We have recently identified the promoter region of
arf-3, a small GTPase implicated in intracellular trafficking, as the tissue-specific driver of the seam cell marker scm::gfp (identified in an enhancer trap screen) commonly used as a fate marker of seam cells in the study of seam cell development. RNAi of
arf-3 leads to variable seam cell numbers suggesting a possible failure in the regulation of seam cell divisions. Furthermore, a deletion mutant of
arf-3,
tm1877, causes embryonic lethality, with escapees dying as larvae with seam abnormalities. The deletion phenotype is difficult to interpret however, because the deletion removes an upstream ncRNA in addition to
arf-3, thus we are currently producing a clean knockout strain by Cas9 mediated deletion of the
arf-3 coding region. Although the
arf-3 enhancer in the context of the scm::gfp marker is expressed exclusively in seam nuclei (due to the presence of a NLS) we found that a translational reporter of
arf-3 is cytoplasmic with ARF-3::GFP/mCherry localised to distinct puncta within the seam cells. We have found that ARF-3::mCherry colocalises with RAB-7::GFP in the seam, suggesting a role for
arf-3 in regulating late endosomes. The seam cells are known to play an important secretory function, giving rise to the cuticle and alae but the cell biology of this is very poorly understood, and we are interested in the potential role of
arf-3 in this process. To identify potential interactors of
arf-3 we performed a targeted RNAi screen to look for mislocalisation of the ARF-3::mCherry translational reporter upon the silencing of genes involved in intracellular trafficking and signalling. We found that knockdown of
apr-1, an orthologue of human APC (a component of the beta -catenin destruction complex) altered the localisation of ARF-3::mCherry. Simultaneous knockdown of
arf-3 and
apr-1 exacerbated the seam cell phenotype and also caused other morphological defects not observed in the single knockdowns. Given the involvement of
apr-1 in Wnt signalling in C. elegans, this may suggest an interaction between
arf-3 and the Wnt pathway. Preliminary evidence suggests that knockdown of
arf-3 in the hypomorphic
apr-1 allele,
bp298, leads to sterility and synthetic lethality with escapees phenocopying the lethal
arf-3(
tm1877) allele. This suggests that
arf-3 may play a role in regulating embryonic as well as seam cell Wnt signalling and supports an important role for intracellular signalling in regulating these processes.