<i>rab-7(ok511)</i> alters LET-23::GFP localization in the VPCs of <i>lin-2(-)</i> animals.

<p>(A-L) Single section confocal images of the VPCs (lateral view) of mid-L3 stage larvae following the first round of VPC division (Pn.px stage) immunostained with anti-GFP to detect LET-23::GFP (A, D, G and J) and the MH27 monoclonal antibody to detect the AJM-1 junctional protein (B, E, H, and K) demarcating the apical/basal boundry, and (C, F, I and L) are merged images with P6.pa and P6.pp cells underlined. (A–C) wild-type larva carrying <i>gaIs27(let-23::GFP)</i> showing LET-23::GFP in both the basal and apical regions of P6.pa and P6.pp cells. (D–F) <i>lin-2(e1309)</i>; <i>gaIs27(let-23::GFP)</i> larva with weak basal cytoplasmic and strong apical LET-23::GFP localization. (G–I) <i>rab-7(ok511)</i>; <i>gaIs27(let-23::GFP)</i> larva with basal cytoplasmic and apical LET-23::GFP expression in P6.pa and P6.pp with LET-23::GFP in cytoplasmic foci. (J–L) <i>rab-7(ok511)</i>; <i>lin-2(e1309)</i>; <i>gaIs27(let-23::GFP)</i> larva with LET-23::GFP localization similar to that in <i>rab-7(ok511)</i>; <i>gaIs27(let-23::GFP)</i> larvae. Bar, 10 µm (C).</p

RNAi of <i>rab-7</i>, <i>hgrs-1</i> and <i>vps-28</i> suppresses the <i>lin-2(e1309)</i> Vul phenotype.

<p>Statistical analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036489#pone-0036489-t001" target="_blank">Table 1</a> comparing each RNAi experiment to the <i>gfp</i> RNAi control. <i>n</i>, number of animals scored.</p>*<p> <i>P<0.05,</i></p>**<p> <i>P<0.01,</i></p>***<p> <i>P<0.001,</i></p>****<p> <i>P<0.0001.</i></p

<i>rab-7(ok511)</i> modulates the vulva phenotypes of mutations affecting components of the EGFR/Ras/MAPK pathway.

<p>Representative DIC images of the vulva phenotypes of <i>rab-7(ok511)/mIn1</i>; <i>let-60(n1046gf)</i> (A), <i>rab-7(ok511)</i>; <i>let-60(n1046gf)</i> (B), <i>rab-7(ok511)/mIn1</i>; <i>let-60(n2021)</i> (C), <i>rab-7(ok511)</i>; <i>let-60(n2021)</i> (D), <i>rab-7(ok511)/mIn1</i>; <i>dpy-20(e1282) ark-1(sy247)</i> (E), <i>rab-7(ok511)</i>; <i>dpy-20(e1282) ark-1(sy247)</i> (F), <i>unc-101(sy108)</i>; <i>rab-7(ok511)/mIn1</i> (G), <i>unc-101(sy108)</i>; <i>rab-7(ok511)</i> (H), <i>rab-7(ok511)/mIn1</i>; <i>lin-2(e1309)</i> (I), and <i>rab-7(ok511)</i>; <i>lin-2(e1309)</i> (J). Open circles mark the normal site of vulva cell invagination, stars mark vulva invaginations due to ectopic induction, vertical lines mark failed vulva cell inductions, and horizontal lines mark ectopic inductions that fail to invaginate. Bar, 10 µm (A).</p

<i>rab-7(−)</i> animals accumulate LET-23::GFP-positive puncta in the hypodermis.

<p>(A and B) Representative epifluorescence images of LET-23::GFP positive foci in the hypodermis in the mid-body of L3 stage <i>rab-7(ok511)/+</i>; <i>xhIs2501</i> (A) and <i>rab-7(ok511)</i>; <i>xhIs2501</i> (B) larvae. (C) A scatter dot plot of the number of LET-23::GFP positive foci within a fixed area of the hypodermis of <i>xhIs2501</i>, <i>rab-7(ok511)/+</i>; <i>xhIs2501</i>, and <i>rab-7(ok511)</i>; <i>xhIs2501</i> L3 larvae. Error bars represent the mean +/− SEM. In an unpaired t test there is a significant difference (P value<0.0001) between the number of LET-23::GFP positive foci in <i>rab-7(ok511)</i> animals as compared to both <i>rab-7(+)</i> and <i>rab-7(ok511)/+</i> animals. <i>n</i> = number of animals scored. Bar, 10 µm (A).</p

<i>rab-7</i> antagonizes LET-60 Ras-mediated vulval cell fate induction.

<p>All experiments were performed at 20°C. Statistical analysis was performed comparing <i>rab-7/+</i> heterozygotes with <i>rab-7(ok511)</i> homozygotes for each vulval mutant background. <i>rab-7(ok511)</i> is balanced in trans by <i>mIn1(+)</i>, <i>rab-7(ok511)</i> is marked in cis with bli-2(e768) in the strain containing let-60(n1876). Fisher's exact test (<a href="http://www.graphpad.com/quickcalcs" target="_blank">www.graphpad.com/quickcalcs</a>) was used to determine statistical significance. n, number of animals scored.</p>*<p> <i>P<0.05,</i></p>**<p> <i>P<0.01,</i></p>****<p> <i>P<0.0001.</i></p

Schematic of the <i>C. elegans</i> EGFR/Ras/MAPK pathway, vulva development, and the <i>rab-7</i> gene structure.

<p>(A) A linear representation (left to right) of the core components of the <i>C. elegans</i> EGFR/Ras/MAPK pathway (black) and some negative regulators (red) of the pathway are shown above or below their presumptive targets. The mammalian homologs are shown in parenthesis below the <i>C. elegans</i> names. (B) The Anchor Cell (AC) in the gonad secretes a LIN-3 EGF signal (green) to the Vulva Precursor Cells (VPCs), P3.p-P8.p, most strongly activating LET-23 EGFR signaling in the closest VPC, P6.p, inducing the 1° vulva cell fate (green). Subsequently, ligands on P6.p activate LIN-12 Notch (N, blue) on the P5.p and P7.p cells inducing them to adopt the 2° vulva cell fate (yellow). P5.p, P6.p. and P7.p give rise to the 22 cells of the vulva, while P3.p, P4.p, and P8.p divide (P3.p only ∼50% of the time) and fuse with the surrounding hypodermis (Hyp). (C) A diagram representing the <i>rab-7 W03C9.3</i> gene and the upstream gene <i>W03C9.5</i>. The genes are oriented with the 5′ end to the left and 3′ to the right with boxes representing the exons and intervening lines as introns and the 3′ UTR. The regions coding for the putative switch and nucleotide binding domains in the RAB-7 protein are shown in blue and red, respectively. The <i>ok511</i> deletion that removes the first three exons of <i>rab-7</i> as well as the 3′ UTR of <i>W03C9.5</i> is marked with a bracket. The indicated lines represent the genomic clones used for RNAi feeding.</p

<i>rab-7(ok511)</i> is synthetic Multivulva with <i>unc-101</i> and <i>ark-1</i> mutants.

<p>Statistical analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036489#pone-0036489-t001" target="_blank">Table 1</a>, comparing <i>rab-7/+</i> heterozygotes with <i>rab-7(ok511)</i> homozygotes for each vulval mutant background, except for in the <i>dep-1(zh34)</i> background where <i>rab-7(ok511)</i> is compared to <i>rab-7(+)</i>. <i>rab-7(ok511)</i> is balanced in trans by <i>mIn1(+)</i>, <i>ark-1(sy247)</i> is marked in cis to <i>dpy-20(e1282)</i>, <i>dep-1(zh34)</i> control is marked in cis to <i>unc-4(e120)</i>. <i>n</i>, number of animals scored.</p>*<p> <i>P<0.05,</i></p>****<p> <i>P<0.0001.</i></p

<i>rab-7(ok511)</i> suppresses the Vul phenotypes of mutations that mislocalize LET-23 but not strong alleles of <i>lin-3</i> and <i>let-23</i>.

<p>Statistical analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036489#pone-0036489-t001" target="_blank">Table 1</a> comparing <i>rab-7/+</i> heterozygotes with <i>rab-7(ok511)</i> homozygotes for each vulval mutant background, except for in the <i>let-23(sy1)</i> and <i>let-23(sy97)</i> backgrounds where <i>rab-7(ok511)</i> is compared to <i>rab-7(+)</i>. <i>rab-7(ok511)</i>; <i>lin-2(e1309)</i>; <i>vhEx1</i> is compared to <i>rab-7(ok511)</i>; <i>lin-2(e1309)</i>. <i>rab-7(ok511)</i> is balanced in trans by <i>mIn1(+)</i>, <i>rab-7(ok511)</i> is marked in cis with <i>bli-2(e768)</i> in the strain containing <i>lin-3(e1417)</i>, <i>let-23(sy1)</i> and <i>let-23(sy97)</i> are marked in cis with <i>unc-4(e120)</i>, and <i>lin-7(e1413)</i> is linked in cis to <i>mIn1</i> on the <i>rab-7(+)</i> chromosome. <i>n</i>, number of animals scored.</p>*<p> <i>P<0.05,</i></p>**<p> <i>P<0.01,</i></p>***<p> <i>P<0.001,</i></p>****<p> <i>P<0.0001.</i></p

AGEF-1 and the AP-1 complex regulate the size of late endosomes/lysosomes in coelomocytes.

<p>(A, B) Confocal images of the coelomocytes of wild-type and <i>agef-1(vh4)</i> 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 <i>agef-1(vh4)</i>. (D, E) Confocal images of the coelomocytes of wild-type and <i>agef-1(vh4)</i> 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 <i>agef-1(vh4)</i>. (G, H) Epifluorescent images of the coelomocytes of <i>apm-1(RNAi) unc-101(sy108)</i> and <i>unc-101(sy108) apg-1(RNAi)</i> 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. *** <i>P<0.001</i>. Shown is the mean vesicle size plus standard error of the mean. All bars, 5 µm.</p

Model of LET-23 EGFR regulation by AGEF-1/Arf/AP-1 and LIN-2/7/10.

<p>(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 <i>agef-1(-)</i> background, there is more LET-23 EGFR on the basal and less apical. (C) In a <i>lin-2(-)</i> 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 <i>agef-1(-); lin-2(-)</i> background, the loss of AGEF-1 partially restores LET-23 EGFR to the basal membrane sufficient to induce vulva cell fates.</p