Fluorescent protein tagging of C. elegans core apoptosis pathway components reveals mitochondrial localization of CED-9 Bcl-2, CED-4 Apaf1 and CED-3 Caspase in non-apoptotic and apoptotic cells

We used CRISPR-Cas-mediated modification of the genomic loci for C. elegans genes ced-9 Bcl-2, ced-4 Apaf1 and ced-3 Caspase to add the coding sequence for the mNeonGreen (mNG) fluorescent protein to the endogenous open reading frames. In each case, the addition of mNG caused little or no apparent alteration of gene function. We found that tagged versions of CED-9, CED-4 and CED-3 proteins colocalize with mitochondria in all cells of live mid-late stage embryos and are distributed along the entire length of mitochondria. However, CED-4 also exhibits localized puncta of ~4-fold enrichment, and these are preferentially oriented toward the nucleus. We do not observe any shift in the localization pattern of tagged CED-4 in cells that are committing to apoptosis during normal development. However, when egl-1 BH3-only is overexpressed or ced-9 removed by mutation, CED-4::mNG is no longer distributed along the entire length of mitochondria and instead becomes enriched in the bright puncta. Finally, localization of CED-3::mNG to mitochondria is independent of both CED-9 and CED-4. This study represents the first analysis of the distribution and sub-cellular localization of endogenous CED-9 Bcl-2, CED-4 Apaf1 and CED-3 Caspase proteins in live embryos. Our results impact the current model of apoptosis commitment in C. elegans.