An AGEF-1/Arf GTPase/AP-1 ensemble antagonizes LET-23 EGFR basolateral localization and signaling during C. elegans vulva induction

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling

TBC-2 Is Required for Embryonic Yolk Protein Storage and Larval Survival during L1 Diapause in Caenorhabditis elegans

C. elegans first stage (L1) larvae hatched in the absence of food, arrest development and enter an L1 diapause, whereby they can survive starvation for several weeks. The physiological and metabolic requirements for survival during L1 diapause are poorly understood. However, yolk, a cholesterol binding/transport protein, has been suggested to serve as an energy source. Here, we demonstrate that C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP) involved in early-to-late endosome transition, is important for yolk protein storage during embryogenesis and for L1 survival during starvation. We found during embryogenesis, that a yolk::green fluorescent protein fusion (YP170::GFP), disappeared much more quickly in tbc-2 mutant embryos as compared with wild-type control embryos. The premature disappearance of YP170::GFP in tbc-2 mutants is likely due to premature degradation in the lysosomes as we found that YP170::GFP showed increased colocalization with Lysotracker Red, a marker for acidic compartments. Furthermore, YP170::GFP disappearance in tbc-2 mutants required RAB-7, a regulator of endosome to lysosome trafficking. Although tbc-2 is not essential in fed animals, we discovered that tbc-2 mutant L1 larvae have strongly reduced survival when hatched in the absence of food. We show that tbc-2 mutant larvae are not defective in maintaining L1 diapause and that mutants defective in yolk uptake, rme-1 and rme-6, also had strongly reduced L1 survival when hatched in the absence of food. Our findings demonstrate that TBC-2 is required for yolk protein storage during embryonic development and provide strong correlative data indicating that yolk constitutes an important energy source for larval survival during L1 diapause

A screen for endocytic regulators of epidermal growth factor receptor signaling in «Caenorhabditis elegans»

Epidermal Growth Factor Receptor (EGFR)/Ras/Mitogen Activated Protein Kinase (MAPK) signaling is involved in regulation of cell proliferation, migration and apoptosis. Activating mutations in EGFR or downstream components of the signaling pathway as well as a loss of a negative regulator of EGFR/Ras/MAPK signaling have been implicated in cancer. EGFR endocytosis and trafficking to the lysosome is an important mechanism of signal downregulation. A small GTPase Rab7 regulates EGFR trafficking and degradation, however its effects on signaling are not known. In C. elegans a highly conserved EGFR/Ras/MAPK signaling pathway is required for vulval cell fate specification. We found that RAB-7 antagonizes LET-23 EGFR signaling during vulval induction suggesting a possible role as a tumor suppressor in mammals. Here I show that a rab-7(ok511) deletion mutant regulates LET-23 localization in the Vulval Precursor Cells (VPCs). Additionally, my data suggest that LET-23 EGFR, similar to mammalian EGFR, traffics through multivesicular bodies (MVBs) en route to lysosome as RNAi knock-down of ESCRT-0 and –I components, HGRS-1 and VPS-28, can suppress the severity of the Vulvaless (Vul) phenotype of a LET-23 mislocalization mutant lin-2(e1309). To identify additional genes that might regulate LET-23 EGFR trafficking and signaling, I conducted a pilot forward genetic screen for suppressors of the lin-2(e1309) Vul phenotype. I identified two suppressor mutants, vh4 and vh22, that are partial embryonic lethal and display phenotypes suggestive of vesicular trafficking defects. I mapped vh4 and vh22 to distinct regions of chromosome I, devoid of previously identified regulators of LET-23 EGFR signaling, suggesting that they are mutations in novel regulators. Additionally, vh4 can enhance the Multivulva (Muv) phenotype of a Ras gain-of-function mutant let-60(n1046) further implicating vh4 as a negative regulator of EGFR/Ras/MAPK signaling. RNAi and genetic complementation data identified AGEF-1, an Arf-GEF, as a potential candidate for vh4. Although I have been unable to identify a corresponding mutation in agef-1, I find that vh4 mutants have phenotypes consistent of a defect in a secretory pathway and affecting AGEF-1 activity. Thus, vh4 and vh22 might represent mutations in novel negative regulators of LET-23 EGFR signaling and possibly regulate trafficking of the receptor.La voie de signalisation EGFR/Ras/MAPK est impliquée dans la régulation de la prolifération cellulaire, la migration et l'apoptose. Les mutations qui activent l'EGFR ou les composants qui sont en aval dans la voie de signalisation ainsi que la perte d'un régulateur négatif de l'EGFR / Ras / MAPK ont été impliquées dans le cancer. L'endocytose et le trafic de l'EGFR vers le lysosome est un important mécanisme de régulation négative du signal. La GTPase Rab7 régule le trafic vésiculaire et la dégradation de l'EGFR qui est un mécanisme important d'atténuation du signal. Toutefois, les effets de Rab7 sur la voie de signalisation EGFR/Ras/MAPK ne sont pas encore connus. La voie de signalisation EGFR/Ras/MAPK qui est très conservée chez C. elegans, est responsable pour la spécification de l'identité cellulaire de la vulve. Nous avons constaté que RAB-7 antagonise la signalisation de LET-23 EGFR pendant l'induction vulvaire, ce qui suggère un rôle possible de Rab7 comme un suppresseur de tumeur chez les mammifères. Ici, je montre que la perte de fonction du mutant de rab-7, rab-7(ok511), régule la localisation du récepteur LET-23 dans les cellules précurseurs vulvaires (CPV). En outre, mes données suggèrent que le récepteur LET-23 (comparable au EGFR chez les mammifères) transitent des corps multivésiculaires (CMVs) vers les lysosomes puisque la réduction d'expression des components du complexe ESCRT-0 et ESCRT-I, HGRS-1 et VPS-28, par ARN interférence supprime la sévérité du phénotype Vul de lin-2(e1309), la mutation qui provoque une mauvaise localisation du LET-23. J'ai mené un projet pilote génétique pour trouver les suppresseurs du phénotype Vul de lin-2(1309) afin d'identifier d'autres gènes qui pourraient aussi réguler le trafic vésiculaire du récepteur LET-23 EGFR. Deux suppresseurs, vh4 et vh22, ont été identifiés. Ils montrent un phénotype de létalité embryonnaire partiel et d'autres phénotypes suggérant un défaut du trafic vésiculaire. J'ai cartographié vh4 et vh22 à des régions distinctes du chromosome I dépourvues de régulateurs du récepteur LET-23 EGFR identifiés précédemment, ce qui suggère que ses mutations sont des nouveaux régulateurs de la voie de signalisation de l'EGFR. En outre, vh4 peut améliorer le phénotype Muv du let-60(n1046), la mutation qui active Ras en permanence, ce qui implique vh4 comme un régulateur négatif de l'EGFR/Ras/MAPK. Des expériences d'ARN interférence ainsi que de la complémentation génétique ont identifié AGEF-1, une Arf1-GEF, comme un candidat potentiel pour vh4. Bien que je n'ai pas pu trouver une mutation correspondante au gène agef-1, je trouve que les mutants vh4 ont des phénotypes cohérente d'un défaut dans une voie de sécrétion et affectant AGEF-1. En conclusion, vh4 et vh22 sont éventuellement des mutations de nouveaux régulateurs négatifs de la voie de signalisation de EGFR/Ras/MAPK qui moduleraient aussi le trafic vésiculaire du récepteur LET-23 EGFR

Regulation of «C. elegans» epidermal growth factor receptor signaling and trafficking by rabs, arfs and motors

A highly conserved Epidermal Growth Factor Receptor (EGFR) signaling pathway specifies vulval cell fate induction during C. elegans development. LET-23 EGFR has to be present on the basolateral membrane of the polarized vulval precursor cells (VPCs) in order to engage and transmit EGF signal. In mammals, EGFR also localizes to basolateral membranes of epithelial cells. Excessive activation of the EGFR signaling pathway has been implicated in cancer and a majority of cancers originate from epithelial cells. Thus, C. elegans VPCs provide a unique in vivo model to study EGFR signaling and localization in polarized epithelium. We conducted a genetic screen for essential regulators of EGFR signalling and identified mutations in the agef-1 and dhc-1, which code for an Arf GTPase guanine nucleotide exchange factor and the heavy chain of the dynein minus-end microtubule motor protein, respectively.A partial loss-of-function mutation in agef-1 enhances EGFR signaling in sensitized backgrounds as well as leads to secretory defects in multiple tissues. Additionally, agef-1 mutant animals have enlarged late endosomes/lysosomes in the coelomocytes. These phenotypes suggest that both secretory and endocytic trafficking pathways are affected in agef-1 mutants. We found that AGEF-1 functions with two Arf GTPases and the AP-1 clathrin adaptor complex to negatively regulate EGFR signaling by antagonizing the basolateral localization of the receptor. Taken together with the recently described role of the AP-1 adaptor in the maintenance of epithelial polarity, AGEF-1 might regulate LET-23 EGFR signaling via multiple mechanisms: directly by antagonizing basolateral localization of the receptor and indirectly by maintaining epithelial polarity in the VPCs. A human homolog of AGEF-1 is mutated in numerous cancer cell lines supporting a tumor suppressive function in humans. Our recent data indicate that a small GTPase RAB-10 is required for multiple agef-1 phenotypes, including negatively regulating LET-23 EGFR signaling in the vulva and affecting the size of late endosomes. This effect is specific to RAB-10 and suggests that AGEF-1 and RAB-10 might function closely in an antagonistic manner.dhc-1(vh22) animals are small and temperature sensitive, with increased embryonic lethality due cell division defects at a restrictive temperature of 20°C. Loss of dhc-1 results in increased LET-23 EGFR signaling in sensitized backgrounds suggesting that DHC-1 is a negative regulator of EGFR signaling. LET-23::GFP accumulates in plasma membrane proximal foci of the VPCs in dhc-1 mutants suggesting that dynein functions at an early step of EGFR endosomal trafficking. Moreover, a late endosomal GTPase RAB-7 also antagonises LET-23 EGFR signaling and its loss leads to LET-23::GFP accumulation in cytoplasmic foci. Given the ability of mammalian Rab7 to engage dynein to promote late endosome trafficking towards the lysosome, it is possible that RAB-7 and DHC-1 might function together at a later step of EGFR trafficking for degradation. Our recent findings identify ZEN-4 KIF23, a kinesin plus-end directed microtubule motor, as a suppressor of dhc-1(vh22) and a positive regulator of LET-23 EGFR signaling in the vulva. Our data indicate that ZEN-4 kinesin is likely to regulate EGFR signaling by its role in epithelial polarity rather than by movement of EGFR-containing vesicles along the microtubule track opposite dynein. Understanding how these genes regulate EGFR signaling and trafficking in C. elegans will inform our understanding of EGFR signaling in human epithelial cells with a potential of uncovering novel tumor suppressors that could serve as therapeutic targets for the treatment of malignancies.Chez C. elegans une voie de signalisation EGFR/Ras/MAPK, qui est très conservée, est responsable pour la spécification du destin des cellules de la vulve. LET-23 EGFR doit être présent sur la membrane basolatérale des cellules précurseurs vulvaires (CPVs) afin d'engager et de transmettre le signal EGF. Chez les mammifères, l'EGFR localise également à membranes basolatérale des cellules épithéliales. Activation excessive de la voie de signalisation de l'EGFR a été impliquée dans le cancer et une majorité des cancers proviennent des cellules épithéliales. Ainsi, CPVs de C. elegans offrent un modèle unique in vivo pour étudier la signalisation et la localisation de l'EGFR dans l'épithélium polarisé.Nous avons mené une étude génétique pour trouver des régulateurs essentiels de la signalisation d'EGFR et nous avons identifié des mutations dans agef-1 et dhc-1, qui code pour un facteur d'échange de nucléotide guanine (GEF) des protéines G Arf et une chaîne lourde de complexe motrice dynéine qui se dirige vers l'extrémité négative des microtubules, respectivement.Une mutation dans agef-1 représente une perte partielle de fonction qui cause l'amélioration de la signalisation d'EGFR ainsi que des défauts de sécrétion dans plusieurs tissus. En outre, agef-1 a d'énormes endosomes tardifs/lysosomes dans les coelomocytes. Ces phénotypes suggèrent que la sécrétion et le trafic vésiculaire sont affectés dans le mutant. Nous avons constaté qu'AGEF-1 fonctionne avec deux protéines G Arf et le complexe AP-1, un adaptateur de clathrine, pour réguler négativement la signalisation d'EGFR. Cet effet est atteint par l'opposition de la localisation basolatérale du récepteur. Le rôle de l'adaptateur AP-1 dans le maintien de la polarité épithéliale a été récemment décrit, donc AGEF-1 peut réguler la signalisation LET-23 EGFR en utilisant de mécanismes multiples: directement en antagonisant la localisation basolatérale du récepteur et indirectement par le maintien de la polarité épithéliale dans les CPVs. Un homologue humain d'AGEF-1 est muté dans de nombreuses lignées de cellules cancéreuses ce qui est favorable à sa fonction comme le suppresseur de tumeur chez les mammifères. Nos données récentes indiquent qu'une protéine G RAB-10 est nécessaire pour plusieurs phénotypes chez agef-1, y compris la régulation négative de la signalisation de LET-23 EGFR dans la vulve et régulation de la taille des endosomes tardifs. Les animaux dhc-1(vh22) sont petits et sensibles à la température, c'est-à-dire que la létalité embryonnaire augmente à une température restrictive de 20°C en raison de défauts de la division cellulaire. Perte de DHC-1 cause l'augmentation de la signalisation de LET-23 EGFR suggérant que DHC-1 est un régulateur négatif de la signalisation d'EGFR. LET-23::GFP accumule dans les structures proches de la membrane plasmique de CPVs en animaux dhc-1, ce qui suggère que dynéine fonction à une étape précoce du trafic vésiculaire de l'EGFR. En outre, une protéine G RAB-7 antagonise également la signalisation de LET-23 EGFR et sa perte mène à l'accumulation de LET-23::GFP dans les structures cytoplasmique. Vu que Rab7 mammifère est capable à engager dynein pour promouvoir le trafic des endosomes vers le lysosome, il est possible que RAB-7 et DHC-1 puissent fonctionner ensemble à une étape précédente la dégradation d'EGFR. Nos récents résultats indiquent que ZEN-4 KIF23, une protéine motrice kinésine qui se déplace en direction de pôle positif des microtubules, est un suppresseur de dhc-1(vh22) et un régulateur positif de la signalisation de LET-23 EGFR dans la vulve. Mieux comprendre comment ces gènes régulent la signalisation et le trafic de LET-23 EGFR chez C. elegans informera notre compréhension de la signalisation d'EGFR dans les cellules épithéliales humaines. Ce qui a un potentiel de découvrir de nouveaux gènes suppresseurs de tumeur qui pourraient servir de cibles thérapeutiques pour le traitement des malignités

RAB-7 Antagonizes LET-23 EGFR Signaling during Vulva Development in Caenorhabditis elegans

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(2) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and-I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity i

<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

<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

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> 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