Figure 1. mNeonGreen (mNG) knock-in site, phenotypes, and localization of mNG:LET-60/Ras:(A) Exon-intron gene structure of
let-60/Ras. Exons are shown in blue and 3'UTR is indicated in grey. The position of the endogenous mNG tag and Cas9 guide RNA sequence used to generate the mNG-tagged
let-60(
qy220) allele are highlighted. The
let-60(
n2021) reduction-of-function allele harbors a point mutation in the first exon that causes the substitution of glycine to serine at codon 75. The C-terminus of LET-60 contains a CAAX motif that is the site of post-translational addition of a farnesyl isoprenoid. (B) Viability of L1 larvae with the genotypes listed. The percentage indicates the proportion of living L2 animals. n.s. (not significant), p > 0.05; Fisher's exact test. (C) Assessment of vulval induction at the L3 larval stage in the listed genotypes (left). The average number of vulval precursor cells (VPCs) that adopt vulval fates in animals examined (middle) and percentage of animals with incomplete vulval induction (right) are shown. The p value was calculated with a Fisher's exact test. (D) Fluorescence images of mNG::LET-60 localization during vulval induction. The corresponding brightfield images indicate the uterine anchor cell (white arrowheads) and the central vulval precursor cell P6.p and its descendants (brackets). Yellow arrows indicate nuclear mNG::LET-60 signal within the anchor cell. (E) Left, mNG::LET-60 localization in VPCs. The white arrowhead and yellow arrow denote the anchor cell in the brightfield and fluorescence images, respectively. Right, boxplot of mean mNG::LET-60 fluorescence intensity in the 1°, 2°, and 3° VPCs (n > 12 for each group). **p < 0.01, n.s. (not significant), p > 0.05; Kruskal-Wallis H test with post hoc Dunn's test. Box edges represent the 25th and 75th percentiles, the line in the box denotes the median value, and whiskers mark the minimum and maximum values. A.U. (arbitrary units). (F) Intracellular and nuclear enrichment of mNG::LET-60 in sex myoblast cells (yellow arrowheads). (G) Prominent mNG::LET-60 localization to the nucleus in intestinal cells at the L3 larval stage (yellow arrow, n = 20/20 animals examined). Note that nucleolar mNG::LET-60 signal was observed in some intestinal cells (black arrow, n = 5/20 animals examined). (H) Intracellular enrichment of mNG::LET-60 at the anterior and posterior regions of the pharynx (arrowheads). (I) mNG::LET-60 is present at high levels intracellularly in the adult spermatheca (arrowhead). (J) Top, prominent localization of mNG::LET-60 in neurons associated with the nerve ring (arrowheads). Bottom, nerve ring region visualized with the pan-neuronal marker
unc-119p::YFP (Calixto et al., 2010). (K) mNG::LET-60 localizes prominently to the plasma membranes of germ cells (cyan arrow) and is abundant in the gonadal sheath cell (yellow arrowhead). (L) mNG::LET-60 localizes to the body wall muscle cell membranes (arrows). (M) mNG::LET-60 localizes to seam cell membranes in larvae (arrows). All fluorescence images shown are representative of the respective developmental stages and tissues (n > 10 animals examined for each stage). Scale bars, 10 µm.