Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte

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 the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted 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.     

RME-8, a Conserved J-Domain Protein, Is Required for Endocytosis in Caenorhabditis elegans

By genetic analysis of Caenorhabditis elegans mutants defective in yolk uptake, we have identified new molecules functioning in the endocytosis pathway. Here we describe a novel J-domain-containing protein, RME-8, identified by such genetic analysis. RME-8 is required for receptor-mediated endocytosis and fluid-phase endocytosis in various cell types and is essential for C. elegans development and viability. In the macrophage-like coelomocytes, RME-8 localizes to the limiting membrane of large endosomes. Endocytosis markers taken up by the coelomocytes rapidly accumulate in these large RME-8-positive endosomes, concentrate in internal subendosomal structures, and later appear in RME-8-negative lysosomes. rme-8 mutant coelomocytes fail to accumulate visible quantities of endocytosis markers. These observations show that RME-8 functions in endosomal trafficking before the lysosome. RME-8 homologues are found in multicellular organisms from plants to humans but not in the yeast Saccharomyces cerevisiae. These sequence homologies suggest that RME-8 fulfills a conserved function in multicellular organisms.     

UEV-1 is an ubiquitin-conjugating enzyme variant that regulates glutamate receptor trafficking in C. elegans neurons

The regulation of AMPA-type glutamate receptor (AMPAR) membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect--a decrease in spontaneous reversals in locomotion--consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking.     

The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila

By screening for mutants exhibiting interactions with a dominant-negative dynamin, we have identified the Drosophila homologue of receptor-mediated endocytosis (Rme) 8, a J-domain-containing protein previously shown to be required for endocytosis in Caenorhabditis elegans. Analysis of Drosophila Rme-8 mutants showed that internalization of Bride of sevenless and the uptake of tracers were blocked. In addition, endosomal organization and the distribution of clathrin were greatly disrupted in Rme-8 cells, suggesting that Rme-8 participates in a clathrin-dependent process. The phenotypes of Rme-8 mutants bear a strong resemblance to those of Hsc70-4, suggesting that these two genes act in a common pathway. Indeed, biochemical and genetic data demonstrated that Rme-8 interacts specifically with Hsc70-4 via its J-domain. Thus, Rme-8 appears to function as an unexpected but critical cochaperone with Hsc70 in endocytosis. Because Hsc70 is known to act in clathrin uncoating along with auxilin, another J-protein, its interaction with Rme-8 indicates that Hsc70 can act with multiple cofactors, possibly explaining its pleiotropic effects on the endocytic pathway.     

Studies of synaptic vesicle endocytosis in the nematode C. elegans

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.     

Lipid droplets as fat storage organelles in Caenorhabditis elegans: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

Lipid droplets are evolutionarily conserved organelles where cellular fat storage and mobilization are exquisitely regulated. Recent studies have defined lipid droplets in C. elegans and explored how they are regulated by genetic and dietary factors. C. elegans offers unique opportunities to visualize lipid droplets at single-cell resolution in live animals. The development of novel microscopy techniques and protein markers for lipid droplets will accelerate studies on how nutritional states and subcellular organization are linked in vivo. Together with powerful tools for genetic and biochemical analysis of metabolic pathways, alteration in lipid droplet abundance, size, and distribution in C. elegans can be readily connected to whole-animal energy homeostasis, behavior, and life span. Therefore, further studies on lipid droplets in C. elegans promise to yield valuable insights that complement our knowledge gained from yeast, Drosophila, and mammalian systems on cellular and organismal fat storage.     

Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans

Unlike apoptosis, necrotic cell death is characterized by marked loss of plasma membrane integrity. Leakage of cytoplasmic material to the extracellular space contributes to cell demise, and is the cause of acute inflammatory responses, which typically accompany necrosis. The mechanisms underlying plasma membrane damage during necrotic cell death are not well understood. We report that endocytosis is critically required for the execution of necrosis. Depletion of the key endocytic machinery components dynamin, synaptotagmin and endophilin suppresses necrotic neurodegeneration induced by diverse genetic and environmental insults in C. elegans. We used genetically encoded fluorescent markers to monitor the formation and fate of specific types of endosomes during cell death in vivo. Strikingly, we find that the number of early and recycling endosomes increases sharply and transiently upon initiation of necrosis. Endosomes subsequently coalesce around the nucleus and disintegrate during the final stage of necrosis. Interfering with kinesin-mediated endosome trafficking impedes cell death. Endocytosis synergizes with autophagy and lysosomal proteolytic mechanisms to facilitate necrotic neurodegeneration. These findings demonstrate a prominent role for endocytosis in cellular destruction during neurodegeneration, which is likely conserved in metazoans.     

Epsin potentiates Notch pathway activity in Drosophila and C. elegans

Endocytosis and trafficking within the endocytosis pathway are known to modulate the activity of different signaling pathways. Epsins promote endocytosis and are postulated to target specific proteins for regulated endocytosis. Here, we present a functional link between the Notch pathway and epsins. We identify the Drosophila ortholog of epsin, liquid facets (lqf), as an inhibitor of cardioblast development in a genetic screen for mutants that affect heart development. We find that lqf inhibits cardioblast development and promotes the development of fusion-competent myoblasts, suggesting a model in which lqf acts on or in fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit essentially identical heart phenotypes, and lqf genetically interacts with the Notch pathway during multiple Notch-dependent events in Drosophila. We extended the link between the Notch pathway and epsin function to C. elegans, where the C. elegans lqf ortholog acts in the signaling cell to promote the glp-1/Notch pathway activity during germline development. Our results suggest that epsins play a specific, evolutionarily conserved role to promote Notch signaling during animal development and support the idea that they do so by targeting ligands of the Notch pathway for endocytosis.     

Cell migration and axon growth cone guidance in Caenorhabditis elegans.

In the past year, several new components involved in cell migration and axon guidance have been identified by genetic analysis in Caenorhabditis elegans, taking us a step closer to being able to trace the pathways which mediate these processes. The completion of the C. elegans genome sequencing project has provided us with the knowledge of the full spectrum of genes that might be involved in cell migration and axon guidance, and can facilitate the analysis of components that have been shown to be important for these processes in other systems.     

Conserved genes act as modifiers of invertebrate SMN loss of function defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.     

A splice variant of the Drosophila vesicular monoamine transporter contains a conserved trafficking domain and functions in the storage of dopamine, serotonin, and octopamine

Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.     

Human brain synembryn interacts with Gsalpha and Gqalpha and is translocated to the plasma membrane in response to isoproterenol and carbachol

Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.     

The Caenorhabditis elegans muscle specific serpin, SRP-3, neutralizes chymotrypsin-like serine peptidases

Members of the intracellular serpin family may help regulate apoptosis, tumor progression, and metastasis. However, their in vivo functions in the context of a whole organism have not been easily defined. To better understand the biology of these serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model organism. Previous in silico analysis suggested that the C. elegans genome harbors nine serpin-like sequences bearing significant similarities to the human clade B intracellular serpins. However, only five genes appear to encode full-length serpins with intact reactive site loops. To determine if this was the case, we have cloned and expressed a putative inhibitory-type C. elegans serpin, srp-3. Analysis of SRP-3 inhibitory activity indicated that SRP-3 was a potent inhibitor of the serine peptidases, chymotrypsin and cathepsin G. Spatial and temporal expression studies using GFP and LacZ promoter fusions indicated that SRP-3 was expressed primarily in the anterior body wall muscles, suggesting that it may play a role in muscle cell homeostasis. Combined with previous studies showing that SRP-2 is an inhibitor of the serine peptidase, granzyme B, and lysosomal cysteine peptidases, these data suggested that C. elegans expressed at least two inhibitory-type serpins with nonoverlapping expression and inhibitory profiles. Moreover, the profiles of these clade L serpins in C. elegans share significant similarities with the profiles of clade B intracellular serpin members in higher vertebrates. This degree of conservation suggests that C. elegans should prove to be a valuable resource in the study of metazoan intracellular serpin function.     

Navigating the cell

Changes in cell shape are associated with a variety of processes including cell migration, axon outgrowth, cell division, and vesicle trafficking. C. elegans UNC-53 and its vertebrate homologs, the Navigators, are required for the migration of cells and the outgrowth of neuronal processes. The identification of novel molecular interactions and live imaging studies have revealed that UNC-53/NAVs are signal transducers associated with actin filaments, microtubules, and intermediate filaments. In addition to modulating cytoskeletal dynamics at the leading edge of migrating or outgrowing cells, both UNC-53 and the navigators are expressed in adult cells, conspicuously those with specialized roles in endocytosis or secretion. Collectively, these results suggest that UNC-53/NAVs may be a central regulator of cytoskeletal dynamics, responsible for integrating signaling cues to multiple components of the cytoskeleton to coordinate rearrangement during cell outgrowth or trafficking.     

Neuronal polarity in C. elegans

Neuronal polarity sets the foundation for information processing and signal transmission within neural networks. However, fundamental question of how a neuron develops and maintains structurally and functionally distinct processes, axons and dendrites, is still an unclear. The simplicity and availability of practical genetic tools makes C. elegans as an ideal model to study neuronal polarity in vivo. In recent years, new studies have identified critical polarity molecules that function at different stages of neuronal polarization in C. elegans. This review focuses on how neurons guided by extrinsic cues, break symmetry, and subsequently recruit intracellular molecules to establish and maintain axon-dendrite polarity in vivo.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants

The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.     

The Caenorhabditis elegans ABL-1 tyrosine kinase is required for Shigella flexneri pathogenesis

Shigellosis is a diarrheal disease caused by the gram-negative bacterium Shigella flexneri. Following ingestion of the bacterium, S. flexneri interferes with innate immunity, establishes an infection within the human colon, and initiates an inflammatory response that results in destruction of the tissue lining the gut. Examination of host cell factors required for S. flexneri pathogenesis in vivo has proven difficult due to limited host susceptibility. Here we report the development of a pathogenesis system that involves the use of Caenorhabditis elegans as a model organism to study S. flexneri virulence determinants and host molecules required for pathogenesis. We show that S. flexneri-mediated killing of C. elegans correlates with bacterial accumulation in the intestinal tract of the animal. The S. flexneri virulence plasmid, which encodes a type III secretory system as well as various virulence determinants crucial for pathogenesis in mammalian systems, was found to be required for maximal C. elegans killing. Additionally, we demonstrate that ABL-1, the C. elegans homolog of the mammalian c-Abl nonreceptor tyrosine kinase ABL1, is required for S. flexneri pathogenesis in nematodes. These data demonstrate the feasibility of using C. elegans to study S. flexneri pathogenesis in vivo and provide insight into host factors that contribute to S. flexneri pathogenesis.