Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling

Grant, Barth D.; Zhang, Yinhua; Paupard, M.-C.; Lin, Sharron X.; Hall, David H.; Hirsh, David

doi:10.1038/35078549

Cited by 246 publications

(314 citation statements)

References 32 publications

(30 reference statements)

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“…As this study shows the applicability of the I-switch in obtaining chemical information within endosomes along the ALBR-mediated endocytosis pathway, we chose two genetic backgrounds that perturb this pathway: a genetic knockout, namely rme-1 (ref. 29), and an RNAi knockdown, namely vha-8 (ref. 30) .…”

Section: Resultsmentioning

confidence: 99%

“…Th e niche of a pH-sensitive DNA nanodevice that is used as an externally introduced probe within a model organism such as C. elegans , where processes are molecularly dissected predominantly by genetics, lies in the demonstration of the nanomachine ' s applicability in various genetic backgrounds. rme-1(b1045) mutants have defects in recycling of receptors 29 , and show a characteristic phenotype of small indistinct cortical puncta in the coelomocytes ( Supplementary Fig. S2c ).…”

Section: Discussionmentioning

confidence: 99%

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An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

et al. 2011

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Structural DNA nanotechnology seeks to build synthetic molecular machinery from DNA. DNA nanomachines are artifi cially designed assemblies that switch between defi ned conformations in response to an external cue. Though it has proved possible to create DNA machines and rudimentary walkers, the function of such autonomous DNA-based molecular devices has not yet been achieved inside living organisms. Here we demonstrate the operation of a pH-triggered DNA nanomachine inside the nematode Caenorhabditis elegans . The nanomachine uses fl uorescence resonance energy transfer to effectively map spatiotemporal pH changes associated with endocytosis in wild type as well as mutant worms, demonstrating autonomous function within the organismal milieu in a variety of genetic backgrounds. From this fi rst demonstration of the independent functionality of a DNA nanomachine in vivo , we observe that rationally designed DNA-based molecular devices retain their in vitro functionality with quantitative precision. This positions DNA nanodevices as exciting and powerful tools to interrogate complex biological phenomena.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

et al. 2011

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“…To determine if the abnormal vacuoles in gum-1 mutant intestines are enlarged basolateral endosomes like those that form in rme-1 mutants, we crossed the gum-1 mutants into the background of a transgene that directs secretion of GFP from body-wall muscle cells into body cavity, from which the secreted GFP is normally endocytosed by the basolateral intestine and then recycled into the body cavity (Fares and Greenwald, 2001a;Grant et al, 2001). We found that the large abnormal vacuoles in gum-1 mutant intestinal cells accumulated GFP from the body cavity, indicating a defect in basolateral recycling similar to that previously found in rme-1 mutants (Figure 1, D-F).…”

Section: Gum-1 Mutants Display Rme-1-like Endocytosis Defects In the mentioning

confidence: 99%

RAB-10 Is Required for Endocytic Recycling in theCaenorhabditis elegansIntestine

Chen

Schweinsberg

Vashist

et al. 2006

MBoC

183

378

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The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. One of the most enigmatic aspects of this process is endocytic recycling, the return of macromolecules (often receptors) and fluid from endosomes to the plasma membrane. We have previously shown that the EH-domain protein RME-1 is a critical regulator of endocytic recycling in worms and mammals. Here we identify the RAB-10 protein as a key regulator of endocytic recycling upstream of RME-1 in polarized epithelial cells of the Caenorhabditis elegans intestine. rab-10 null mutant intestinal cells accumulate abnormally abundant RAB-5-positive early endosomes, some of which are enlarged by more than 10-fold. Conversely most RME-1-positive recycling endosomes are lost in rab-10 mutants. The abnormal early endosomes in rab-10 mutants accumulate basolaterally recycling transmembrane cargo molecules and basolaterally recycling fluid, consistent with a block in basolateral transport. These results indicate a role for RAB-10 in basolateral recycling upstream of RME-1. We found that a functional GFP-RAB-10 reporter protein is localized to endosomes and Golgi in wild-type intestinal cells consistent with a direct role for RAB-10 in this transport pathway. INTRODUCTIONEndocytosis and endocytic trafficking controls the uptake and sorting of extracellular macromolecules as well as the components of the cell membrane itself, counterbalancing secretion and allowing a complex interplay between cells and their environment that is important for a myriad of cellular activities (Brodsky et al., 2001;Maxfield and McGraw, 2004). The steps involved in the uptake and trafficking of cargo within the endosomal system have been well described, but many of the components mediating these steps at the molecular level remain to be identified (Brodsky et al., 2001;Maxfield and McGraw, 2004). Many receptors and their associated ligands cluster into clathrincoated pits, whereas other types of cargo utilize clathrinindependent uptake mechanisms (Nichols, 2003;Gesbert et al., 2004). Plasma membrane invaginations pinch off into vesicles, uncoat, and then fuse with one another and with early endosomes. In early endosomes some ligand-receptor complexes dissociate because of the reduced pH of the endosomal lumen (Mukherjee et al., 1997). Many receptors then recycle to the plasma membrane (PM) either directly or indirectly via recycling endosomes (Mukherjee et al., 1997;Maxfield and McGraw, 2004). Many ligands do not recycle but instead are transported from early to late endosomes and eventually to lysosomes for degradation (Mukherjee et al., 1997). In polarized epithelial cells such as cultured Madin-Darby canine kidney (MDCK) cells, an additional layer of complexity in the endocytic pathway contributes to formation and/or maintenance of the specialized apical and basolateral domains (Nelson and Yeaman, 2001;Mostov et al., 2003;Hoekstra et al., 2004). Both the apical and basolateral membranes deliver cargo ...

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“…Experiments using FM4-64 and labeled BSA were conducted as described by Grant et al (2001). To assess uptake of lactosylceramide, animals were incubated for 2 h in 5 M BODIPY-tagged lactosylceramide (BODIPY FL C5 LacCer; Invitrogen, Paisley, UK).…”

Section: Analysis Of Endocytosismentioning

confidence: 99%

“…Recent work using genetic and in vivo approaches has led to the development of the C. elegans intestine as a system for the study of trafficking (Fares and Grant, 2002;Grant and Sato, 2006). Grant et al (2001) showed that exposure of the apical surface to lipid markers, such as FM4-64, and fluid-phase markers, such as Texas Red-BSA, results in accumulation of the markers in gut granules. Application of FM4-64 to the basolateral membrane has the same result, but basolaterally applied fluidphase markers, such as Texas Red-BSA or ssGFP (GFP secreted into the body cavity; Fares and Greenwald, 2001b), undergo recycling.…”

Section: Introductionmentioning

confidence: 99%

Caveolin-2 Is Required for Apical Lipid Trafficking and Suppresses Basolateral Recycling Defects in the Intestine ofCaenorhabditis elegans

Parker

Walker

et al. 2009

MBoC

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Caveolins are plasma membrane-associated proteins that colocalize with, and stabilize caveolae. Their functions remain unclear although they are known to be involved in specific events in cell signaling and endocytosis. Caenorhabditis elegans encodes two caveolin genes, cav-1 and cav-2. We show that cav-2 is expressed in the intestine where it is localized to the apical membrane and in intracellular bodies. Using the styryl dye FM4-64 and BODIPY-labeled lactosylceramide, we show that the intestinal cells of cav-2 animals are defective in the apical uptake of lipid markers. These results suggest parallels with the function of caveolins in lipid homeostasis in mammals. We also show that CAV-2 depletion suppresses the abnormal accumulation of vacuoles that result from defective basolateral recycling in rme-1 and rab-10 mutants. Analysis of fluorescent markers of basolateral endocytosis and recycling suggest that endocytosis is normal in cav-2 mutants and thus, that the suppression of basolateral recycling defects in cav-2 mutants is due to changes in intracellular trafficking pathways. Finally, cav-2 mutants also have abnormal trafficking of yolk proteins. Taken together, these data indicate that caveolin-2 is an integral component of the trafficking network in the intestinal cells of C. elegans. INTRODUCTIONCaveolae are 50 -100-nm invaginations located on the plasma membrane of many cell types (Anderson, 1998). They are enriched in lipid raft-associated molecules: cholesterol, sphingolipids, glycosphingolipids, and many signaling and receptor proteins. Although identified more than 50 years ago, their functions have remained elusive. However, they are known to play roles in specific cell-signaling processes, and it is clear that endocytosis through caveolae is an important clathrin-independent endocytic pathway (Parton and Richards, 2003;Parton and Simons, 2007). A range of molecules including glycosphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, cholera toxin B, and SV40 virus have been shown to traffic through caveolaedependent endocytosis pathways (see Marsh and Helenius, 2006;Mayor and Pagano, 2007; Parton and Simons, 2007 for reviews).Caveolae require caveolin proteins for formation so that depletion of caveolins results in loss of morphologically detectable caveolae (Drab et al., 2001;Galbiati et al., 2001). Furthermore, introduction of caveolin into cells that do not produce caveolae stimulates caveolar biogenesis (Lipardi et al., 1998). The majority of caveolin appears to traffic to the plasma membrane and is relatively immobile; however, internal caveolin-containing structures, named caveosomes, have been visualized (Parton and Simons, 2007) and appear to be an integral part of the caveolin-dependent endocytic machinery (Nichols, 2003). Mammalian cells have three caveolin subtypes: caveolin 1 is widely expressed, but is found at particularly high levels in adipocytes, endothelial cells, and fibroblasts. Caveolin 2 interacts with caveolin 1, whereas caveolin 3 is expressed in myocytes ...

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Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling

Cited by 246 publications

References 32 publications

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism

RAB-10 Is Required for Endocytic Recycling in theCaenorhabditis elegansIntestine

Caveolin-2 Is Required for Apical Lipid Trafficking and Suppresses Basolateral Recycling Defects in the Intestine ofCaenorhabditis elegans

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