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Significance In neurodegenerative disease, protein aggregates spread to neighboring cells to promote pathology. The in vivo regulation of toxic material transfer remains poorly understood, although mechanistic understanding should reveal previously unrecognized therapeutic targets. Proteostressed Caenorhabditis elegans neurons can concentrate protein aggregates and extrude them in membrane-encased vesicles called exophers. We identify specific systemic stress conditions that enhance exopher production, revealing stress-type, stress-level, and temporal constraints on the process. We identify three pathways that promote fasting-induced exophergenesis: lipid synthesis, FGF/RAS/MAPK, and EGF/RAS/MAPK. In establishing an initial molecular model for transtissue requirements for fasting-induced exopher elevation in neurons, we report molecular insights into the regulation of aggregate transfer biology, relevant to the fundamental mysteries of neurodegenerative disease., In human neurodegenerative diseases, neurons can transfer toxic protein aggregates to surrounding cells, promoting pathology via poorly understood mechanisms. In Caenorhabditis elegans, proteostressed neurons can expel neurotoxic proteins in large, membrane-bound vesicles called exophers. We investigated how specific stresses impact neuronal trash expulsion to show that neuronal exopher production can be markedly elevated by oxidative and osmotic stress. Unexpectedly, we also found that fasting dramatically increases exophergenesis. Mechanistic dissection focused on identifying nonautonomous factors that sense and activate the fasting-induced exopher response revealed that DAF16/FOXO-dependent and -independent processes are engaged. Fasting-induced exopher elevation requires the intestinal peptide transporter PEPT-1, lipid synthesis transcription factors Mediator complex MDT-15 and SBP-1/SREPB1, and fatty acid synthase FASN-1, implicating remotely initiated lipid signaling in neuronal trash elimination. A conserved fibroblast growth factor (FGF)/RAS/MAPK signaling pathway that acts downstream of, or in parallel to, lipid signaling also promotes fasting-induced neuronal exopher elevation. A germline-based epidermal growth factor (EGF) signal that acts through neurons is also required for exopher production. Our data define a nonautonomous network that links food availability changes to remote, and extreme, neuronal homeostasis responses relevant to aggregate transfer biology.

Despite the importance of fertilization, the molecular basis of sperm-egg interaction is not well understood. In a forward genetics screen for fertility mutants in Caenorhabditis elegans we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell-autonomously and localizes to the surface of the spermatozoa. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments., Author summary P-type ATPases are a large family of transporters that are conserved from unicellular eukaryotes and plants to metazoans. Structurally and functionally, they fall into five subfamilies, P1 to P5, of which the latter is further divided into P5A and P5B-type ATPases. Unlike for other P-type ATPases, no mutant phenotypes for the P5A-type ATPases have been described in metazoans. Here, we show that the catp-8/P5A-ATPase in the nematode Caenorhabditis elegans is involved in multiple aspects of neuronal patterning, including neuronal migrations as well as axon guidance and dendrite patterning. A functional fluorescent reporter fusion shows the CATP-8/P5A-ATPase is expressed in most, if not all, tissues in the endoplasmic reticulum and that catp-8 can function both in neurons and surrounding tissues from where it orchestrates neuronal development. Genetically, catp-8 acts in multiple pathways during these processes, including the Wnt signaling and the Menorin pathway. Imaging studies suggest that the catp-8/P5A-ATPase is necessary for proper localization of cell-surface transmembrane proteins to dendrites of sensory neurons, but likely not for their trafficking. In summary, we propose that CATP-8/P5A-ATPase serves a function in the ER during development of select neurons, by localizing certain transmembrane, and possibly, secreted proteins.

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease. C. elegans adult neurons that express aggregating proteins can extrude large (~4 µm) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called "exophers" and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases. While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons. Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Tetraspanins are a unique family of 4-pass transmembrane proteins that play important roles in a variety of cell biological processes. We have previously shown that 2 paralogous tetraspanins in Caenorhabditis elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling. The underlying molecular mechanisms, however, are not fully understood. In this study, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple cell types, and that both proteins are localized on the cell surface and in various types of endosomes, including early, late, and recycling endosomes. Animals lacking both TSP-12 and TSP-14 exhibit reduced cell-surface levels of the BMP type II receptor DAF-4/BMPRII, along with impaired endosome morphology and mislocalization of DAF-4/BMPRII to late endosomes and lysosomes. These findings indicate that TSP-12 and TSP-14 are required for the recycling of DAF-4/BMPRII. Together with previous findings that the type I receptor SMA-6 is recycled via the retromer complex, our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors and highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. As TSP-12 and TSP-14 are conserved in mammals, our findings suggest that the mammalian TSP-12 and TSP-14 homologs may also function in regulating transmembrane protein recycling and BMP signaling.

After endocytosis, transmembrane cargoes such as signaling receptors, channels, and transporters enter endosomes where they are sorted to different destinations. Retromer and ESCRT (endosomal sorting complex required for transport) are functionally distinct protein complexes on endosomes that direct cargo sorting into the recycling retrograde transport pathway and the degradative multivesicular endosome pathway (MVE), respectively. Cargoes destined for degradation in lysosomes are decorated with K63-linked ubiquitin chains, which serve as an efficient sorting signal for entry into the MVE pathway. Defects in K63-linked ubiquitination disrupt MVE sorting and degradation of membrane proteins. Here, we unexpectedly found that UBC-13, the E2 ubiquitin-conjugating enzyme that generates K63-linked ubiquitin chains, is essential for retrograde transport of multiple retromer-dependent cargoes including MIG-14/Wntless. Loss of ubc-13 disrupts MIG-14/Wntless trafficking from endosomes to the Golgi, causing missorting of MIG-14 to lysosomes and impairment of Wnt-dependent processes. We observed that retromer-associated SNX-1 and the ESCRT-0 subunit HGRS-1/Hrs localized to distinct regions on a common endosome in wild type but overlapped on ubc-13(lf) endosomes, indicating that UBC-13 is important for the separation of retromer and ESCRT microdomains on endosomes. Our data suggest that cargo ubiquitination mediated by UBC-13 plays an important role in maintaining the functionally distinct subdomains to ensure efficient cargo segregation on endosomes.

Members of the superfamily of solute carrier (SLC) transmembrane proteins transport diverse substrates across distinct cellular membranes. Three SLC protein families transport distinct neurotransmitters into synaptic vesicles to enable synaptic transmission in the nervous system. Among them is the SLC17A6/7/8 family of vesicular glutamate transporters, which endows specific neuronal cell types with the ability to use glutamate as a neurotransmitter. The genome of the nematode Caenorhabditis elegans encodes three SLC17A6/7/8 family members, one of which, eat-4/VGLUT, has been shown to be involved in glutamatergic neurotransmission. Here, we describe our analysis of the two remaining, previously uncharacterized SLC17A6/7/8 family members, vglu-2 and vglu-3. These two genes directly neighbor one another and are the result of a recent gene duplication event in C. elegans, but not in other Caenorhabditis species. Compared to EAT-4, the VGLU-2 and VGLU-3 protein sequences display a more distant similarity to canonical, vertebrate VGLUT proteins. We tagged both genomic loci with gfp and detected no expression of vglu-3 at any stage of development in any cell type of both C. elegans sexes. In contrast, vglu-2::gfp is dynamically expressed in a restricted set of distinct cell types. Within the nervous system, vglu-2::gfp is exclusively expressed in a single interneuron class, AIA, where it localizes to vesicular structures in the soma, but not along the axon, suggesting that VGLU-2 may not be involved in synaptic transport of glutamate. Nevertheless, vglu-2 mutants are partly defective in the function of the AIA neuron in olfactory behavior. Outside the nervous system, VGLU-2 is expressed in collagen secreting skin cells where VGLU-2 most prominently localizes to early endosomes, and to a lesser degree to apical clathrin-coated pits, the trans-Golgi network, and late endosomes. On early endosomes, VGLU-2 colocalizes most strongly with the recycling promoting factor SNX-1, a retromer component. Loss of vglu-2 affects the permeability of the collagen-containing cuticle of the worm, and based on the function of a vertebrate VGLUT1 protein in osteoclasts, we speculate that vglu-2 may have a role in collagen trafficking in the skin. We conclude that C. elegans SLC17A6/7/8 family members have diverse functions within and outside the nervous system.

Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases., Author summary In order to function properly, cells must continually import materials from the outside. This process, termed endocytosis, is necessary for the uptake of nutrients and for interpreting signals coming from the external environment or from within the body. These signals are critical during animal development but also affect many types of cell behaviors throughout life. In our current work, we show that several highly conserved proteins in the nematode Caenorhabditis elegans, NEKL-2 and NEKL-3, regulate endocytosis. The human counterparts of NEKL-2 and NEKL-3 have been implicated in cardiovascular and renal diseases as well as many types of cancers. However, their specific functions within cells is incompletely understood and very little is known about their role in endocytosis or how this role might impact disease processes. Here we use several complementary approaches to characterize the specific functions of C. elegans NEKL-2 and NEKL-3 in endocytosis and show that their human counterparts likely have very similar functions. This work paves the way to a better understanding of fundamental biological processes and to determining the cellular functions of proteins connected to human diseases.

Syndapin/pascin-family F-BAR domain proteins bind directly to membrane lipids and are associated with actin dynamics at the plasma membrane. Previous reports also implicated mammalian syndapin 2 in endosome function during receptor recycling, but precise analysis of a putative recycling function for syndapin in mammalian systems is difficult because of its effects on the earlier step of endocytic uptake and potential redundancy among the three separate genes that encode mammalian syndapin isoforms. Here we analyze the endocytic transport function of the only Caenorhabditis elegans syndapin, SDPN-1. We find that SDPN-1 is a resident protein of the early and basolateral recycling endosomes in the C. elegans intestinal epithelium, and sdpn-1 deletion mutants display phenotypes indicating a block in basolateral recycling transport. sdpn-1 mutants accumulate abnormal endosomes positive for early endosome and recycling endosome markers that are normally separate, and such endosomes accumulate high levels of basolateral recycling cargo. Furthermore, we observed strong colocalization of endosomal SDPN-1 with the F-actin biosensor Lifeact and found that loss of SDPN-1 greatly reduced Lifeact accumulation on early endosomes. Taken together, our results provide strong evidence for an in vivo function of syndapin in endocytic recycling and suggest that syndapin promotes transport via endosomal fission.

Macroautophagy/autophagy involves the formation of an autophagosome, a double-membrane vesicle that delivers sequestered cytoplasmic cargo to lysosomes for degradation and recycling. Closely related, endocytosis mediates the sorting and transport of cargo throughout the cell, and both processes are important for cellular homeostasis. However, how endocytic proteins functionally intersect with autophagy is not clear. Mutations in the DAF-2/insulin-like IGF-1 (INSR) receptor at the permissive temperature result in a small increase in GFP::LGG-1 foci, i.e. autophagosomes, but a large increase at the nonpermissive temperature, allowing us to control the level of autophagy. In a RNAi screen for endocytic genes that alter the expression of GFP::LGG-1 in daf-2 mutants, we identified RAB-10, a small GTPase that regulates basolateral endocytosis. Loss of rab-10 in daf-2 mutants results in more GFP::LGG-1-positive foci at the permissive, but less GFP::LGG-1 or SQST-1::GFP foci at the nonpermissive temperature. As previously reported, loss of rab-10 alone resulted in an increase of GFP:LGG-1 foci. Exposure of rab-10 mutant animals to chloroquine, a known inhibitor of autophagic flux, failed to increase the number of GFP::LGG-1 foci. Moreover, colocalization between LMP-1::tagRFP and GFP::LGG-1 (the lysosome and autophagosome reporters) was decreased in daf-2; rab-10 dauers at the nonpermissive temperature. Intriguingly, RAB-10 was required to maintain the normal size of GFP::ATG-9-positive structures in daf-2 mutants at both the permissive and nonpermissive temperature. Finally, we found that RAB-10 GTPase cycling was required to control the size of GFP::ATG-9 foci. Collectively, our data support a model where rab-10 controls autophagic flux by regulating autophagosome formation and maturation.

After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1-labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8 microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenon we observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.

The transforming growth factor β (TGFβ) superfamily of signaling pathways, including the bone morphogenetic protein (BMP) subfamily of ligands and receptors, controls a myriad of developmental processes across metazoan biology. Transport of the receptors from the plasma membrane to endosomes has been proposed to promote TGFβ signal transduction and shape BMP-signaling gradients throughout development. However, how postendocytic trafficking of BMP receptors contributes to the regulation of signal transduction has remained enigmatic. Here we report that the intracellular domain of Caenorhabditis elegans BMP type I receptor SMA-6 (small-6) binds to the retromer complex, and in retromer mutants, SMA-6 is degraded because of its missorting to lysosomes. Surprisingly, we find that the type II BMP receptor, DAF-4 (dauer formation-defective-4), is retromer-independent and recycles via a distinct pathway mediated by ARF-6 (ADP-ribosylation factor-6). Importantly, we find that loss of retromer blocks BMP signaling in multiple tissues. Taken together, our results indicate a mechanism that separates the type I and type II receptors during receptor recycling, potentially terminating signaling while preserving both receptors for further rounds of activation.

After endocytosis, a selective endocytic recycling process returns many endocytosed molecules back to the plasma membrane. The RAB-10/Rab10 GTPase is known to be a key recycling regulator for specific cargo molecules. New evidence, focused on C. elegans RAB-10 in polarized epithelia, points to a key role of RAB-10 in the regulation of endosomal phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) levels. In turn, PI(4,5)P2 levels strongly influence the recruitment of many peripheral membrane proteins, including those important for vesicle budding through their membrane bending activities. Part of the effect of RAB-10 on endosomal PI(4,5)P2 is through its newly identified effector CNT-1, a predicted GTPase activating protein (GAP) of the small GTPase ARF-6/Arf6. In mammals PI(4,5)P2 generating enzymes are known Arf6 effectors. In C. elegans we found that RAB-10, CNT-1 and ARF-6 are present on the same endosomes, that RAB-10 recruits CNT-1 to endosomes, and that loss of CNT-1 or RAB-10 leads to overaccumulation of endosomal PI(4,5)P2, presumably via hyperactivation of endosomal ARF-6. In turn this leads to over-recruitment of PI(4,5)P2-dependent membrane-bending proteins RME-1/Ehd and SDPN-1/Syndapin/PACSIN. Conversely, in arf-6 mutants, endosomal PI(4,5)P2 levels were reduced and endosomal recruitment of RME-1 and SDPN-1 failed. This work makes an unexpected link between distinct classes of small GTPases that control endocytic recycling, and provides insight into how this interaction affects endosome function at the level of lipid phosphorylation.

EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation., Author Summary Endosomes are intracellular organelles that sort protein and lipid components integral to the membrane, as well as more loosely associated lumenal content, for delivery to distinct intracellular destinations. Endosomes associated with recycling cargo back to the plasma membrane are often tubular in morphology, and this morphology is thought to be essential for recycling function. Our previous work identified a particularly dramatic network of endosomal tubules involved in membrane protein recycling in the basolateral intestinal epithelial cells of C. elegans. Our subsequent genetic analysis of basolateral recycling in this system identified a number of key regulators of these endosomes, including the small GTPase RAB-10 and its effector EHBP-1. Our new work presented here shows that EHBP-1 promotes endosomal tubulation by linking the membrane lipid PI(4,5)P2 to the actin cytoskeleton, and that the linkage of EHBP-1 to actin is enhanced by the interaction of EHBP-1 with RAB-10. This work has broad implications for how endosomal tubulation occurs in all cells, and has specific implications for the role of EHBP-1 in related processes such as insulin-stimulated recycling of glucose transporters in human adipocytes, a process intimately linked to type II diabetes.

RAB-6.2, its effector LIN-10, and the retromer complex maintain synaptic strength by recycling postsynaptic glutamate receptors along the retrograde transport pathway., Regulated membrane trafficking of AMPA-type glutamate receptors (AMPARs) is a key mechanism underlying synaptic plasticity, yet the pathways used by AMPARs are not well understood. In this paper, we show that the AMPAR subunit GLR-1 in Caenorhabditis elegans utilizes the retrograde transport pathway to regulate AMPAR synaptic abundance. Mutants for rab-6.2, the retromer genes vps-35 and snx-1, and rme-8 failed to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. In contrast, expression of constitutively active RAB-6.2 drove the retrograde transport of GLR-1 from dendrites back to cell body Golgi. We also find that activated RAB-6.2 bound to and colocalized with the PDZ/phosphotyrosine binding domain protein LIN-10. RAB-6.2 recruited LIN-10. Moreover, the regulation of GLR-1 transport by RAB-6.2 required LIN-10 activity. Our results demonstrate a novel role for RAB-6.2, its effector LIN-10, and the retromer complex in maintaining synaptic strength by recycling AMPARs along the retrograde transport pathway.

SNARE domain proteins are key molecules mediating intracellular fusion events. SNAP25 family proteins are unique target-SNAREs possessing two SNARE domains. Here we report the genetic, molecular, and cell biological characterization of C. elegans SNAP-29. We found that snap-29 is an essential gene required throughout the life-cycle. Depletion of snap-29 by RNAi in adults results in sterility associated with endomitotic oocytes and pre-meiotic maturation of the oocytes. Many of the embryos that are produced are multinucleated, indicating a defect in embryonic cytokinesis. A profound defect in secretion by oocytes and early embryos in animals lacking SNAP-29 appears to be the underlying defect connecting these phenotypes. Further analysis revealed defects in basolateral and apical secretion by intestinal epithelial cells in animals lacking SNAP-29, indicating a broad requirement for this protein in the secretory pathway. A SNAP-29-GFP fusion protein was enriched on recycling endosomes, and loss of SNAP-29 disrupted recycling endosome morphology. Taken together these results suggest a requirement for SNAP-29 in the fusion of post-Golgi vesicles with the recycling endosome for cargo to reach the cell surface.

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. Here we show binding of RAB-10 to EHBP-1, a CH-domain protein, and demonstrate a requirement for EHBP-1 in RAB-10–regulated transport in both of these tissues., Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

The RME1 ATPases are implicated in endocytic recycling. C. elegans RME1 interacts with Amphiphysin to regulate endocytic recycling in vivo and the two proteins cooperate in the generation of cargo carriers in vitro. The interaction is conserved in other eukaryotes.

SummaryThe molecular underpinnings of the oocyte-to-embryo transition are poorly understood. Here we show that two protein tyrosine phosphatase-like (PTPL) family proteins, EGG-4 and EGG-5, are required for key events of the oocyte-to-embryo transition in Caenorhabditis elegans. The predicted EGG-4 and EGG-5 amino acid sequences are 99% identical and their functions are redundant. In embryos lacking EGG-4 and EGG-5, we observe defects in meiosis, polar body formation, the block to polyspermy, F-actin dynamics, and eggshell deposition. During oogenesis, EGG-4 and EGG-5 assemble at the oocyte cortex with the previously identified regulators or effectors of the oocyte-to-embryo transition EGG-3, CHS-1, and MBK-2 [1, 2]. All of these molecules share a complex interdependence with regards to their dynamics and subcellular localization. Shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the cortex during meiotic anaphase I. Therefore, EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle.

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG-14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome-associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J-domain protein RME-8 associates with the retromer component SNX-1. Loss of SNX-1, RME-8, or the clathrin chaperone Hsc70/HSP-1 leads to over-accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG-14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME-8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.

Endocytic recycling is coordinated with endocytic uptake to control the composition of the plasma membrane. Although much of our understanding of endocytic recycling has come from studies on the transferrin receptor, a protein internalized through clathrin-dependent endocytosis, increased interest in clathrin-independent endocytosis has led to the discovery of new endocytic recycling systems. Recent insights into the regulatory mechanisms that control endocytic recycling have focused on recycling through tubular carriers and the return to the cell surface of cargo that enters cells through clathrin-independent mechanisms. Recent work emphasizes the importance of regulated recycling in such diverse processes as cytokinesis, cell adhesion and morphogenesis, cell fusion, and learning and memory.

How endosomes contribute to the maintenance of vesicular structures at presynaptic terminals remains controversial and poorly understood. Here, we have investigated synaptic endosomal compartments in the presynaptic terminals of C. elegans GABAergic motor neurons. Using RAB reporters, we find that several subsynaptic compartments reside in, or near, presynaptic regions. Loss of function in the C. elegans JIP3 protein, UNC-16, causes a RAB-5-containing compartment to accumulate abnormally at presynaptic terminals. Ultrastructural analysis shows that synapses in unc-16 mutants contain reduced number of synaptic vesicles, accompanied by an increase in the size and number of cisternae. FRAP analysis revealed a slow recovery of RAB-5 in unc-16 mutants, suggestive of an impairment of RAB-5 activity state and local vesicular trafficking. Overexpression of RAB-5:GDP partially suppresses, whereas overexpression of RAB-5:GTP enhances, the synaptic defects of unc-16 mutants. Our data demonstrate a novel function of UNC-16 in the regulation of synaptic membrane trafficking and suggest that the synaptic RAB-5 compartment contributes to synaptic vesicle biogenesis or maintenance.

Clathrin is a coat protein involved in vesicle budding from several membrane-bound compartments within the cell. Here we present an analysis of a temperature-sensitive (ts) mutant of clathrin heavy chain (CHC) in a multicellular animal. As expected Caenorhabditis elegans chc-1 ( b1025ts) mutant animals are defective in receptor-mediated endocytosis and arrest development soon after being shifted to the restrictive temperature. Steady-state clathrin levels in these mutants are reduced by more than 95% at all temperatures. Hub interactions and membrane associations are lost at the restrictive temperature. chc-1 ( b1025ts ) animals become paralyzed within minutes of exposure to the restrictive temperature because of a defect in the nervous system. Surprisingly synaptic vesicle number is not reduced in chc-1(b1025ts ) animals. Consistent with the normal number of vesicles, postsynaptic miniature currents occur at normal frequencies. Taken together, these results indicate that a high level of CHC activity is required for receptor-mediated endocytosis in nonneuronal cells but is largely dispensable for maintenance of synaptic vesicle pools.

The evolutionarily conserved Eps15 homology domain (EHD)/receptor-mediated endocytosis (RME)-1 family of C-terminal EH domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these adenosine triphosphatases function on endosomal membranes in mammals and Caenorhabditis elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large guanosine triphosphatases.

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

We previously identified a novel type of caveolin-enriched secretory vesicle in Caenorhabditis elegans oocytes. These vesicles undergo synchronous fusion with the plasma membrane immediately after fertilization, suggesting that they could be cortical granules that have been described in diverse animal species. Here, we report that these vesicles are indeed cortical granules, delivering essential chondroitin proteoglycans and mucin-like glycoproteins to the early embryonic extracellular matrices (ECMs). Furthermore, we have found that the small GTPase RAB-11 and the target-SNARE SYN-4 are required for cortical granule excoytosis after fertilization. In oocytes, SYN-4 localizes mainly to the plasma membrane, whereas GFP::RAB-11 accumulates transiently on the cortical granules during ovulation, immediately prior to fertilization. Importantly, cytokinesis defects in early embryos are commonly observed after depletion of either rab-11 or syn-4, producing a phenotype very similar to that observed after blockade of chondroitin synthesis. Taken together, our results indicate that at least part of the essential role for RAB-11 and SYN-4 in early embryogenesis is in the targeting of cortical granules to the plasma membrane during the precisely regulated secretion of ECM components.

BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

Summary Fertilization triggers egg activation and converts the egg into a developing embryo. The events of this egg-to-embryo transition typically include the resumption of meiosis, the reorganization of the cortical actin cytoskeleton, and the remodeling of the oocyte surface [1–3]. The factors that regulate sperm-dependent egg-activation events are not well understood. Caenorhabditis elegans EGG-3, a member of the protein tyrosine phosphatase-like (PTPL) family [4], is essential for regulating cell-surface and cortex rearrangements during egg activation in response to sperm entry. Although fertilization occurred normally in egg-3 mutants, the polarized dispersal of F-actin is altered, a chitin eggshell is not formed, and no polar bodies are produced. EGG-3 is associated with the oocyte plasma membrane in a pattern that is similar to CHS-1 and MBK-2. CHS-1 is required for eggshell deposition [5–7], whereas MBK-2 is required for the degradation of maternal proteins during the egg-to-embryo transition [8–12]. The localization of CHS-1 and EGG-3 are interdependent and both genes were required for the proper localization of MBK-2 in oocytes. Therefore, EGG-3 plays a central role in egg activation by influencing polarized F-actin dynamics and the localization or activity of molecules that are directly involved in executing the egg-to-embryo transition.

In a genome-wide RNA-mediated interference screen for genes required in membrane traffic - including endocytic uptake, recycling from endosomes to the plasma membrane, and secretion - we identified 168 candidate endocytosis regulators and 100 candidate secretion regulators. Many of these candidates are highly conserved among metazoans but have not been previously implicated in these processes. Among the positives from the screen, we identified PAR-3, PAR-6, PKC-3 and CDC-42, proteins that are well known for their importance in the generation of embryonic and epithelial-cell polarity. Further analysis showed that endocytic transport in Caenorhabditis elegans coelomocytes and human HeLa cells was also compromised after perturbation of CDC-42/Cdc42 or PAR-6/Par6 function, indicating a general requirement for these proteins in regulating endocytic traffic. Consistent with these results, we found that tagged CDC-42/Cdc42 is enriched on recycling endosomes in C. elegans and mammalian cells, suggesting a direct function in the regulation of transport.

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions., Author Summary When cargo is internalized from the cell surface by endocytosis, it enters a series of intracellular organelles called endosomes. Endosomes sort cargo, such that some cargos are sent to the lysosome for degradation, while others are recycled to the plasma membrane. Small GTPase proteins of the Rabs family are master regulators of endosomes, functioning by acting as molecular switches. As cargo moves through the endosomal system, it must pass from the domain controlled by one Rab-GTPase to the domain controlled by another. Little is known about how transitions along the recycling pathway are controlled. Here we analyze a group of protein interactions that act along the early-to-recycling pathway. Our work shows that RAB-5 deactivation mediated by TBC-2 and its recruiters RAB-10 and AMPH-1 is important for cargo recycling. This work provides mechanistic insight into how Rab proteins controlling different steps of trafficking interact during endocytic recycling.

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

The microscopic nematode Caenorhabditis elegans (C. elegans) serves as an excellent animal model for studying membrane traffic. This is due in part to its highly advanced genetics and genomics, and a transparent body that allows the visualization of fluorescently tagged molecules in the physiologically relevant context of the intact organism. Notably, C. elegans oocytes, coelomocytes, and intestinal epithelia have been established as facile cellular models to explore nonpolarized and polarized cell membrane trafficking mechanisms. In this chapter, we describe in vivo C. elegans assays, utilizing fluorescent dyes or proteins, to examine the molecular mechanisms that control endocytosis and endocytic recycling. Tissue-specific, steady-state imaging and associated quantitative analysis allow the identification and interpretation of subcellular events in the intact animal. To better understand the kinetic characteristics of recycling tubules that mediate membrane protein recycling, we describe recently developed dynamic-imaging assays in intestinal epithelial cells. Such methods bring new clarity to the system, helping to elucidate the functional roles of recycling mediators.

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

A fusion of cutting-edge research in cell biology, developmental biology and immunology made the recent workshop on Membrane Dynamics in Endocytosis an outstanding success. Members of an increasingly diverse community converged upon the small town of Sant Feliu de Guixols on the coast of Spain, between September 17–22, 2005, to discuss common themes emerging from their studies on membrane transport. Organized by Margaret Robinson (Cambridge, UK) and Howard Riezman (Geneva, Switzerland), the meeting covered diverse topics that highlighted essential roles for endocytosis during cell growth, development and parasitic invasion.

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin–EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.

Myotubularins (MTMs) constitute a large family of lipid phosphatases that specifically dephosphorylate phosphatidylinositol (3)P. MTM1 and MTM2 are mutated in X-linked myotubular myopathy and Charcot-Marie-Tooth disease (type 4B), respectively, although the mechanisms whereby MTM dysfunction leads to these diseases is unknown. To gain insight into MTM function, we undertook the study of MTMs in the nematode Caenorhabditis elegans, which possesses representative homologues of the four major subgroups of MTMs identified in mammals. As in mammals, we found that C. elegans MTMs mediate distinct functions. let-512 (vps34) encodes the C. elegans homologue of the yeast and mammalian homologue of the phosphatidylinositol 3-kinase Vps34. We found that reduction of mtm-6 (F53A2.8) function by RNA inhibition rescued the larval lethality of let-512 (vps34) mutants and that the reduction of mtm-1 (Y110A7A.5) activity by RNA inhibition rescued the endocytosis defect of let-512 animals. Together, these observations provide genetic evidence that MTMs negatively regulate phosphatidylinositol (3)P levels. Analysis of MTM expression patterns using transcriptional green fluorescence protein reporters demonstrated that these two MTMs exhibit mostly non-overlapping expression patterns and that MTM-green fluorescence protein fusion proteins are localized to different subcellular locations. These observations suggest that some of the different functions of MTMs might, in part, be a consequence of unique expression and localization patterns. However, our finding that at least three C. elegans MTMs play essential roles in coelomocyte endocytosis, a process that also requires VPS34, indicates that MTMs do not simply turn off VPS34 but unexpectedly also function as positive regulators of biological processes.

We discuss in this review recent studies using the worm Caenorhabditis elegans to decipher endocytic trafficking in a multicellular organism. Recent advances, including in vivo assay systems, new genetic screens, comparative functional analysis of conserved proteins, and RNA-mediated interference (RNAi) in C. elegans, are being used to study the functions of known membrane trafficking factors and to identify new ones. The ability to monitor endocytosis in vivo in worms allows one to test current endocytosis models and to demonstrate the physiological significance of factors identified by genetic and biochemical methods. The available human genome sequence facilitates comparative studies where human homologs of new factors identified in C. elegans can be quickly assayed for similar function using traditional cell biological methods in mammalian cell systems. New studies in C. elegans have used a combination of these techniques to reveal novel metazoan-specific trafficking factors required for endocytosis. Many more metazoan-specific trafficking factors and insights into the mechanisms of endocytosis are likely to be uncovered by analysis in C. elegans.

The counterbalancing action of the endocytosis and secretory pathways maintains a dynamic equilibrium that regulates the composition of the plasma membrane, allowing it to maintain homeostasis and to change rapidly in response to alterations in the extracellular environment and/or intracellular metabolism. These pathways are intimately integrated with intercellular signaling systems and play critical roles in all cells. Studies in Caenorhabditis elegans have revealed diverse roles of membrane trafficking in physiology and development and have also provided molecular insight into the fundamental mechanisms that direct cargo sorting, vesicle budding, and membrane fisson and fusion. In this review, we summarize progress in understanding membrane trafficking mechanisms derived from work in C. elegans, focusing mainly on work done in non-neuronal cell-types, especially the germline, early embryo, coelomocytes, and intestine.

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures. (J Histochem Cytochem 49:949–956, 2001)

Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1–L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol by C. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.

RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis genes in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.

In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Eps15-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-1 mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by theC. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. eleganspredicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme(receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.

Previous work indicated that sel-1 functions as a negative regulator of lin-12 activity, and predicted that SEL-1 is a secreted or membrane associated protein. In this study, we describe cell ablation experiments that suggest sel-1 mutations elevate lin-12 activity cell autonomously. We also use transgenic approaches to demonstrate that the predicted signal sequence of SEL-1 can direct secretion and is important for function, while a C-terminal hydrophobic region is not required for SEL-1 function. In addition, by analyzing SEL-1 localization using specific antisera we find that SEL-1 is localized intracellularly, with a punctate staining pattern suggestive of membrane bound vesicles. We incorporate these observations, and new information about a related yeast gene, into a proposal for a possible mechanism for SEL-1 function in LIN-12 turnover.

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001, eLife digest The cell membrane is a busy place, with cell-surface proteins continually added and removed according to the needs of the cell. Each protein extends a polypeptide tail into the cell cytoplasm. When a protein is to be removed from the cell surface, its tail recruits a protein complex known as the AP2 adaptor to the membrane. AP2 then recruits a coat protein called clathrin, which forms a spherical scaffold around the adaptor, the target protein and the surrounding membrane, enclosing them inside a vesicle that breaks off from the membrane and enters the cell. Endocytosis is particularly common in neurons, which use it as a means of recycling proteins at synapses—the contact points between nerve cells. However, it is unclear whether synaptic-vesicle recycling also involves clathrin and AP2. To address this question, Gu et al. examined mutant nematode worms (C. elegans) in which the composition of AP2 had been altered. AP2 has four subunits, called α, β2, μ2 and σ2, and Gu et al. produced worms that lack either the α- or μ2-subunit, or both. Few worms that lacked both subunits survived. Surprisingly, however, worms that lacked just one subunit were viable, despite previous evidence that AP2 requires all four subunits to be functional. Nevertheless, these single mutants produced 30% fewer synaptic vesicles compared to wild-type worms. To examine the consequences of both subunits being absent, Gu et al. rescued the double mutants by selectively expressing AP2 in their skin. These animals—which still lack AP2 in their nervous systems—produced 70% fewer synaptic vesicles than their wild-type counterparts. The results show that AP2 does not need all four of its subunits and that it can exist as two semi-independent hemicomplexes. Moreover, Gu et al. show that C. elegans uses at least two endocytotic mechanisms (AP2-dependent and independent) to recycle vesicles and so maintain synaptic function. DOI: http://dx.doi.org/10.7554/eLife.00190.002

Neurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons. rab -5 and rab -10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other’s GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi–endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting.