Figure 1.
agef-1(vh4) suppresses the lin-2(e1309) Vul phenotype, has large vesicles in coelomocytes and a dumpy body morphology.
(A–D) Representative Differential Interference Contrast (DIC) images of vulvas of wild-type, lin-2(e1309), agef-1(vh4) and agef-1(vh4); lin-2(e1309) L4 stage larvae. The lin-2(e1309) larva lacks a vulva while agef-1(vh4); lin-2(e1309) has a second vulval invagination and hence is Muv. agef-1(vh4) single mutants have a wild-type vulva. Bar, 10 µm. (E, F) DIC images of coelomocyte pairs (white arrows) of wild-type and agef-1(vh4) L4 larvae. agef-1(vh4) coelomocytes have enlarge vesicles as compared to wild-type. Bar, 10 µm. (G, H) DIC images of adult wild-type and agef-1(vh4) animals; agef-1(vh4) mutants have a smaller body length compared to wild-type. Bar, 100 µm.
Table 1.
AGEF-1 is a negative regulator of EGFR/Ras/MAPK signaling during vulva induction.
Figure 2.
vh4 is a missense mutation in agef-1 and homology between C. elegans and human Arf GTPases.
(A) Schematic representation of the right end of chromosome I (LGI). The SNPs used for interval mapping are indicated on top with their chromosomal locations (map units). The number of recombinant animals positive for the Hawaiian SNP out of the total number of animals tested for each SNP is indicated below. The two chromosomal deficiencies used for mapping are dxDf2 and eDf3 shown in red and blue, respectively. A bracket indicates the 0.9 map unit interval between the haw14137 SNP and the left end of eDf3 to which vh4 maps. (B) Homology domains of the AGEF-1 protein that are common with the human BIG1 and BIG2 proteins and S. cerevisiae Sec7 Arf GEFs: Dimerization Cyclophilin Binding (DCB), Homology Upstream of Sec7 (HUS), catalytic GEF domain (SEC7), Homology Downstream of Sec7 (HDS1-4). HDS1-4 are not homologous to each other. Below shows the alignment of the sequences around the amino acid E1028 which is substituted for a Lysine (K) in agef-1(vh4). E1028 is conserved in the human and yeast homologs. Amino acid (aa) identities are highlighted in black and similarities in grey. (C) A phylogenetic tree showing the evolutionary relationship between the four C. elegans Arf GTPases and the three classes of human Arf GTPases as well as the closely related Arl1 GTPases. The human and C. elegans Arfs are depicted in black and red, respectively. Bootstraps are shown in blue.
Figure 3.
AGEF-1 and the AP-1 complex regulate the size of late endosomes/lysosomes in coelomocytes.
(A, B) Confocal images of the coelomocytes of wild-type and agef-1(vh4) L4 larvae expressing the early endosomal marker 2×FYVE::GFP. (C) Quantification of the diameter of the largest 2×FYVE::GFP-positive vesicle per coelomocyte in wild-type and agef-1(vh4). (D, E) Confocal images of the coelomocytes of wild-type and agef-1(vh4) L4 larvae expressing the late endosomal/lysosomal marker LMP-1::GFP. (F) Quantification of the diameter of the largest LMP-1::GFP-positive vesicle per coelomocyte in wild-type and agef-1(vh4). (G, H) Epifluorescent images of the coelomocytes of apm-1(RNAi) unc-101(sy108) and unc-101(sy108) apg-1(RNAi) L4 larvae. (I) Quantification of the diameter of the largest LMP-1::GFP-positive vesicle per coelomocyte upon depletion of multiple AP-1 subunits. Prism 5 (GraphPad Software, Inc., La Jolla, CA) was used for statistical analysis; unpaired t-test was performed to compare changes in the vesicle size. *** P<0.001. Shown is the mean vesicle size plus standard error of the mean. All bars, 5 µm.
Figure 4.
Secretion defect from the body wall muscle cells in agef-1(vh4) mutants.
(A–D) Representative DIC and epifluorescent images of ssGFP in the coelomocytes of wild-type and agef-1(vh4) mutants. Coelomocytes are outlined with white circles. (E) Quantification of mean ssGFP pixel intensity in the coelomocytes. Images were acquired at an exposure time of 25 ms. (F–I) Representative DIC and epifluorescent images of wild-type and agef-1(vh4) body wall muscle cells expressing ssGFP. Arrows indicate nuclei of the muscle cells. (J) Quantification of mean ssGFP pixel intensity in the body wall muscle cells. Images were acquired at an exposure time of 100 ms. Prism 5 (GraphPad Software, Inc., La Jolla, CA) was used for statistical analysis; unpaired t-test was performed to compare mean GFP intensities and standard error of the mean between wild-type and agef-1(vh4) animals. All bars, 10 µm.
Table 2.
Class I and II Arf mutants suppress the lin-2(e1309) Vul phenotype.
Figure 5.
AGEF-1 and UNC-101 AP-1μ antagonize basolateral localization of LET-23 EGFR.
(A–F′) Representative confocal images of LET-23::GFP localization in P6.p (A–F) and P6.px (A′–F′) vulval cells of zhIs035 transgene-carrying animals. (A–B′) The LET-23::GFP is localized to both apical and basolateral membranes in wild-type and agef-1(vh4) animals. (C, C′) The basolateral receptor localization is lost in lin-2(e1309) mutants. (D, D′) agef-1(vh4); lin-2(e1309) mutants with faint LET-23::GFP expression on the basolateral membrane of P6.p and P6.px. (E, E′) unc-101(RNAi); lin-2(e1309) animals with faint LET-23::GFP expression on the basolateral membrane of P6.p and P6.px. (F, F′) unc-101(RNAi) in agef-1(vh4); lin-2(e1309) mutants results in more LET-23::GFP on the basolateral membranes. (G, G′) Percent animals with LET-23::GFP on both the basolateral and apical membranes or apical only localization. Because some animals have no or little basolateral membrane localization, basolateral localization of LET-23::GFP in the P6.p and P6.px cells was determined by measuring the intensity at the basal membrane versus background. If the GFP intensity on the basal membrane was twice that of the background the cell was considered to have basolateral membrane localization. Bar, 5 µm.
Table 3.
AGEF-1 antagonizes basal membrane localization of LET-23 EGFR in P6.p.
Figure 6.
Model of LET-23 EGFR regulation by AGEF-1/Arf/AP-1 and LIN-2/7/10.
(A) LET-23 EGFR is localized to both basal and apical membranes in the VPCs of wild-type animals. The LIN-2/7/10 complex promotes the basal localization while the AGEF-1/Arf/AP-1 ensemble either inhibits basal or promotes apical localization. (B) In an agef-1(-) background, there is more LET-23 EGFR on the basal and less apical. (C) In a lin-2(-) background, most all of the LET-23 EGFR is apical with presumably residue LET-23 EGFR at the basal membrane, but not enough to induce vulva cell fates. (D) In an agef-1(-); lin-2(-) background, the loss of AGEF-1 partially restores LET-23 EGFR to the basal membrane sufficient to induce vulva cell fates.