The small GTPase ARF-1.2 is a regulator of unicellular tube formation in Caenorhabditis elegans
The membrane trafficking events that regulate unicellular tube formation and maintenance are not well understood. Here, using an RNAi screen, we identified the small GTPase ARF1 homolog ARF-1.2 as a regulator of excretory tube formation in Caenorhabditis elegans. RNAi-mediated knockdown and knockout of the arf-1.2 gene resulted in the formation of large intracellular vacuoles at the growth sites (varicosities) of the excretory canals. arf-1.2 mutant animals were sensitive to hyperosmotic conditions. arf-1.2 RNAi affected the localization of the anion transporter SULP-8, which is expressed in the basal plasma membrane of the excretory canals, but did not affect the expression of SULP-4, which is expressed in the apical membrane. The phenotype of arf-1.2 mutants was suppressed by mutation of the small Rho GTPase CDC-42, a regulator of apical/basal traffic balance. These results suggest that ARF-1.2 plays an essential role in basal membrane traffic to regulate the formation of the unicellular excretory tube.
KeywordsCaenorhabditis elegans Unicellular tube formation Small GTPase ARF-1.2 CDC-42
Epithelial tubes that enable nutrition uptake and fluid transport are essential in all metazoans. Tube formation requires cell polarization and the maintenance of two distinct domains, namely, the apical and basal membranes. The small GTPase family members play essential roles in the multiple steps required for polarized membrane traffic and multicellular tube formation. For example, the small GTPase RAB11, a member of the Rab family, has been implicated in the regulation of the apical recycling pathway . In addition, a member of the small GTPase Arf/Sar family, ARF6, has been proposed to act in clathrin-dependent endocytosis at the apical  and basolateral  membranes of polarized epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells. CDC42, a member of the Rho family of small GTPases, has been shown to function in a pathway that defines the apical membrane. Specifically, CDC42 has been shown to recruit the Par complex to the apical membrane  and is required for the multicellular apical polarization that drives and maintains multicellular tubes .
Contrary to multicellular tubes, unicellular tubes, including capillaries, are composed of individual cells with a hollow lumen. The Caenorhabditis elegans (C. elegans) excretory system provides a simple model of unicellular tube morphogenesis [6, 7, 8]. The excretory cell, a single cell that forms the major tubular component, extends branched processes along the length of the body to regulate fluid osmolarity and ion content. WNK kinases, CLIC-like proteins, Patched-related proteins, mucins, and aquaporins have all been reported to participate in the development and function of the excretory cell [9, 10, 11]. Transcription factors, such as CEH-6, NHR-31, and PROX-1, have also been demonstrated to control downstream genes to form the excretory cell [12, 13, 14].
Several genes that are related to membrane traffic have also been proposed to play roles in excretory tube formation. For example, mutants for rdy-1/vha-5, which encodes a vacuolar H+-ATPase α-subunit, display less extension of the excretory tubes . In addition, loss-of-function mutations in exc-5, which encodes a homolog of FDG1 RhoGEF (guanine exchange factor), cause abnormalities in the apical membrane of the excretory cell . However, the molecular mechanisms that regulate intracellular polarized transport in unicellular tubes are still largely unknown. In the present study, we identified the small GTPase ARF-1.2 as a regulator of basal trafficking in the excretory tube of C. elegans.
Results and discussion
The small GTPase ARF-1.2 is a regulator of excretory tube formation and function
To determine whether the arf-1.2 gene is responsible for this phenotype, we assayed arf-1.2(ok796) deletion mutants for the morphology of the excretory cell. Notably, arf-1.2 mutants displayed a phenotype that is indistinguishable from that of arf-1.2 RNAi-treated animals (Fig. 1). These results strongly suggest that arf-1.2 is required for proper tube formation of the excretory canals. To further characterize the abnormal excretory cell of the arf-1.2 mutants, transmission electron microscopy (TEM) was performed. Although vacuoles failed to be captured, we noticed that the arf-1.2 mutant exhibited squashed excretory canals, in which the lumen and canaliculi were poorly defined (Fig. S1). One possibility is that the abnormal formation of the arf-1.2 excretory canals may cause reduced fluid excretion to the lumen, resulting in the squashed lumen in the process of TEM, although other possible explanations cannot be ruled out.
Subcellular localization of ARF-1.2
arf-1.2 RNAi affected the localization of the basal membrane protein
The vacuolar phenotype of arf-1.2 mutants was suppressed by mutation of the small Rho GTPase CDC-42
ARF1 has been proposed to play a role in polarized transport through the dynamic control of AP1 and AP4 coat assembly in the trans-Golgi network (TGN) . Because of the lack of an AP4 complex in C. elegans, AP1-clathrin components were knocked down and assayed for phenotypic abnormalities. However, we were not able to determine whether RNAi of the AP1-clathrin components (chc-1, apg-1, aps-1, apb-1) exhibited canal defects due to the severe Gro or Let phenotypes. ARF-1.2 and the ArfGEF GBF-1 have been proposed to act in ER-mitochondrial contacts to regulate mitochondrial morphology and function . Skorobogata et al. has reported that ARF-1.2 and the ArfGEF AGEF-1 antagonize LET-23 EGFR basolateral membrane localization and signaling in the vulva . As vacuoles in the excretory canals were not detected following RNAi against gbf-1 or agef-1, other ArfGEF(s) may participate in the function of ARF-1.2 in the excretory canals. However, we were not able to rule out the possibility that RNAi against gbf-1 and agef-1 were not sufficiently effective.
Model of membrane traffic in the excretory unicellular tube
Mattingly and Buechner described a model of membrane traffic in the excretory canals, with organelle markers GRIP (for Golgi bodies), RAB-5 (for early endosomes), RAB-7 (for late endosomes), RAB-11 and RME-1(for recycling endosomes), and GLO-1 (for lysosomes), in which CDC-42 regulates the transport from the recycling endosome to apical plasma membrane . In the present study, subcellular compartments were labeled with those markers, except for Golgi marker (AMAN-2 in this study) and lysosome marker (LMP-1 in this study), suggesting that ARF-1.2 localizes, at least in part, to the Golgi bodies in the excretory canals. We showed that the inactivation of arf-1.2 caused accumulation of intracellular vacuoles that are likely to be related to basal membrane trafficking. In addition, the vacuolar phenotype of arf-1.2 mutants was suppressed by mutation of the cdc-42 gene. The result implicates an interplay between ARF-1.2 and CDC-42, in which ARF-1.2 suppresses or interact with CDC-42 directly or indirectly to balance of the apical and basal transport. Based on these data, we propose a working model, in which ARF-1.2 regulates basal membrane traffic of the excretory canals (Fig. 6b).
ARF1 has been reported to function in epidermal cell polarity in Arabidopsis . However, the role of ARF1 in polarized transport has been less explored than that of ARF6. In the present study, we showed that the C. elegans ARF1 homolog ARF-1.2 plays a role in basal membrane traffic in the excretory unicellular tube and in the morphology of the canals. Our findings provide new insights into the function of ARF1 in polarized transport and unicellular tube formation.
C. elegans strains were cultured using standard techniques . Bristol strain N2 was used as the wild-type C. elegans strain. The following strains were obtained from the Caenorhabditis Genetics Center: VC567 arf-1.2(ok796) III, VC898 cdc-42(gk388)/mIn1 [mIs14 dpy-10(e128)] II.
Constructs and transgenic lines
tmIs806[hmit-1.2p::egfp] and tmIs807[hmit-1.2p::egfp] was generated in the previous report . To generate the vha-8p::EGFP plasmid, the vha-8 upstream genomic fragment (approximately 1.5 kb) was PCR amplified and cloned into the BamHI/NotI sites of the pFX_EGFPT expression vector . To generate the vha-8p::ARF-1.2::EGFP plasmid, arf-1.2 cDNA was cloned into the NotI site of the vha-8p::EGFP plasmid. The expression vectors vha-8p::ARF-1.2(T31N)::EGFP (dominant negative form) and vha-8p::ARF-1.2(Q71L)::EGFP (constitutively active form) were generated by site-directed mutagenesis with the In-fusion system (Clontech and Takara). The full-length cDNA of lmp-1 and partial cDNA of aman-2 (containing the coding sequence of the first 88 amino acids) were cloned into the NotI site of the vha-8p::EGFP plasmid to generate vha-8p::LMP-1::EGFP (lysosome/basal membrane) and vha-8p::AMAN-2(82aa)::EGFP (Golgi), respectively. To generate N-terminal fusion constructs, pFX_vha-8p_VenusT(N) was constructed by subcloning the vha-8 promoter into pFX_VenusT(N), and the full-length cDNA of rab-5, rab-7, rab-11.1, and rme-1d were cloned into the pFX_vha-8p_VenusT(N) to generate vha-8p::VENUS::RAB-5 (early endosome), vha-8p::VENUS::RAB-7 (late endosome), vha-8p::VENUS::RAB-11.1 (recycling endosome), and vha-8p::VENUS::RME-1d (recycling endosome), respectively. To construct sulp-4p::SULP-4::EGFP and sulp-8p::SULP-8::EGFP plasmids, 7.4- and 6.6-kb genomic DNA fragment containing the 5′ upstream promoter and the entire CDS were amplified, and cloned into the pFX_EGFPT plasmid. To generate extrachromosomal (Ex) transgenic animals, these plasmids were injected into N2 at 10 ng/μl with an injection marker myo-2p::dsredm (at 20 ng/μl) and pBluescript (at 170 ng/μl).
Bacterial RNAi feeding
RNA interference (RNAi) was carried out by feeding animals dsRNA-producing bacteria, as previously described , with some modifications. Briefly, P0 animals at the L4 stage were transferred to plates containing RNAi-bacteria grown on NGM containing 100 µg/ml ampicillin and 1 mM isopropyl-beta-d-thiogalactopyranoside (IPTG). The animals were cultured at 20 °C (or 15 °C in cold tolerance assays) until the F1 animals developed into young adults. F1 animals were used for the subsequent assays so that the knockdown was effective from embryonic stages. For the feeding RNAi screen, post-embryonic RNAi was simultaneously performed. In this case, synchronized animals at L1–L2 stage were transferred to the feeding RNAi plates and cultured until the transferred animals became young adults. For the screening using tmIs807[hmit-1.2p::egfp] transgenic animals, modified NGM plates that contained ampicillin, IPTG, and fourfold NaCl (200 mM final) were used to induce EGFP expression. The RNAi sub-library (Table S1) for membrane traffic-associated genes was prepared from the Ahringer Library.
Microscopy and the size measurement of vacuoles
Differential interference contrast (DIC) and fluorescence images were obtained using a BX51 microscope that was equipped with a DP30BW CCD camera (Olympus, Japan). The size measurement of vacuoles was examined by processing fluorescence micrographs with ImageJ (Rasband, W.S., US National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/). Investigators were not blinded to the treatment groups performed the image analysis.
Assays for osmotic stress sensitivity
Synchronized L1 animals (day 1) were cultured on nematode growth medium (NGM) plates containing 51 mM NaCl (normal condition) or 255 mM NaCl (hyperosmotic condition) at 20 °C until they reached 5 days of age (day 5). Body size was determined every day by measuring the projected area of the worm body. The images were taken using a BX51 microscope that was equipped with a DP73 CCD camera (Olympus, Japan) and analyzed using ImageJ software (Rasband, W.S., US National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/).
Transmission electron microscopy
Wild-type or mutant young adults were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 100 mM cacodylate buffer at 4 °C. Transmission electron microscopy was performed by the Hanaichi Ultrastructure Research Institute Co. (Okazaki, Japan). Briefly, fixed samples were postfixed for 2 h with 2% osmium tetroxide in 100 mM cacodylate buffer, followed by dehydration and infiltration with epoxy resin (TAAB, UK). Ultrathin sections of the surface area were analyzed using an electron microscope (H-7600, HITACHI, Japan).
We thank the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, USA; supported by the National Institutes of Health-National Center for Research Resources) for providing C. elegans strains. This work was supported partly by a Grant-in-Aid for Scientific Research from JSPS (to S.M.), and by a Grant-in-Aid for young scientists from JSPS, The Kato Memorial Bioscience Foundation, the Astellas Foundation for Research on Metabolic Disorders, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Takeda Science Foundation, and The Naito Foundation (to E.K-N.).
EK-N performed most of the experiments. SS performed osmotic stress assay. SI and SY performed double mutant analyses of arf-1.2 and cdc-42. All authors discussed the results and designed the experimental approaches. EK-N and SM wrote the manuscript.
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