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.

L´apoptosi, o mort cel.lular programada, és un procés actiu que mobilitza els recursos cel.lulars amb l´objectiu de mantenir l´homeostasi de l´organisme a expenses del suïcidi de cèl.lules individuals. Diferents estudis han mostrat un increment de l´activiat d´algunes cdk, especialment Cdk1 i Cdk2, en correlació amb la progressió dels primers estadis apoptòtics. En el nostre laboratori l´estudi de l´apoptosi en timòcits, els quals no tenen una activitat cdk significativa degut a l´aturada del cicle cel.lular en G1, demostren que la inducció de l´activitat de Cdk2 després del tractament amb radiació gamma o amb glucocorticoides és necessària per l´inici de l´apoptosi. Mentre cap de les ciclines conegudes sembla ser la proteïna activadora de Cdk2 en apoptosi, en el nostre laboratori hem identificat un nou membre de la família de les ciclines, denominada Ciclina O, capaç d´activar aquesta kinasa in vivo en línies cel.lulars. L´expressió d´aquesta nova ciclina en el timus, i altres teixits, s´indueix ràpidament després del tractament amb radiació gamma i coincideix amb l´aparició de l´apoptosi. Aquests resultats posicionen la Ciclina O com a millor candidat a ser l´activador de Cdk2 necessari per induïr la mort cel.lular programada en el timus, i probablement també en altres òrgans. / The apoptosis, also called programmed cell death, is an active process able to use the cellular mechanisms to kill individual cells in order to keep the functional homeostasis of the whole organism. Different studies had shown a correlation between the first apoptotic events and the induction of some cdk proteins, particularly Cdk1 and Cdk2. The studies of thymocytes in our laboratory, wich lacks the most amount of cdk activity related to the cell cycle because of its arrest in G1, had shown that the induction of Cdk2 activity after the treatment with gamma radiation or glucocorticoids is a necessary step for the apoptosis induction. While any of the cyclins described at the moment seems to be the Cdk2 activator for apoptosis a new member of the cyclin family able to activate the kinase Cdk2 in vivo in cell lines has been identified in our laboratory. The expresion of this cyclin, known as Cyclin O, is quickly induced in the thymus after the treatment with gamma radiation and correlates with the induction of apoptosis. These results position Cyclin O as the best candidate to activate Cdk2 and inuce the programmed cell death in the thymus, and probably other tissues.