, including control of protein translation, via the mTOR branch of the pathway. To date, most work on the human ALS2 disease phenotypes has focused on the role of alsin in membrane trafficking, and neglected alsin's potential role in signalling via its Rho-GEF domain. The focus of this project was to study the role of alsin in signalling during early Xenopus development, a period rich in well-characterised cell-cell signalling.I have shown that alsin is maternally loaded and zygotically expressed in the early Xenopus embryo. In cell culture, alsin is localised to early endosomes¬. Knockdown of alsin protein through the use of mopholinos (MO), resulted in a gastrulation defect, in particular, failure to close the blastopore caused by disrupted mesoderm induction and convergent extension movements. An animal cap assay was used to study mesoderm induction in the presence of als2-MO and activin protein, a potent mesoderm inducer. These animal caps extended normally, indicating proper mesoderm induction. By contrast, als2-MO animal caps failed to extend when co-injected with activin mRNA suggesting that alsin is important for the production and/or secretion of the activin ligand in the source cell. Subsequently it was determined that knockdown of alsin reduced the precursor protein levels of TGF-β family members activin and Xnr-2. These results suggest a novel role for alsin in mRNA stability, translational regulation or post-translational control of specific mesoderm-inducer mRNAs.
|Date of Award||1 Aug 2012|
- The University of Manchester
|Supervisor||Nancy Papalopulu (Supervisor)|
- mRNA stabilty
- post-translational control