Developmentally Regulated Cleavage and Localization of MYRF-1.

(A) Domain structure and cleavage of endogenously tagged MYRF-1. GFP was inserted at Ala172 (ybq14 allele) or mEGFP at Gly651 (syb3705 allele). Self-cleavage is mediated by the trimerized ICA domain, with key catalytic residues S483 and K488, producing an N-terminal fragment (1–P482). (B) GFP::MYRF-1(ybq14) localization during L1–L2 transition correlated with seam cell divisions, visualized with Pscm::RFP::PH(ybqSi296). Seam cells categorized by division/fusion state: Pre-1st division; 1st division only; 1st and 2nd divisions; Post-2nd division (prior to fusion); and Fusion/Elongation (elongated daughter cells). Right: quantification of MYRF-1 localization in each category. (C) Stage-specific GFP::MYRF-1(ybq14) localization in L1 larvae. Localization shifts from membrane-only (early L1) to nuclear (mid/late L1), and back to membrane during lethargus. Lethargus was defined by cessation of pharyngeal pumping. Three sagittal sections shown: mid-head (pharynx, head neurons), lateral trunk (epidermis, seam), and mid-trunk (intestine, ventral nerve cord). See (C) for schematic. (D) Schematic of major cell types visible in (B) images. Top: head region (pharynx, neurons); middle: lateral trunk (epidermis, seam); bottom: midline trunk (intestine, VNC). Muscle and gonad not shown. (E) Western blot detection of MYRF-1 cleavage. Protein extracts from early (6 h) and late (14 h) L1 larvae of GFP::MYRF-1(ybq14) and N2 controls. Cleaved N-terminal (81 kDa) band appears only in late L1; full-length (131 kDa) detected in both. (F) Cleavage-defective mutant gfp::myrf-1(syb1487[S483A, K488A]). GFP signal remains membrane-localized throughout L1, with no nuclear enrichment. (G) N-terminal only mutant gfp::myrf-1(syb1491[1–482]). GFP is consistently nuclear, with no membrane signal. (H) C-terminal tagged GFP in gfp::myrf-1(syb3705[GFP::G651]). GFP signal is detected on the membrane throughout L1. (I) Oscillatory MYRF-1 localization from L2 to adulthood. GFP::MYRF-1 shows membrane-localized signal in early stages, nuclear signal in mid/late stages, and membrane-signal again during lethargus. Signal disappears in young adults. Early larval stages were defined as 3 h post-lethargus; late L2, L3, and L4 as 25 h, 35 h, and 44 h, respectively. (J) Quantification of GFP::MYRF-1 subcellular localization. (K) Percentage of nuclear-enriched GFP::MYRF-1 at each stage.

MYRF-1 Cleavage Is Suppressed at Nutrient-Dependent Developmental Checkpoints.

(A) GFP::MYRF-1 localization under starvation initiated at late L1, L2, L3, or L4. In all groups, GFP remains membrane-associated, except in late L4-starved animals, where signal is lost. Right: Quantification of GFP localization. (B) GFP::MYRF-1 localization is altered by nutritional status. In late L1, it is enriched in the nucleus under feeding conditions. After 8 hours of food deprivation starting in late L1, GFP::MYRF-1 becomes localized to the cell membrane, while control animals that continue feeding show high nuclear localization. (C) Representative GFP::MYRF-1 localization and seam cell pattern 24 hours after late L1 food removal. Below: Quantification of seam cell categories from (B); Quantification of GFP::MYRF-1 localization patterns in late L1 starvation assay. (D) IP–MS analysis of MYRF-1 under nutrient-rich and food-deprived conditions using myrf-1::gfp (syb3705). Left: Schematic of the C-terminal GFP fusion at Gly651. Middle: Experimental design for culture and starvation treatment. Right: Mass-spectrometry reads covering residues S620 or S623 that contain identified phosphorylation in well-fed versus food-deprived samples. Only phosphopeptide-containing reads are shown. Bottom table: Percentage of phosphopeptide reads among those covering S620/S623, and the proportion of reads covering these residues relative to total MYRF-1 reads.

The Juxtamembrane Region of MYRF-1 Inhibits Cleavage and Regulates Development

(A) Schematic of gfp::myrf-1(ybq212[ΔJM]) allele, with deletion of the juxtamembrane (JM) region (K601–C650). (B) GFP localization upon overexpression of NLS::GFP::MYRF-1[G477-C’]. GFP remains cytoplasmic with intact JM; nuclear enrichment is observed when the JM region (K601–C650) is deleted. Nuclei marked by his-24::mCherry. (C) Subcellular localization of GFP::MYRF-1[ΔJM]. GFP is detected at both the membrane and nucleus in early-mid L1 (6 h), with increased nuclear accumulation by late L1 (14 h). Right: Quantification of GFP localization in wild-type and myrf-1[ΔJM] animals at early-mid and late L1 stages. (D) Reduced GFP intensity in myrf-1[ΔJM] mutants. Mean signal intensity in the head region ± SEM; ***p < 0.001, t-test. (E) lin-4p::gfp reporter expression in myrf-1[ΔJM] mutants vs. controls. Reporter activated precociously in mutants at early late L1. Right: quantification of GFP intensity (mean ± SEM; ***p < 0.001, t-test). (F) Body length over time in myrf-1[ΔJM] vs. wild-type. Mean ± SD from L1 to young adult (n = 25 per genotype per stage. (G) Total progeny counts (± SEM) of wild-type and myrf-1[ΔJM] at 192 h post-hatching; each data point represents progeny count from an individual animal. (H) Lifespan analysis of myrf-1[ΔJM] vs. wild-type. Kaplan-Meier survival curve monitored from day 1 post-L4 (n ≥ 200 per genotype).

MYRF-1[ΔJM] Partially Bypasses Late-Stage Nutrient Checkpoints

(A) Seam cell divisions (marked by heIs63) 24 h after late L1 starvation. myrf-1[ΔJM] mutants resemble controls, with most seam cells rounded and between the first and second divisions. Right: Quantification of seam cell categories. (B) Sex myoblast division assessed using hlh-8p::gfp at late L2 and 24 h post-food removal. myrf-1[ΔJM] mutants show advanced divisions. Right: quantification at late L2; 8h, 24h and 48h starvation. (C) Vulval development 24 h after late L3 starvation. myrf-1[ΔJM] mutants show adult vulvae and cuticle shedding, while controls remain in early L4. Right: stage quantification based on vulva morphology.

PAN-1 CCT Inhibits MYRF-1 Cleavage and Controls Developmental Progression.

(A) Schematic of the pan-1(ybq225[ΔCCT]) allele with deletion of the cytoplasmic tail (CCT); also shows extracellular interaction between PAN-1 and MYRF-1. (B) GFP::MYRF-1(ybq14) localization in pan-1 mutants: in pan-1(gk142) null mutants MYRF-1 is degraded in the cytoplasm; in pan-1[ΔCCT] mutants GFP::MYRF-1 is strongly nuclear at 6 h and 14 h. Right: quantification of GFP localization. (C) Western blot analysis of MYRF-1 cleavage in pan-1[ΔCCT] mutants; extracts from gfp::myrf-1(ybq14); pan-1[ΔCCT]/mT1 progeny show an 81 kDa cleaved N-terminal MYRF-1 band (∼10% homozygous mutants). (D) C-terminal GFP fusion myrf-1::gfp(syb3705) in pan-1[ΔCCT] shows membrane localization at early L1, indicating intact trafficking. (E) Developmental arrest in pan-1[ΔCCT] mutants: Left: representative animals at 3 days post-hatching showing arrest between L1–L3; Right: body length over time (mean ± SD). (F) lin-4p::gfp expression is prematurely activated in pan-1[ΔCCT] at 6 h and 14 h. Right: quantification (mean ± SEM; ****p < 0.0001, t-test). (G) M-cell lineage progression assessed with hlh-8p::gfp(ayIs6); classification based on division and migration status; dot size reflects population proportion. (H) Endogenous lin-4 reporter (umn84) expression in control, myrf-1(ju1121), pan-1[ΔCCT], and myrf-1; pan-1[ΔCCT] double mutants at 6 h; premature lin-4 activation in pan-1[ΔCCT] is suppressed in the double mutant. Right: Quantification of reporter intensity (mean ± SEM; ***p < 0.001, ****p < 0.0001; t-test).

Disruption of L1 arrest by MYRF-1[ΔJM] and PAN-1[ΔCCT].

(A) Schematic of the L1 diapause assay. Embryos hatched on food-less plates enter diapause and resume development upon refeeding. (B) GFP::MYRF-1 localization after 24 h starvation. Controls show membrane localization; pan-1[ΔCCT] and myrf-1[ΔJM]; pan-1[ΔCCT] mutants show predominant nuclear localization. Right: quantification of GFP::MYRF-1 localization. (C) Larvae at 16 and 48 h starvation. myrf-1[ΔJM]; pan-1[ΔCCT] mutants are longer than controls. Right: body length quantification (mean ± SD). (D) lin-4p::GFP expression at 24 h starvation; it increases in mutants. Right: quantification of reporter intensity at 24, 48, and 72 h starvation (mean ± SEM). (E) M-cell lineage progression in control and pan-1[ΔCCT] mutants at 14, 24, 48, and 72 h starvation; hlh-8p::gfp used for staging. (F) Summary model. Left: MYRF cleavage peaks oscillate during larval stages. Right: cleavage is suppressed by the MYRF JM region and PAN-1 CCT; inhibition is likely relieved by a systemic cue enabling trimerization and self-cleavage.

Cleavage pattern and localization dynamics of MYRF-1

(A) Overexpressed GFP::MYRF-1 in DD neurons localizes to the cytoplasm and overlaps with the Golgi marker manII::mCherry. (B) GFP::MYRF-1(syb1487[S483A, K488A]) shows membrane localization in the trunk (top: epidermis, seam; bottom: intestine, VNC) across L1. Complements head-region images in Figure 1E. (C) GFP::MYRF-1(syb1491[1–482]) shows nuclear localization in trunk across L1. Complements Figure 1F. (D) C-terminal GFP fusion in myrf-1::gfp(syb3705) localizes to the cell membrane in the trunk across L1. Complements Figure 1G. Right: Schematic shows GFP insertion at Gly651.

IP–MS identifies a potential regulatory region in MYRF-1.

(A) Peptide coverage of MYRF-1 across three IP–MS samples. The juxtamembrane (JM) region is highlighted in peach. Each blue line represents an identified peptide. (B) Representative localization of GFP::MYRF-1(ybq215[S623A]) at 6 h and 14 h post-hatching. Right: Quantification of subcellular localization.

Characterization of myrf-1(ybq212[ΔJM]) mutants.

(A) Representative myrf-1[ΔJM] mutants during larval development. Mutants are shorter than controls; adults exhibit bagging. Right: Developmental progression of gfp::myrf-1(ybq14) controls and myrf-1[ΔJM] mutants at 48 h and 72 h post-hatching. (B) Schematic of M-cell lineage: hlh-8p::GFP marks undifferentiated M-cell progeny; expression is lost upon muscle differentiation. (C) hlh-8p::GFP expression in M-cell lineages of control and myrf-1[ΔJM] mutants at 6, 16, and 27 h. Right: quantification of M-cell division stages.

Characterization of pan-1(ybq225[ΔCCT]) mutants.

(A) In pan-1[ΔCCT] mutants, MYRF-1(syb3705[GFP::G651]) remains membrane-localized at 6 h. Sagittal sections show mid-head, lateral trunk, and mid-trunk regions. Complements Figure 5D. (B) Body length measurements of control, myrf-1[ΔJM], pan-1[ΔCCT], and myrf-1[ΔJM]; pan-1[ΔCCT] animals post-hatching (mean ± SD; **p < 0.01; ****p < 0.0001; t-test). (C) Co-labeling of GFP::MYRF-1 (ybq14), gfp::myrf-1[ΔJM], or GFP::MYRF-1 in pan-1[ΔCCT] with endogenous lin-4 reporter (umn84) at 6 h. lin-4 is not activated in wild type, but precociously activated in both mutants, colocalizing with nuclear MYRF-1. (D) hlh-8p::GFP(ayIs6) marking M-cell divisions in control and pan-1[ΔCCT] mutants at 6, 16, and 27 h. (E) In myrf-1[ΔJM]; pan-1[ΔCCT] double mutants, GFP is predominantly nuclear. Right: quantification of GFP localization.

Disruption of L1 diapause by relieving MYRF-1 cleavage inhibition.

(A) Representative pan-1[ΔCCT] animals under early L1 starvation (24 h and 48 h), with M cells labeled by hlh-8p::GFP(ayIs6). M-cell divisions occur during diapause in mutants but not in controls. By 48 h, mutants show 16–18 M-lineage cells with weak hlh-8p::GFP expression. Complements Figure 6E. (B) myrf-1[ΔJM] mutants under early L1 starvation for 24 h and 48 h. M-cell divisions are not advanced. Right: Quantification of M-lineage patterns. (C) Recovery ability after 48 h of early L1 starvation. Animals were either starved post-bleaching followed by 72h recovery or cultured with food continuously. Right: Body length measurements (mean ± SD; >20 animals/group).