mNG::LET-60 was present at high levels in VPCs during VPC induction in the L3 larval stage, where it localized predominantly to the intracellular region-cytosol and possibly endomembranes. We also observed that LET-60 levels were significantly higher in the 1° VPCs compared to the 2° and 3° VPCs during VPC induction where LET-60/Ras activity is higher (Shin & Reiner, 2018). Interestingly, mNG::LET-60 Ras was present at low levels in the uterine anchor cell and during early stages of VPC induction was largely found in the anchor cell nucleus. High levels of mNG::LET-60 were observed in the sex myoblast cells in the intracellular region and nucleus during the L3 and L4 larval stages (Figure 1F), where LET-60/Ras regulates sex myoblast migration and differentiation (Sundaram et al., 1996; Sundaram, 2013). High levels of mNG::LET-60 was also observed in tissues where LET-60/Ras is not known to function, such as nuclear and occasional nucleolar localization of mNG::LET-60 in intestinal cells in early larval stages, regionalized intracellular localization in the pharynx, and high levels of intracellular mNG::LET-60 accumulation in the spermatheca. Enriched intracellular mNG::LET-60 was detected in neurons associated with the nerve ring, consistent with
let-60 mRNA expression and LET-60 function in olfactory neurons whose cell bodies and axons are located within the nerve ring (Hirotsu et al., 2000; Menini et al., 2010; Taylor et al., 2021). Finally, plasma membrane localization of mNG::LET-60 was found throughout the germline, in body wall muscle cells, and hypodermal seam cells, which are tissues where LET-60/Ras has known functions (Sundaram, 2013). We noted high levels of intracellular and nuclear mNG::LET-60 in the somatic sheath cells of the gonad, where LET-60 might regulate germline morphogenesis (Lu et al., 2008).