kri-1 promotes radiation-induced apoptosis in the C. elegans germline by a cell non-autonomous mechanism that functions independently of the
p53-like gene
cep-1 (Ito et al., 2010). KRI-1 is the homologue of human KRIT1, which when mutated leads to the neurovascular disease in humans Cerebral Cavernous Malformations (CCM). To understand how
kri-1 promotes germline apoptosis, I am taking a candidate approach with known apoptosis genes, as well as an unbiased mutagenesis suppressor screen. The C. elegans ERK homologue, MPK-1, is phosphorylated and activated in the germline upon irradiation, and this activation is necessary for promoting both
cep-1-dependent and
cep-1-independent apoptosis (Rutkowski et al., 2011, Eberhard et al. 2013). To determine if
kri-1 regulates the Ras/MAPK pathway, I immunostained germlines for activated p-MPK-1 and observed reduced levels in
kri-1(0) worms compared to wild type controls. To determine if
kri-1 is regulating apoptosis through Ras/MAPK signalling, I ablated GLA-3, an inhibitor of MPK-1 by RNAi and found that apoptosis was restored. Furthermore, apoptosis is restored in
kri-1;
lip-1 double mutant worms. LIP-1 is a germline phosphatase that negatively regulates MPK-1. Since KRI-1 is expressed in the soma, this suggests that
kri-1 is somehow able to non-autonomously activate Ras/MAPK signalling in the germline. In order to identify additional downstream targets of
kri-1 and to determine how
kri-1 is sending signals from the soma to the germline, I performed an EMS mutagenesis suppressor screen. To isolate candidates from many mutagenized ­
kri-1 worms, I first selected for mutations that restore other easy to screen
kri-1 phenotypes. From the candidates that made it past this first round of selection, I have sent fourteen candidates, with restored sensitivity to radiation-induced germline apoptosis, for whole genome sequencing. I am now investigating various genes mutated in my suppressor strains to determine the causal mutations that restore apoptosis. .