Walker, Aidan et al. (2017) International Worm Meeting "Investigating the role of the ARF GTPase arf-3 in regulating seam cell development and secretion."

Woollard, Alison, Walker, Aidan

[International Worm Meeting, 2017]

We have recently identified the promoter region of arf-3, a small GTPase implicated in intracellular trafficking, as the tissue-specific driver of the seam cell marker scm::gfp (identified in an enhancer trap screen) commonly used as a fate marker of seam cells in the study of seam cell development. RNAi of arf-3 leads to variable seam cell numbers suggesting a possible failure in the regulation of seam cell divisions. Furthermore, a deletion mutant of arf-3, tm1877, causes embryonic lethality, with escapees dying as larvae with seam abnormalities. The deletion phenotype is difficult to interpret however, because the deletion removes an upstream ncRNA in addition to arf-3, thus we are currently producing a clean knockout strain by Cas9 mediated deletion of the arf-3 coding region. Although the arf-3 enhancer in the context of the scm::gfp marker is expressed exclusively in seam nuclei (due to the presence of a NLS) we found that a translational reporter of arf-3 is cytoplasmic with ARF-3::GFP/mCherry localised to distinct puncta within the seam cells. We have found that ARF-3::mCherry colocalises with RAB-7::GFP in the seam, suggesting a role for arf-3 in regulating late endosomes. The seam cells are known to play an important secretory function, giving rise to the cuticle and alae but the cell biology of this is very poorly understood, and we are interested in the potential role of arf-3 in this process. To identify potential interactors of arf-3 we performed a targeted RNAi screen to look for mislocalisation of the ARF-3::mCherry translational reporter upon the silencing of genes involved in intracellular trafficking and signalling. We found that knockdown of apr-1, an orthologue of human APC (a component of the beta -catenin destruction complex) altered the localisation of ARF-3::mCherry. Simultaneous knockdown of arf-3 and apr-1 exacerbated the seam cell phenotype and also caused other morphological defects not observed in the single knockdowns. Given the involvement of apr-1 in Wnt signalling in C. elegans, this may suggest an interaction between arf-3 and the Wnt pathway. Preliminary evidence suggests that knockdown of arf-3 in the hypomorphic apr-1 allele, bp298, leads to sterility and synthetic lethality with escapees phenocopying the lethal arf-3(tm1877) allele. This suggests that arf-3 may play a role in regulating embryonic as well as seam cell Wnt signalling and supports an important role for intracellular signalling in regulating these processes.

Talavera, K. et al. (2009) International Worm Meeting "The GRP-1 Arf GEF and the CNT-2 Arf GAP proteins define two Arf cycles that regulate asymmetric neuroblast divisions in C. elegans."

Garriga, G., Cordes, S., Talavera, K., Singhvi, A.

[International Worm Meeting, 2009]

During asymmetric cell division (ACD) a mother cell divides forming two daughter cells that adopt different fates to generate cellular diversity. We have been studying genes required for the asymmetric division of the Q.p neuroblast, which divides to produce a cell that dies and the A/PVM-SDQ precursor. Loss of GRP-1 or CNT-2 causes Q.p to divide symmetrically to generate two precursors. This transformation results in an extra A/PVM and an extra SDQ. GRP-1 is the C. elegans ortholog of the cytohesin family of ARF guanine nucleotide exchange factors (GEFs) for Arf6. CNT-2 is a member of the AGAP family of Arf GAP proteins that stimulate the GTPase activity of Arf1. ADP ribosylation factors (Arfs) are a family of small GTPases that regulate vesicular trafficking. We have shown that the GRP-1 GEF activity and CNT-2 GAP activity is essential for their roles in asymmetric cell division. We find that ARF-1 (class I Arf) and ARF-6 (class III Arf) are also involved in the Q.p division. Loss of either arf alone does not cause an obvious Q.p division defect, but arf-1; arf-6 double mutants produce extra A/PVMs about 30% of the time. The simplest model would be that GRP-1 and CNT-2 define a single Arf cycle for the two Arfs that act redundantly, but genetic interactions suggest that the situation is more complex. Loss of ARF-1, but not ARF-6, enhances the Q.p defect of grp-1 mutants, consistent with GRP-1 acting as a GEF for ARF-6. Loss of ARF-1, but not ARF-6, suppresses the Q.p defect of cnt-2 mutants. We rationalize this result by proposing that the cnt-2 mutant phenotype is caused by its cognate Arfs being trapped in an activated GTP bound state resulting in the mutant phenotype. Eliminating an Arf is predicted to decrease the amount of activated Arf activity and suppress the defect. Consistent with this interpretation, loss of ARF-1 but not ARF-6 enhances the Q.p division defect caused by a CNT-2 mutant protein with unregulated GAP activity. These results suggest that GRP-1 and ARF-6 define one cycle and that CNT-2 and ARF-1 define another. They also predict that genetic approaches can identify additional molecules that are part of the two cycles.

Karla Gutierrez et al. (2006) Development & Evolution Meeting "The GRP-1/cytohesin signaling pathway regulates asymmetric neuroblast divisions in C. elegans"

Shaun Cordes, Karla Gutierrez, Aakanksha Singhvi, Gian Garriga

[Development & Evolution Meeting, 2006]

We are studying asymmetric cell division (ACD), the process by which a mother cell divides to produce daughter cells that adopt different fates. In a screen for mutations that disrupt ACD in C. elegans neuroblast lineages, we isolated an allele of grp-1. GRP-1 is the C. elegans ortholog of the cytohesin family of ARF guanine nucleotide exchange factors (GEFs) that regulate cell adhesion, actin cytoskeletal dynamics and membrane trafficking. Cytohesins have an N-terminal coiled-coil domain, a SEC7 domain that contains ARF GEF activity, and a C-terminal pleckstrin-homology domain that binds to phosphatidylinositides. Cytohesins are recruited to the plasma membrane in response to receptor tyrosine kinase activation of phosphatidylinositol-3-kinase (PI3K) where they activate ARF6. ARF6 stimulates molecules that control actin dynamics and phospholipid metabolism. We have found that mutations in C. elegans grp-1 disrupt ACD in certain neuroblasts, resulting in the transformation of the apoptotic daughters of these neuroblasts into their sisters and the production of extra neurons. GRP-1 acts cell non-autonomously in at least one of these divisions, suggesting that it controls a signal. To determine whether GRP-1 functions with other components of the cytohesin pathway, we used RNAi to screen candidate genes for Grp-1 phenotypes. This approach identified arf-1.2, arf-3, age-1, cnt-2, and rng-1. arf-1.2 and arf-3 encode the C. elegans orthologs of Class I and II ARFs, respectively. age-1 encodes the C. elegans ortholog of Class I PI3K that is activated by receptor tyrosine kinases. cnt-2 encodes the worm ortholog of centaurin gamma, a protein that contains an ARF GTPase activating domain and functions in protein trafficking and the regulation of nuclear PI3K activity. rng-1 encodes a conserved protein containing a ring zinc finger motif. Mutational analysis verified the results obtained by RNAi for arf-1.2, arf-3 and age-1. Analysis of an arf-6 deletion allele has also implicated this Class III ARF in ACD. Our results suggest that C. elegans GRP-1 functions in a cytohesin-like signaling pathway. Current efforts are directed towards characterizing the cnt-2 and rng-1 phenotypes, and determining whether these genes function in the GRP-1 pathway.

Murtaugh J et al. (1991) International C. elegans Meeting "GUANINE NUCLEOTIDE-DEPENDENT SIGNALING PATHWAYS IN C. Elegans: ISOLATION OF TWO GENES ENCODING 20 kDa ADP-RIBOSYLATION FACTORS."

Fire A, Moss J, Fleming JT, Murtaugh J, Lee C, Vaughan M, Takada T, Mishima K

[International C. elegans Meeting, 1991]

ADP-ribosylation factors (ARFs) comprise a highly conserved family of guanine nucleotidebinding proteins initially identified by their ability to stimulate, in a GTP-dependent process, the ADP- ribosyltransferase activity of cholera toxin, and are believed to function in golgimediated vesicular transport and secretion. We sought to determine whether one or multiple ARF genes are present in C. elegans. Strategies using either polymerase chain reaction (PCR), or hybridization of a mixed degenerate oligonucleotide encoding a conserved ARF sequence to lambda DNA libraries identified two genes, designated ARF-a and ARF-b. These genes were found to have 543 and 552 bp open reading frames, coding for proteins of 181 and 184 amino acids, respectively. Conservation of sequence between C. elegans ARFs and those of other eukaryotes was noted throughout the coding region and, in particular, in putative GTP binding and hydrolyzing domains. Nothern analysis revealed three ARF mRNA species. Southern analysis of C. elegans genomic DNA using both oligonucleotide and PCR-derived probes was consistent with the presence of these two ARF genes. ARF-a was physically mapped by Paul Coulson to a central contig on chromosome III. A partially purified low molecular weight ( 20 kDa) factor from C. elegans stimulated, in the presence of GTP, cholera toxin-catalyzed ADP-ribosylation of proteins and auto-ADP-ribosylation of the toxin catalytic unit, consistent with the expression of a GTP- dependent ARF- like protein in C. elegans.

Teuliere, Jerome et al. (2009) International Worm Meeting "Two ARF GTPase cycles and a Frizzled pathway collaborate to regulate the asymmetric cell division of the Q.p neuroblast."

Garriga, Gian, Singhvi, Aakanksha, Ou, Guangshuo, Talavera, Karla, Vale, Ron, Cordes, Shaun, Teuliere, Jerome

[International Worm Meeting, 2009]

Much of the cellular diversity in the metazoan nervous system is generated by asymmetric cell divisions (ACDs), in which a mother cell divides to produce daughters that adopt different fates. While the regulation of asymmetric neuroblast divisions has been well studied in the fruit fly Drosophila melanogaster, little is known about the regulation of these divisions in other organisms. Since all neurons in the nematode Caenorhabditis elegans are produced from asymmetric divisions, the worm is an attractive system to address this question. The Q.p neuroblast normally divides to generate a smaller cell that dies (Q.pp) and a larger precursor (Q.pa) that divides to produce the A/PVM and SDQ neurons. We have identified several genes required for the Q.p division. Mutations in these genes cause Q.p to divide symmetrically to produce two precursors that are more similar in size. Several of these genes encode components of the Arf GTPase cycle, which is involved in membrane trafficking. The Arf GEF GRP-1, the Arf GAP CNT-2 and the Arfs ARF-1 and ARF-6 are involved in the Q.p ACD. While these genes function cell autonomously in the Q.p lineage, genetic interactions indicate that CNT-2 is an ARF-1 GAP and GRP-1 is an ARF-6 GEF, suggesting that two separate Arf cycles regulate the Q.p ACD. We are currently testing whether a more centrally positioned mitotic spindle in these mutants can explain the equivalent sizes of the Q.p daughters. We have also implicated the LIN-17 and MOM-5 Frizzled receptors for Wnt secreted glycoproteins in the Q.p ACD: lin-17 mom-5 double mutants but not lin-17 or mom-5 single mutants display Q.p lineage defects. Because loss of a single Frizzled can suppress the Q.p defects of either cnt-2 and grp-1 mutants, we propose that the function of the Arf cycles is to negatively regulate the Frizzled pathway. One surprising finding is that various Wnt mutant combinations or mig-14 mutants, which are defective in Wnt secretion, do not display Q.p defects, suggesting that the role of Frizzleds in this division might be Wnt independent. We are now investigating the mechanistic relationships between the Arf and Wnt signaling pathways in the Q.p ACD.

Gauthier, K. et al. (2017) International Worm Meeting "LIN-10 can promote LET-23 EGFR signalling independently of LIN-2 and LIN-7."

Gauthier, K., Rocheleau, C.

[International Worm Meeting, 2017]

The spatial organization of signal transduction cascades is critical for regulating cell signalling and function. This is particularly evident in Caenorhabditis elegans vulva precursor cells (VPCs), where Epidermal Growth Factor Receptor (LET-23 EGFR) signalling from the basolateral membrane is required for vulva cell fate induction. Basolateral localization of LET-23 is dependent on the LIN-2 CASK/LIN-7 Veli/LIN-10 Mint1 complex. Disruption of this complex results in exclusive apical localization of LET-23 and a vulvaless (Vul) phenotype; however, it is unclear how this complex regulates LET-23 localization. We previously identified a pathway consisting of ARF-1.2 and ARF-3 GTPases, AGEF-1 (a putative Arf guanine exchange factor), and the AP-1 clathrin adaptor complex that antagonizes basolateral localization of LET-23 and negatively regulates signalling. Interestingly, mammalian Mint proteins (LIN-10) bind Golgi-associated Arf GTPases and may serve as an adaptor protein analogous to the AP-1 complex. I hypothesize that the LIN-2/7/10 complex competes with the AGEF-1/ARF/AP-1 ensemble at the trans-Golgi network or recycling endosomes for LET-23 binding to mediate basolateral transport of LET-23. I found that GFP::LIN-10 localizes to cytoplasmic foci in the VPCs that may represent Golgi mini-stacks or recycling endosomes independently of LIN-2, LIN-7, and ARF GTPases. This localization is mediated by the C-terminal domains of LIN-10 that also mediate Arf binding in mammalian Mint proteins. Preliminary analysis suggests that LIN-10 partially colocalizes with ARF-1.2 in the VPCs. Unexpectedly, I found that overexpression of LIN-10 rescues the lin-2 and lin-7 Vul phenotypes, and partly restores basolateral LET-23 localization in a lin-2 mutant, indicating that LIN-10 promotes signalling independently of its complex. Overexpression of LIN-7, but not LIN-2, can also partly rescue mutations in the complex components. Going forward, I will test the hypothesis that LIN-10 promotes the LET-23 signalling pathway by interacting with ARFs. The results will offer new insight into how trafficking and scaffolding components work together to carefully regulate cellular events by controlling the spatial organization of signalling proteins in polarized cells.

Gauthier, Kimberley et al. (2015) International Worm Meeting "Functional analysis of the LIN-2/7/10 complex in LET-23 EGFR localization and signalling during vulval development."

Gauthier, Kimberley, Skorobogata, Olga, Rocheleau, Christian

[International Worm Meeting, 2015]

The LET-23 EGFR/ LET-60 RAS/ MPK-1 ERK signalling pathway is activated in the vulva precursor cells (VPCs) to initiate vulval development. The LIN-2 CASK/ LIN-7 Veli/ LIN-10 Mint1 (LIN-2/7/10) complex is necessary to maintain basolateral localization of LET-23 where it can engage the LIN-3 ligand and initiate downstream signaling. Disruption of this complex results in the apical only localization of LET-23 and a vulvaless (Vul) phenotype. This complex has been well-defined biochemically, however the cellular mechanisms by which it regulates LET-23 localization are not understood. To test if the LIN-2/7/10 complex was involved in endocytosis of LET-23 at the plasma membrane, I used RNAi to knockdown key endocytosis genes. My results suggest that the LIN-2/7/10 complex is not involved in promoting or inhibiting internalization of LET-23 from the plasma membrane. This supports a model in which LIN-2/7/10 is involved in targeting LET-23 to the basolateral membrane from the Golgi. We identified the ARF-1.2 and ARF-3 GTPases, the AGEF-1 Arf GEF, and the AP-1 clathrin adaptor complex as antagonizing LET-23 basolateral localization and hence signalling likely in opposition to the LIN-2/7/10 complex. Interestingly, the PTB and PDZ domains of mammalian LIN-10 homolog Mint1 interact with Arf GTPases, and Mint1 has been proposed to serve as an adaptor protein analogous to the AP-1 complex. I hypothesize that LIN-10 and the AP-1 complex function antagonistically at the trans-Golgi network or recycling endosomes to regulate its transport of LET-23 to the basolateral membrane. Consistent with this data, I found that GFP::LIN-10 and ARF-1.2::GFP, localize to punctate structures in the VPCs, which may represent Golgi ministacks or recycling endosomes. The C-terminal PTB and PDZ domains of LIN-10 regulate its localization in the VPCs, however this localization appears to be independent of the ARF-1.2 and ARF-3 GTPases. I also found that GFP::LIN-10 localization is independent of its complex components LIN-2 and LIN-7. Unexpectedly I found that overexpression of LIN-10 is sufficient to rescue both the lin-2 and lin-7 Vul phenotypes. Going forward, I will see if LIN-10 colocalizes and interacts with ARF-1.2, and I will test if this interaction is necessary for the rescue of lin-2 and lin-7 Vul phenotypes and to restore LET-23 to the basolateral membrane. Our results suggest that LIN-10 might have LIN-2 and LIN-7 independent functions to promote LET-23 EGFR basolateral localization in the VPCs.

Walker, Amy et al. (2019) International Worm Meeting "Disruption of intracellular membrane lipids activates a dual transcriptional program to increase lipogenesis and bypass organelle dysfunction."

Ding, Wei, Walker, Amy

[International Worm Meeting, 2019]

Preserving membrane homeostasis is essential for cell structure, organelle function, and signaling. This homeostasis is disrupted when low phosphatidylcholine (PC) causes cells to overproduce stored lipids. We previously discovered that a master regulator of lipogenic transcription, SREBP-1 (C. elegans sbp-1), is proteolytically activated in low PC, stimulating fat storage in C. elegans, murine liver, and human cell lines. SREBPs are stored in the ER membrane and released by cleavage by Golgi-resident proteases. Subsequently, we found that the phospholipid imbalances activating SBP-1/SREBP-1 are likely to act by disrupting the function of the ARF-1.2/ARF1 GTPase, a key regulator of Golgi/ER traffic. Thus, imbalances in PC can feedback to activate SBP-1/SREBP-1 and lipid production by altering trafficking pathways. We noted that imbalances in phospholipid levels through interference with PC production (sams-1(RNAi)), blocking the conversion of phosphatidic acid to diaglycerol (lpin-1(RNAi), limiting ARF1 function (by arf-1.2(RNAi) or brefeldinA) or sbp-1(RNAi) drive high-level expression of an ARF1 paralog, arf-1.1. We hypothesized that arf-1.1 might be part of a transcriptional program compensating for the loss of ARF-1.2/ARF1 function when phospholipid levels are imbalanced. Indeed, we found that reduction of arf-1.1 expression in sams-1(loss of function) animals decreased survival. Strikingly, the arf-1.1 paralog is required for arf-1.2/ARF1 RNAi animals to grow past initial larval stages, showing that this paralog biological compensates for the reduction in ARF1 function. Finally, analysis of gene expression patterns in arf-1.2/ARF1 animals shows that an entire suite of trafficking regulators is activated. This activation requires the activity of CREB transcription factors, which may be proteolytically activated and cleaved from ER membranes, just as SREBPs. We hypothesize that blockage of ARF1 activity initiates a bipartite program, stimulating SBP-1/SREBP-1 dependent lipogenesis and activating a protective transcriptional program including a compensatory ARF1 paralog. Since this program can also be stimulated by the loss of SBP-1/SREBP-1, we suggest the primary function is as a feedback loop to drive SBP-1/SREBP-1 proteolytic activation. Finally, our discovery of this regulatory loop linking ARF1 dysfunction to transcriptional of a compensatory program represents an example of how cells use gene regulatory pathways to sense and respond to changes in lipid levels, linking metabolic changes to cellular pathways that can restore homeostasis.

Sobhani, Aida et al. (2021) International Worm Meeting "RAB-10 functions opposite of the AGEF-1/Arf GTPase/AP-1 pathway to regulate vesicle trafficking"

Skorobogata, Olga, Rocheleau, Christian E, Gauthier, Kimberley, Fazlollahi, Ali, Sobhani, Aida

[International Worm Meeting, 2021]

Cell polarity is critical for the function of the many cell types. In C. elegans, basolateral localization of the LET-23 Epidermal Growth Factor Receptor (EGFR) in the vulval precursor cells (VPCs) is required for the development of the vulva. A class I/II Arf GTPase pathway regulated by AGEF-1, a homolog of the mammalian BIG1/2 Arf GEFs, antagonizes LET-23 EGFR signaling by promoting its apical trafficking. Loss of the AGEF-1/Arf GTPase/AP-1 pathway results in increased basolateral LET-23 EGFR and over induction of vulva tissue. Thus, this pathway is required to maintain the proper levels of LET-23 EGFR at the basolateral membrane to ensure proper vulva induction. In human, the EGFR signaling pathway is frequently over-activated in many cancers and studying the mechanisms regulating EGFR signaling is crucial to understand the development of EGFR-related diseases. We found that RAB-10 GTPase has novel antagonistic interactions with the Arf GTPase pathway. RAB-10 GTPase is required for agef-1(vh4) trafficking phenotypes in several tissues including increased basolateral localization of LET-23 EGFR in the VPCs, but it is not required for LET-23 EGFR localization in a wild-type background. RAB-10, AGEF-1 as well as the Arf GTPase pathway localize to Golgi and recycling endosomes to regulate polarized trafficking in epithelial cells, however, it is not known how their functions are coordinated. The focus of my project is to find how RAB-10 interacts with the Arf GTPase pathway and which effectors and regulators function with RAB-10 to regulate LET-23 EGFR localization and signaling. Our genetic epistasis indicates that rab-10(+) is required downstream of agef-1(vh4) and arf-1(ok796), but upstream or in parallel with unc-101(sy101) AP-1 m subunit. Thus, RAB-10 might function with the AP-1 clathrin adaptor complex in the absence of Arf GTPase activity to promote basolateral transport of LET-23 EGFR. Additionally, we screened RAB-10 effectors for interactions with the AGEF-1. Two RAB-10 effectors, CNT-1, an Arf6 GAP, and SEC-15, a subunit of the exocyst complex, are suppressors of agef-1(vh4) embryonic phenotypes. Unexpectedly, we found that CNT-1 functions opposite of RAB-10 in the VPCs and thus might function in a distinct pathway. We are still testing the role of SEC-15 in regulation of LET-23 EGFR trafficking. These studies will determine how RAB-10 and the AGEF-1/Arf/AP-1 trafficking intersects to regulate polarized transport in epithelial cells.

Aakanksha Singhvi et al. (2007) International Worm Meeting "Two ARF GTPase cycles and two modes of regulating asymmetric cell divisions."

Aakanksha Singhvi, Shaun Cordes, Gian Garriga, Karla Guitterez

[International Worm Meeting, 2007]

Asymmetric cell divisions in the developing C.elegans nervous system generate both apoptotic cells and differentiated neurons. The mechanisms that specify distinct apoptotic versus neural fates in these dividing cells are largely unknown. We have identified the cytohesin GRP-1, a Guanine Nucleotide Exchange Factor (GEF) for the ARF family of small GTPases; two GTPases, ARF-1 and ARF-6; and the ARF GTPase Activating Protein (GAP) CNT-2 as regulators of the asymmetric division of the Q.p neuroblast. Loss of these molecules results in the survival of Q.pp, a cell normally fated to die, as well as its transformation into the A/PVM-SDQ precursor, its sister cell, resulting in extra A/PVM and SDQ neurons. Homologs of these molecules have been shown to regulate membrane trafficking and cell signaling. Our structure-function analyses show that the GEF activity of GRP-1 is both necessary and sufficient for its function and that the GAP domain of CNT-2 is necessary for its function. Expression of GRP-1 from specific promoters suggests that it functions cell non-autonomously and thus may regulate a signal that controls the Q.p division. GRP-1 localizes to the midbody, raising the interesting possibility that this structure might be involved in cell signaling. Based on several criteria, we come to the surprising conclusion that GRP-1 and CNT-2 define two independent ARF GTPase cycles that regulate asymmetric neuroblast divisions. First, while grp-1 mutations do not alter the sizes of the Q.p daughter cells, cnt-2 mutations do. Second, GRP-1 and CNT-2 have distinct sub-cellular localization patterns. Third, genetic interactions suggest that ARF-1 functions in parallel to GRP-1. Finally, the most compelling evidence indicating that GRP-1 and CNT-2 play different roles in the Q.p division comes from the finding that CNT-2 acts autonomously in the Q.p division, revealing that GRP-1 and CNT-2 act in different cells. Taken together, our studies have for the first time shown in an in vivo context that ARF-mediated pathways regulate asymmetric cell divisions, and that they do so both cell autonomously as well as by regulating a signal.