Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans.

Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates.

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans.

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5-trisphosphate (IP3) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP3 signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up-regulating IP3 signalling decreases sensitivity. Tissue-specific rescue experiments suggest IP3 functions in the intestine. We also exploit IP3 signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.

Genetic analysis of IP3 and calcium signalling pathways in C. elegans.

BACKGROUND: The nematode, Caenorhabditis elegans is an established model system that is particularly well suited to genetic analysis. C. elegans is easily manipulated and we have an in depth knowledge of many aspects of its biology. Thus, it is an attractive system in which to pursue integrated studies of signalling pathways. C. elegans has a complement of calcium signalling molecules similar to that of other animals. SCOPE OF REVIEW: We focus on IP3 signalling. We describe how forward and reverse genetic approaches, including RNAi, have resulted in a tool kit which enables the analysis of IP3/Ca2+ signalling pathways. The importance of cell and tissue specific manipulation of signalling pathways and the use of epistasis analysis are highlighted. We discuss how these tools have increased our understanding of IP3 signalling in specific developmental, physiological and behavioural roles. Approaches to imaging calcium signals in C. elegans are considered. MAJOR CONCLUSIONS: A wide selection of tools is available for the analysis of IP3/Ca2+ signalling in C. elegans. This has resulted in detailed descriptions of the function of IP3/Ca2+ signalling in the animal's biology. Nevertheless many questions about how IP3 signalling regulates specific processes remain. GENERAL SIGNIFICANCE: Many of the approaches described may be applied to other calcium signalling systems. C. elegans offers the opportunity to dissect pathways, perform integrated studies and to test the importance of the properties of calcium signalling molecules to whole animal function, thus illuminating the function of calcium signalling in animals. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.

Disruption of the ATP-binding cassette B7 (ABTM-1/ABCB7) induces oxidative stress and premature cell death in Caenorhabditis elegans.

X-linked sideroblastic anemia with ataxia (XLSA/A) is a rare inherited disorder characterized by mild anemia and ataxia. XLSA/A is caused by mutations in the ABCB7 gene, which encodes a member of the ATP-binding cassette transporter family. Studies in yeast, mammalian cells, and mice have shown that ABCB7 functions in the transport of iron-sulfur (Fe-S) clusters into the cytoplasm. To further investigate the mechanism of this disease, we have identified and characterized the Caenorhabditis elegans homologue of the ABCB7 gene, abtm-1. We have studied the function of abtm-1 using mutants and RNAi. abtm-1-depleted animals produce arrested embryos that have morphogenetic defects and unusual premature, putative apoptotic events. abtm-1(RNAi) animals also show accumulation of ferric iron and increased oxidative stress. Despite the increased level of oxidative stress in abtm-1(RNAi) animals, they have an increased life span. We observed accumulation of DAF-16/FOXO in the nuclei of affected animals and elevation of the expression of SOD-3, a well established target of DAF-16, which may explain the increased life span extension of these animals. abtm-1 is strongly expressed in tissues with a high energy demand, and abtm-1(RNAi) animals have phenotypes that reflect the need for abtm-1 in these tissues. Finally, we show that reducing the function of other genes involved in Fe-S cluster production produces similar phenotypic consequences to abtm-1 loss of function. Therefore, ablation of abtm-1 in C. elegans provides a model in which to investigate the mechanism underlying XLSA/A.

Inositol 1,4,5-trisphosphate signalling regulates the avoidance response to nose touch in Caenorhabditis elegans.

When Caenorhabditis elegans encounters an unfavourable stimulus at its anterior, it responds by initiating an avoidance response, namely reversal of locomotion. The amphid neurons, ASHL and ASHR, are polymodal in function, with roles in the avoidance responses to high osmolarity, nose touch, and both volatile and non-volatile repellents. The mechanisms that underlie the ability of the ASH neurons to respond to such a wide range of stimuli are still unclear. We demonstrate that the inositol 1,4,5-trisphosphate receptor (IP(3)R), encoded by itr-1, functions in the reversal responses to nose touch and benzaldehyde, but not in other known ASH-mediated responses. We show that phospholipase Cbeta (EGL-8) and phospholipase Cgamma (PLC-3), which catalyse the production of IP(3), both function upstream of ITR-1 in the response to nose touch. We use neuron-specific gene rescue and neuron-specific disruption of protein function to show that the site of ITR-1 function is the ASH neurons. By rescuing plc-3 and egl-8 in a neuron-specific manner, we show that both are acting in ASH. Imaging of nose touch-induced Ca(2+) transients in ASH confirms these conclusions. In contrast, the response to benzaldehyde is independent of PLC function. Thus, we have identified distinct roles for the IP(3)R in two specific responses mediated by ASH.

Global urban environmental change drives adaptation in white clover.

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale.

Phospholipase C-epsilon regulates epidermal morphogenesis in Caenorhabditis elegans.

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP(3))-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP(3) production is stimulated is unknown. IP(3) is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-epsilon produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP(3) receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-gamma and EGL-8/PLC-beta can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-epsilon.

Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision.

Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-delta in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.

Reverse genetic strategies in Caenorhabditis elegans: towards controlled manipulation of the genome.

Caenorhabditis elegans has a complete annotated genome sequence that is augmented by increasing quantities of data from high-throughput postgenomic analyses. This has led to an increasing need to identify the biological functions of specific genes using reverse genetics, i.e., moving from gene to phenotype. Fundamental to this aim is the ability to alter the structure of particular genes by means that are not accessible to classical genetic strategies. Thus, one dream of C. elegans researchers is to establish a toolkit for the controlled manipulation of any loci within the genome. Although C. elegans is amenable to a wide variety of genetic and molecular manipulations, controlled manipulation of endogenous genes by, for example, gene targeting has proved elusive until relatively recently. In this review, we describe and discuss the different methods available for the inactivation and modification of endogenous loci with a focus on strategies that permit some measure of control in this process. We describe methods that use random mutagenesis to isolate mutations in specific genes. We then focus on techniques that allow controlled manipulation of the genome: gene modification by transposon mobilisation, gene knock-out mediated by zinc-finger nucleases, and gene targeting by biolistic transformation.

TRPM channels are required for rhythmicity in the ultradian defecation rhythm of C. elegans.

BACKGROUND: Ultradian rhythms, rhythms with a period of less than 24 hours, are a widespread and fundamental aspect of life. The mechanisms underlying the control of such rhythms remain only partially understood. Defecation in C. elegans is a very tightly controlled rhythmic process. Underlying the defecation motor programme is an oscillator which functions in the intestinal cells of the animal. This mechanism includes periodic calcium release and subsequent intercellular calcium waves which in turn regulate the muscle contractions that make up the defecation motor programme. Here we investigate the role of TRPM cation channels in this process. RESULTS: We use RNA interference (RNAi) to perturb TRPM channel gene expression. We show that combined knock down of two of the TRPM encoding genes, gon-2 and gtl-1, results in an increase in the variability of the cycle but no change in the mean, in normal culture conditions. By altering the mean using environmental (temperature) and genetic approaches we show that this increase in variability is separable from changes in the mean. We show that gon-2 and gtl-1 interact with components of the calcium signalling machinery (itr-1 the C. elegans inositol 1,4,5-trisphosphate receptor) and with plasma membrane ion channels (flr-1 and kqt-3) which are known to regulate the defecation oscillator. Interactions with these genes result in changes to the mean period and variability. We also show that knocking down a putative transcription factor can suppress the increased variability caused by reduction of gon-2 and gtl-1 function. We also identify a previously unrecognised tendency of the defecation cycle to compensate for cycles with aberrant length by adjusting the length of the following cycle. CONCLUSION: Thus TRPM channels regulate the variability of the defecation oscillator in C. elegans. We conclude that the mean and the variability of the defecation oscillator are separable. Our results support the notion that there is a strong underlying pacemaker which is able to function independently of the observable defecation rhythm and is not perturbed by increases in the variability of the cycle. The interaction of gon-2 and gtl-1 with other components of the oscillator shows that TRPM channels play an important role in the oscillator machinery. Such a role may be through either regulation of cation levels or membrane properties or both. Specifically our results support previous proposals that gon-2 and gtl-1 regulate IP3 signalling and that kqt-3 may act by altering calcium influx. Our results provide novel insights into the properties of the defecation oscillator and thus to our understanding of ultradian rhythms.

Inositol 1,4,5-trisphosphate signaling regulates mating behavior in Caenorhabditis elegans males.

Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP(3)) is required at several points during mating. Disruption of IP(3) receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-beta, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.

Decoding the intensity of sensory input by two glutamate receptors in one C. elegans interneuron.

How neurons are capable of decoding stimulus intensity and translate this information into complex behavioral outputs is poorly defined. Here, we demonstrate that the C. elegans interneuron AIB regulates two types of behaviors: reversal initiation and feeding suppression in response to different concentrations of quinine. Low concentrations of quinine are decoded in AIB by a low-threshold, fast-inactivation glutamate receptor GLR-1 and translated into reversal initiation. In contrast, high concentrations of quinine are decoded by a high-threshold, slow-inactivation glutamate receptor GLR-5 in AIB. After activation, GLR-5 evokes sustained Ca2+ release from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores and triggers neuropeptide secretion, which in turn activates the downstream neuron RIM and inhibits feeding. Our results reveal that distinct signal patterns in a single interneuron AIB can encode differential behavioral outputs depending on the stimulus intensity, thus highlighting the importance of functional mapping of information propagation at the single-neuron level during connectome construction.

A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C. elegans.

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R). We show that C. elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. We defined the interaction sites on both the IP(3)R and MYO-1. To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C. elegans. This decreased the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP(3)-mediated process [15]. Other IP(3)-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C. elegans and probably more generally.

Reduction of Caenorhabditis elegans frataxin increases sensitivity to oxidative stress, reduces lifespan, and causes lethality in a mitochondrial complex II mutant.

Friedreich ataxia is an autosomal recessive neurological disorder caused by deficiency of the mitochondrial protein frataxin. Studies in patient cells, mouse knockout animals, and Saccharomyces cerevisiae models have suggested several hypotheses on the frataxin function, but the full physiology of frataxin in mitochondria has not been well established yet. We have characterized the genomic structure of frh-1, the Caenorhabditis elegans frataxin gene, and we have developed a transient knockdown model of C. elegans frataxin deficiency by RNA interference. frh-1(RNAi) worms show a consistent pleiotropic phenotype that includes slow growth, lethargic behavior, egg laying defects, reduced brood size, abnormal pharyngeal pumping, and altered defecation. Lifespan is significantly reduced, and worms have increased sensitivity to oxidative stress that, in turn, might explain the reduction of longevity of the worms. We also demonstrate synthetic genetic interaction between frh-1 and mev-1, the gene encoding the succinate dehydrogenase cytochrome b subunit of complex II in mitochondria, suggesting a possible role of the C. elegans frataxin in the electron transport chain; thus, the respiratory chain might be involved in the pathogenesis of the disease. We propose that this C. elegans model may be a useful biological tool for drug screening in Friedreich ataxia.

Muscular dystrophy associated mutations in caveolin-1 induce neurotransmission and locomotion defects in Caenorhabditis elegans.

Mutations in human caveolin-3 are known to underlie a range of myopathies. The cav-1 gene of Caenorhabditis elegans is a homologue of human caveolin-3 and is expressed in both neurons and body wall muscles. Within the body wall muscle CAV-1 localises adjacent to neurons, most likely at the neuromuscular junction (NMJ). Using fluorescently tagged CAV-1 and pre- and post-synaptic markers we demonstrate that CAV-1 co-localises with UNC-63, a post-synaptic marker, but not with several pre-synaptic markers. To establish a model for human muscular dystrophies caused by dominant-negative mutations in caveolin-3 we created transgenic animals carrying versions of cav-1 with homologous mutations. These animals had increased sensitivity to levamisole, suggesting a role for cav-1 at the NMJ. Animals carrying a deletion in cav-1 show a similar sensitivity. Sensitivity to levamisole and locomotion were also perturbed in animals carrying a dominant-negative cav-1 and a mutation in dynamin, which is a protein known to interact with caveolins. Thus, indicating an interaction between CAV-1 and dynamin at the NMJ and/or in neurons.

IRI-1, a LIN-15B homologue, interacts with inositol-1,4,5-triphosphate receptors and regulates gonadogenesis, defecation, and pharyngeal pumping in Caenorhabditis elegans.

Inositol-1,4,5-triphosphate receptors (IP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores. They are central to a wide range of cellular responses. IP(3)Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP(3) and IP(3)Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP(3)R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.

Inositol 1,4,5-trisphosphate signaling regulates rhythmic contractile activity of myoepithelial sheath cells in Caenorhabditis elegans.

Intercellular communication between germ cells and neighboring somatic cells is essential for reproduction. Caenorhabditis elegans oocytes are surrounded by and coupled via gap junctions to smooth muscle-like myoepithelial sheath cells. Rhythmic sheath cell contraction drives ovulation and is triggered by a factor secreted from oocytes undergoing meiotic maturation. We demonstrate for the first time that signaling through the epidermal growth factor-like ligand LIN-3 and the LET-23 tyrosine kinase receptor induces ovulatory contractions of sheath cells. Reduction-of-function mutations in the inositol 1,4,5-trisphosphate (IP(3)) receptor gene itr-1 and knockdown of itr-1 expression by RNA interference inhibit sheath contractile activity. itr-1 gain-of-function mutations increase the rate and force of basal contractions and induce tonic sheath contraction during ovulation. Sheath contractile activity is disrupted by RNAi of plc-3, one of six phospholipase C-encoding genes in the C. elegans genome. PLC-3 is a PLC-gamma homolog and is expressed in contractile sheath cells of the proximal gonad. Maintenance of sheath contractile activity requires plasma membrane Ca(2+) entry. We conclude that IP(3) generated by LET-23 mediated activation of PLC-gamma induces repetitive intracellular Ca(2+) release that drives rhythmic sheath cell contraction. Calcium entry may function to trigger Ca(2+) release via IP(3) receptors and/or refill intracellular Ca(2+) stores.

Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis.

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.

Overexpression of caveolins in Caenorhabditis elegans induces changes in egg-laying and fecundity.

Caveolae are small plasma membrane-associated invaginations that are enriched in proteins of the caveolin family in addition to, sphingolipids, glycosphingolipids and cholesterol. Caveolae have been implicated in several endocytic and trafficking mechanisms. Mutations in caveolins have been shown to cause disease and caveolae offer one site for pathogen entry. The Caenorhabditis elegans genome encodes two caveolins (cav-1 and cav-2); we have shown that these two proteins have distinct expression patterns. CAV-1 is found in the majority of cells in embryos and in the body-wall muscles, neurons and germ line of adult worms. CAV-2 is expressed in the intestine and is required for apical lipid trafficking. In the course of our studies, we generated several constructs to overexpress caveolins in C. elegans. Here we show that overexpression of cav-1 protects against the decrease in brood size associated with the effects of heat shock and the presence of extrachromosomal arrays in heat-shocked animals. Furthermore, we show that overexpression of cav-2 in the nervous system increases the rate of egg-laying and total number of eggs laid.

Caveolin-2 is required for apical lipid trafficking and suppresses basolateral recycling defects in the intestine of Caenorhabditis elegans.

Caveolins are plasma membrane-associated proteins that colocalize with, and stabilize caveolae. Their functions remain unclear although they are known to be involved in specific events in cell signaling and endocytosis. Caenorhabditis elegans encodes two caveolin genes, cav-1 and cav-2. We show that cav-2 is expressed in the intestine where it is localized to the apical membrane and in intracellular bodies. Using the styryl dye FM4-64 and BODIPY-labeled lactosylceramide, we show that the intestinal cells of cav-2 animals are defective in the apical uptake of lipid markers. These results suggest parallels with the function of caveolins in lipid homeostasis in mammals. We also show that CAV-2 depletion suppresses the abnormal accumulation of vacuoles that result from defective basolateral recycling in rme-1 and rab-10 mutants. Analysis of fluorescent markers of basolateral endocytosis and recycling suggest that endocytosis is normal in cav-2 mutants and thus, that the suppression of basolateral recycling defects in cav-2 mutants is due to changes in intracellular trafficking pathways. Finally, cav-2 mutants also have abnormal trafficking of yolk proteins. Taken together, these data indicate that caveolin-2 is an integral component of the trafficking network in the intestinal cells of C. elegans.