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Vol. 19, Issue 7, 2682-2695, July 2008
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Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston MA 02114
Submitted November 8, 2007;
Revised April 8, 2008;
Accepted April 11, 2008
Monitoring Editor: Sandra Lemmon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors. Work from several laboratories suggests that regulation of the abundance of AMPA receptors is a cellular mechanism for producing activity-dependent changes in synaptic strength, e.g., long-term potentiation (LTP), long-term depression, and homeostatic plasticity (Malinow and Malenka, 2002
; Song and Huganir, 2002; Bredt and Nicoll, 2003; Park et al., 2004). For these reasons, there is great interest in defining the mechanisms that regulate the trafficking of AMPA receptors.
AMPA receptors at synapses are derived from a recycling pool of receptors (Ehlers, 2000; Lin et al., 2000). The rate of AMPA receptor exocytosis and endocytosis is regulated by synaptic activity (Ehlers, 2000). After endocytosis, AMPA receptors are sorted into either of two postendocytic pathways: the recycling endosome (from which receptors are recycled to the plasma membrane) or late endosomes/lysosomes (where receptors are degraded) (Ehlers, 2000; Lee et al., 2004). Recycling endosomes are thought to be the source of AMPA receptors involved in activity-dependent potentiation of synapses (Park et al., 2004). Although substantial progress has been made in understanding the endocytosis of AMPA receptors, much remains to be learned about the mechanisms by which AMPA receptors are sorted between these two postendocytic pathways.
Rab GTPases are thought to play a pivotal role in orchestrating the specificity of membrane trafficking events (Chavrier and Goud, 1999; Zerial and McBride, 2001; Grosshans et al., 2006). Rabs comprise the largest class of Ras-like GTPases, and they have been implicated in many aspects of intracellular membrane trafficking, such as vesicle budding from donor compartments, and vesicle tethering and fusion with target membranes. Each Rab seems to function in specific membrane transport steps, and this specificity arises from the selective recruitment and activation of Rabs on specific membranes. Association and dissociation of Rabs with their cognate membranes is coupled to the hydrolysis of their bound guanine nucleotides. Thus, nucleotide hydrolysis is required for Rabs to perform their membrane transport functions (Grosshans et al., 2006).
The nematode Caenorhabditis elegans has been used as a genetic model to study the trafficking of an AMPA-type glutamate receptor GLR-1. GLR-1 is expressed in ventral cord interneurons (Hart et al., 1995; Maricq et al., 1995), where it is localized to sensory–interneuron and interneuron–interneuron synapses (Rongo and Kaplan, 1999; Burbea et al., 2002). We previously showed that the synaptic abundance of GLR-1 receptors is regulated by clathrin-mediated endocytosis and by ubiquitin ligases (Burbea et al., 2002; Juo and Kaplan, 2004; Dreier et al., 2005), as are mammalian AMPA receptors (Carroll et al., 1999; Man et al., 2000; Colledge et al., 2003; Patrick et al., 2003). In previous studies, mammalian Rab2 has been proposed to regulate retrograde trafficking between pre-Golgi intermediate compartment (ERGIC) and the endoplasmic reticulum (ER) (Tisdale and Balch, 1996; Tisdale and Jackson, 1998). Here, we show that UNC-108/Rab2 plays a role in postendocytic trafficking of GLR-1 and other cargo in neurons. We also show that UNC-108/Rab2 is necessary for proper trafficking of postendocytic cargo in coelomocytes. Thus, our results identify a new function for Rab2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The following strains were used in this study: N2 Bristol, unc-108(nu415), unc-67(e713), unc-108(n501), unc-108(n777), nuIs24 (Pglr-1::GLR-1::GFP), bIs33 (Prme-8::RME-8::GFP), nuEx1324 (Psnb-1::UNC-108::FLAG), nuEx1325 (Pglr-1::UNC-108::FLAG), nuEx1326 (Punc-17::UNC-108::FLAG), nuEx1327 (Pmyo-3::UNC-108::FLAG), nuIs125 (Pglr-1::GFP::SNB-1), nuEx1328 (ssVenus::KDEL), nuEx1328 (ssVenus::AAAA), nuEx1329 (Pglr-1::ssTomato::KDEL), nuEx1330 (Pglr-1::RFP::BET-1), nuEx1331 (Pglr-1::RFP::GOS-28), nuEx1332 (Pglr-1::RFP::Syntaxin-16), nuEx1333 (Pglr-1::Cherry::Rab10), nuEx1334 (Pglr-1::Cherry::Rab8), nuEx1335 (Pglr-1::Cherry::Rab14), nuEx1336 (Pglr-1::Cherry::Rab11), nuEx1337 (Pglr-1::RFP::Syntaxin-13), nuEx1338 (Pglr-1::Cherry::Rab5), nuEx1339 (Pglr-1::Cherry::Rab7), nuIs232 (Pglr-1::GFP::Syntaxin-13), nuEx1340 (Pglr-1::RFP::Syntaxin-5), nuEx1341 (Punc-122::UNC-108::FLAG), nuIs145 (vps-4(DN)), nuIs89 (Pglr-1::MUb) nuEx1345 (Pglr-1::GFP::BET-1) nuEx1351 (Punc-129::GFP::UNC-64, Punc-129::RFP::KDEL), nuEx1350 (Pglr-1::UNC- 108::FLAG, Pglr-1::RFP::BET-1), and nuEx1352 (Pglr-1::UNC-108 (Q65L)::FLAG).
Cloning of unc-108(nu415)
The unc-108(nu415) allele was mapped based on the Uncoordinated (Unc) phenotype after back crosses revealed close linkage to the GLR-1::GFP phenotype. unc-108(nu415) was initially isolated using the nuIs25 (Pglr-1::GLR-1::GFP) strain, and it was linked to the nuIs25 transgene demonstrating that nu415 is on chromosome I. Five Unc genes located on chromosome I were tested for complementation based on similar Unc phenotypes: unc-40, unc-14, unc-67, unc-87, and unc-59. unc-108(nu415) and unc-67(e713) failed to complement, indicating that they are allelic. unc67(e713) was a previously uncloned Unc gene.
Mapping of nu415, was performed by crossing into CB4856 Hawaiian worms and using polymorphism-based Snip-single-nucleotide polymorphism (SNP) mapping (Wicks et al., 2001). unc-108(nu415) was positioned between two SNPs at positions –3.16 and –1.65 on chromosome I. This region of 1.51 map units was spanned by 14 cosmids. Cosmid injections identified that cosmid F53F10 was sufficient to rescue the Unc phenotype. Fragments of this cosmid were injected in order to test for single gene rescue and a BamHI/Eag I (4.7-kb) fragment of F53F10 that contained only the unc-108 genomic region was able to rescue the Unc phenotype.
Constructs and Transgenes
The unc-108 rescuing promoter consisted of 2.3 kb upstream of the unc-108 start codon. This promoter fragment was amplified to add SphI and BamHI sites and cloned into the Fire lab vector pPD 95.75 to place it upstream of soluble GFP. unc-108 cDNA was isolated from wild-type worms and cloned into the Fire vector pPD 49.26 with either the glr-1 or the unc-108 promoter. To observe UNC-108 localization, GFP was added to the N terminus of UNC-108, but this construct was unable to rescue the unc-108(nu415) Unc or the GLR-1::GFP phenotype. Therefore, a rescuing construct was made by inserting a FLAG epitope after amino acid 191 (at the end of homology with mammalian Rab2). The UNC-108::FLAG construct was cloned into pPD 49.26 and the following promoters were then inserted: Punc-108, Psnb-1, Pglr-1, Punc-17, and Pmyo-3. UNC-108 with the point mutation Q65L contains a FLAG epitope at amino acid 191 and was placed under the Pglr-1 promoter.
Subcellular markers consisting of either soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNARE proteins or Rab proteins were generated by insertion of a NotI restriction site immediately following the start codon ATG and insertion of a NotI-flanked RFP (mCherry) or GFP. ssVenus::KDEL and ssVenus::AAAA were generated by fusing the Pat-3 signal sequence upstream of the Venus (yellow fluorescent protein [YFP] variant) and adding the sequence encoding either the KDEL signal or four Alanines before the Venus stop codon. ssTomato::KDEL was generated by creating a sequence that consisted of the Pat-3 signal sequence a NotI restriction site encoding three alanines (GCGGCCGCC) followed by the KDEL sequence, and a NotI-flanked Tomato was then cloned in. These markers were then cloned into pPD 49.26 containing the glr-1 promoter. Dominant-negative VPS-4 (vps-4(DN)) was generated by making a single amino acid change from K164 to Q. vps-4 (K164Q) was coned into pPD 49.26 containing the glr-1 promoter. Injection of all constructs into worms was performed as described previously to generate transgenic lines. Both vps-4(DN) and GFP::Syntaxin-13 were integrated into chromosomes using a UV cross-linker (Fisher Science, Pittsburgh, PA). The RME-8::GFP strain was a gift from Barth Grant (Rutgers University, Piscataway, NJ).
Immunolabeling, Endoglycosidase H (Endo H) and peptide-N–-(N-acetyl-β-glucosaminyl)asparagine Amidase (PNGase F) Treatment, and Western Blot Analysis
For immunolabeling experiments, nuEx1324 and nuEx1350 worms were fixed using Bouin's fixative and labeled with antibodies to the FLAG epitope (M2 antibody; Sigma-Aldrich, St. Louis, MO) and red fluorescent protein (RFP) (DsRed polyclonal antibody; Clontech, Palo Alto, CA) at a concentration of 1:500, and subsequently, Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were then applied at a concentration of 1:500. Animals were mounted on slides and imaged. Endogenous UNC-108 protein was visualized on Western blot by boiling wild-type and unc-108(nu415) worms in 5x SDS sample buffer and loading the lysates onto an SDS denaturing gel. Western blots were performed using an antibody against full-length mouse Rab2 (Santa Cruz Biotechnology, Santa Cruz, CA). UNC-108::FLAG protein was analyzed in extracts prepared from nuEx1323 (Punc-108::UNC-108::FLAG) worms by immunoblotting with an anti-FLAG antibody (Sigma-Aldrich).
For Endo H and PNGase F assays, lysates were made by boiling worms in 5x denaturing buffer (2.5% SDS and 5% beta-mercaptoethanol) followed by sonication. Lysates were then diluted to 1x denaturing buffer in water plus protease inhibitors (10 µg/ml leupeptin, 5 µg/ml chymostatin, 3 µg/ml elastinal, 1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). Digests were then performed as recommended by the enzyme manufacturer (New England Biolabs, Ispwich, MA). Treated and untreated lysates were run on SDS denaturing gels. Western blots were performed on samples using an anti-GFP antibody (JL-8; Clontech). GLR-1::GFP levels were measured by boiling a mixed population of worms in 5X SDS sample buffer and loading on a SDS denaturing gel. Blots were then probed with a GFP antibody (JL-8; Clontech). Samples were normalized for protein levels by measuring actin levels by using an anti-actin antibody (MP Biomedicals, Irvine, CA). For Endo H and protein level measurements, five independent populations were measured for each genotype. Quantitative imaging of Western blots was performed using a Typhoon Trio Plus variable mode imager and ImageQuant TL version 2005 software (GE Healthcare, Chalfont St. Giles, United Kingdom).
Imaging
Fluorescent imaging of the ventral nerve cord was performed using an Axiovert 100 microscope (Carl Zeiss, Jena, Germany) and PlanApo 100x (1.4 numerical aperture [NA]) (Olympus, Tokyo, Japan) objective equipped with fluorescein isothiocyanate/GFP or RFP filters and an ORCA100 charge-coupled device camera (Hamamatsu, Bridgewater, NJ). Antibody stained animals were mounted on agarose pads and imaged. Live animals were immobilized with 30 mg/ml butanedione monoxime (BDM; Sigma-Aldrich). Image stacks were captured and maximum intensity projections were obtained using MetaMorph 4.5 software (Molecular Devices, Sunnyvale, CA). Identical camera gain, exposure settings, and fluorescence filters were used for all live animal imaging.
Line scans of ventral nerve cord fluorescence were analyzed in IGOR Pro (WaveMetrics, Portland, OR) using custom-written software (Burbea et al., 2002; Sieburth et al., 2005). Arc lamp output was normalized by measuring fluorescence of 0.5 µm FluoSphere beads (Invitrogen) for each day. Fluorescence amplitude for puncta peak and cord values was normalized to wild-type controls to facilitate comparison. Statistical significance was calculated using a Student's t test as described previously.
Fluorescent imaging of neuronal cell bodies and coelomocytes was performed on an Olympus Fluoview FV1000 confocal laser scanning microscope by using a Olympus PlanApo N 60x objective (1.45 NA). Animals imaged for neuronal cell bodies were immobilized with 30 mg/ml BDM (Sigma-Aldrich). Coelomocyte images were captured after injection of Texas Red-bovine serum albumin (TR-BSA) as described previously (Zhang et al., 2001): animals were put on ice for 10 min to arrest endocytosis then fixed in 1% paraformaldehyde for 10 min before imaging. For both neuronal cell bodies and coelomocytes, Z-stacks were taken of the entire cell, and a representative single plane was selected.
For quantification of GLR-1::GFP colocalization with RFP-tagged compartment markers in unc-108(nu415) mutant neuronal cell bodies, a region of interest was drawn around a GLR-1::GFP tubulovesicular structure, and this image was thresholded so that all pixels below 1 SD of the mean pixel intensity calculated for the region of interest were excluded. The percent area colocalization of the thresholded GLR-1::GFP images and the various RFP-tagged compartment markers were then measured (using MetaMorph software), and statistical significance was calculated using a Student's t test as described previously. To compare quantitatively the colocalization of GLR-1::GFP with a particular RFP-tagged marker in wild type versus unc-108 mutant neuronal cell bodies, a slightly different strategy for thresholding was used. The pixel intensity corresponding to the cut-off for the brightest 5% of pixels in individual images of GLR-1::GFP in both wild-type and unc-108(nu415) neuronal cell bodies was calculated by plotting the cumulative percentage of total pixel count against pixel intensity. The pixel intensity corresponding to the brightest 5% of pixels in the GLR-1::GFP image was then used as the threshold for the image, and these thresholded GLR-1::GFP images were subsequently used to determine the percent area colocalization with their respective RFP-tagged compartment markers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Pfeffer, 2005). The uncoordinated locomotion defect of unc-108(nu415) animals was rescued by the expression of FLAG-tagged UNC-108 in all neurons (using the snb-1 promoter) (Figure 1C). Rescue was not observed when unc-108 was expressed in cholinergic neurons, ventral cord interneurons, or in body muscles. Consistent with function in neurons, an unc-108 transcriptional reporter construct (Punc-108::GFP) was expressed strongly in many, and perhaps all neurons (Figure 1D). Weaker expression was also observed in the gut, seam cells, and hypodermis. Finally, unc-108(nu415) mutants were slightly hypersensitive to the paralytic effects of aldicarb, an acetylcholinesterase inhibitor (Supplemental Figure S1), a phenotype that is often associated with changes in synaptic transmission at neuromuscular junctions (Miller et al., 1996; Loria et al., 2004; Sieburth et al., 2005). Collectively, these results argue that UNC-108 acts in neurons to promote normal locomotion.
The unc-108 gene was originally defined by two gain-of-function alleles (n501dm and n777dm), both of which cause a dominantly inherited uncoordinated locomotion phenotype (Park and Horvitz, 1986). Both dominant unc-108 alleles are predicted to increase the abundance of GTP-liganded Rab2. The n501dm (D122N) and n777dm (S149F) mutations alter conserved residues in the GTPase domain (Simmer et al., 2003) (Figure 1A). A dominant-negative missense mutation in the corresponding residue of Ras increases the spontaneous guanine nucleotide exchange rate, thereby producing a constitutively active Ras protein (Cool et al., 1999). The S149 residue is conserved among most Ras related GTPases and falls in the G5 loop, which is also required for guanine nucleotide binding (Paduch et al., 2001). To confirm the gain-of-function nature of these alleles, we expressed mutant unc-108/Rab2 cDNAs containing these mutations, and we found that both produced an uncoordinated locomotion phenotype similar to that seen in the unc-108(dm) mutants (data not shown).
Together, these results suggest that the unc-67 and unc-108 genes are allelic, and that gain and loss-of-function unc-108 alleles produce similar locomotion defects. Previous studies showed that the function of Rab proteins in intracellular membrane fusion requires GTP hydrolysis (Walworth et al., 1992); therefore, it was not surprising that similar locomotion defects were produced by gain and loss-of-function unc-108 mutations. Hereafter, we refer to this gene as unc-108/Rab2.
UNC-108/Rab2 Regulates GLR-1 Distribution in the Ventral Nerve Cord
In unc-108(nu415) mutants, significant increases were observed in the intensity (24% increase, p < 0.001) and width (41% increase, p < 0.001) of GLR-1::GFP puncta in the ventral nerve cord (Figure 2, A and B, and E and F) We showed previously that GLR-1::GFP puncta correspond to receptors clustered at postsynaptic elements (Rongo et al., 1998; Burbea et al., 2002). A similar increase in GLR-1::GFP fluorescence was also observed in unc-108(n501dm) and unc-108(e713) mutants, although the defects caused by e713 were less severe (Figure 2, D–F; data not shown). Thus, similar changes in GLR-1::GFP distribution were observed in both loss and gain-of-function unc-108 mutants, as was the case for the locomotion defects. The increased GLR-1::GFP punctal fluorescence in unc-108(nu415) mutants was rescued by expression of a FLAG-tagged UNC-108 construct under the glr-1 promoter (Figure 2C, E, and F), demonstrating that UNC-108/Rab2 activity in ventral cord interneurons is required for proper localization of GLR-1::GFP. An anti-FLAG antibody detected a 23-kDa protein in extracts of transgenic animals, which is consistent with the predicted molecular weight for the UNC-108::FLAG protein (Supplemental Figure S2A). FLAG-tagged UNC-108 was expressed in glr-1 interneurons, and immunostaining of animals expressing this construct indicated that UNC-108::FLAG is diffusely distributed in neurites, with occasional small puncta (Supplemental Figure S2, B and C). The total abundance of GLR-1::GFP in unc-108(nu415) mutants and wild-type controls were not significantly different (wild type 24.8 ± 6.0 AU vs. unc-108 24.5 ± 4.4 AU, p = 0.57) (Figure 3A), suggesting that changes in GLR-1::GFP puncta fluorescence observed in unc-108 mutants were unlikely to be caused by changes in total GLR-1 abundance.
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; Burbea et al., 2002; Juo and Kaplan, 2004, Schaefer and Rongo, 2006; Juo et al., 2007). The uncoordinated locomotion of unc-108(nu415) mutants precludes their use in a reversal assay; therefore, we used transgenic animals expressing a GTPase-defective mutant UNC-108(Q65L) in the ventral cord interneurons. Animals expressing UNC-108(Q65L) had significantly higher reversal frequencies compared with wild-type controls (Figure 2G), consistent with increased glutamatergic signaling. Thus, UNC-108 regulates both GLR-1::GFP abundance in the ventral cord, and produces corresponding changes in a GLR-1–mediated behavior.
To determine whether UNC-108/Rab2 regulates the trafficking of other synaptic proteins, we examined the distribution of a GFP-tagged synaptic vesicle protein, Synaptobrevin (GFP::SNB-1) in the ventral cord interneurons. We found that neither the GFP::SNB-1 punctal fluorescence nor the diffuse axon fluorescence were altered in unc-108(nu415) mutants (Figure 3B-E). We also analyzed several other presynaptic markers in ventral cord motor neurons (including SYD-2/-Liprin, UNC-10/RIM-1, SNN-1/Synapsin, Gelsolin, and INS-22/Insulin), and we found that all were distributed in a normal pattern in unc-108 mutants (data not shown). Thus, UNC-108/Rab2 is not generally required for trafficking of synaptic proteins, nor is it required for normal synapse formation or synapse morphology. Instead, these results suggest that UNC-108/Rab2 plays a relatively specific role in the trafficking of a subset of synaptic proteins, including GLR-1.
UNC-108/Rab2 Is Not Required for ER Retention of KDEL-containing Proteins
To determine the mechanism for the altered trafficking of GLR-1::GFP in unc-108 mutants, we first tested the known secretory functions of Rab2. Previous studies in mammalian systems suggested that Rab2 plays an important role in retrograde trafficking between the Golgi and the ER (Tisdale and Balch, 1996). To assay COPI-mediated retrograde transport, we expressed a secreted form of YFP (ssVenus) containing the ER retention signal KDEL (ssVenus::KDEL). Proteins containing the KDEL retention signal are constitutively retrieved from the Golgi by COPI-mediated retrograde transport, resulting in their retention in the ER (Pidoux and Armstrong, 1992; Terasaki et al., 1996). In PVC interneuron cell bodies, ssVenus::KDEL was localized in a diffuse network consistent with retention in the ER in both wild-type and unc-108(nu415) mutants (Figure 4, A and B). In both wild type and unc-108 neurons, the ssVenus::KDEL signal did not colocalize with the Golgi marker RFP::GOS-28 (Volchuk et al., 2004) (Figure 4, E and F). Mutations that disrupt COPI-mediated transport often alter the morphology of the ERGIC (Donaldson et al., 1990; Guo et al., 1994; Gaynor and Emr, 1997); however, the morphology of the ERGIC (labeled with RFP::BET-1, Volchuk et al., 2004) was unaltered in unc-108 mutants (Figure 4, G and H).
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; Andag et al., 2001). Therefore, as an independent measure of ER retention, we analyzed secretion of ssVenus::KDEL. Secreted fluorescent proteins are endocytosed and degraded by coelomocytes, which are macrophage-like scavenger cells in the body cavity (Fares and Greenwald, 2001; Sieburth et al., 2007; Speese et al., 2007). Thus, failure to retain ssVenus::KDEL in the ER should result in its secretion by interneurons, which can be detected as fluorescence in postendocytic structures of coelomocytes. ssVenus::KDEL fluorescence was dim in the coelomocytes of both wild-type and unc-108(nu415) mutants (Figure 4, A and B inset). In fact, decreased coelomocyte ssVenus::KDEL fluorescence was observed in unc-108(nu415) mutants, which is likely due to a defect in postendocytic trafficking in coelomocytes (detailed below). As expected, proteins lacking ER retention signals (e.g., ssVenus::AAAA) were efficiently secreted from both unc-108(nu415) mutants and wild-type interneurons (Figure 4, C and D, inset). Collectively, these results suggest that COPI-mediated retrograde transport remains functional in unc-108 mutants, although we cannot exclude a modest defect in this pathway.
Anterograde Trafficking Occurs Normally in unc-108/Rab2 Mutants
Disrupting Rab2 function in cultured mammalian cells is also associated with defects in anterograde transport of cargo proteins, e.g., VSV-G (Tisdale and Balch, 1996). Similar anterograde trafficking defects are often observed in yeast mutants with disrupted COPI retrograde transport (Gaynor and Emr, 1997). Therefore, we did several experiments to test the idea that the altered distribution of GLR-1::GFP in unc-108 mutants was caused by anterograde trafficking defects. GLR-1::GFP accumulated in the ventral cord of unc-108 mutants, suggesting that anterograde trafficking out of the cell body occurs normally in these mutants. If early secretory components (e.g., ER or Golgi membranes) were mislocalized to the ventral cord in unc-108 mutants, this could account for the altered GLR-1::GFP distribution in these mutants. However, we did not find increased ventral cord abundance for several ER and Golgi markers in unc-108 mutants, including ssVenus::KDEL, BET-1 (ERGIC), Syntaxin-5 (TGN), and Syntaxin-16 (TGN) (Figure 5, A–D; data not shown). Mutations disrupting COPII-mediated anterograde transport in yeast often result in accumulation of ER membranes (Kaiser and Schekman, 1990). Again, we did not observe any obvious change in the ER network, as visualized by ssVenus::KDEL (Figure 4, A and B).
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). Endo H removes N-glycans containing immature carbohydrate chains, which are typically found in the ER. In the Golgi, carbohydrate chains undergo further modifications, rendering them resistant to Endo H. The Endo H-sensitive fractions of GLR-1::GFP observed in wild-type (65%, SEM ± 2.3%) and unc-108(nu415) mutants (58%, SEM ± 4.6%) were similar (Figure 5E), suggesting that anterograde delivery of GLR-1::GFP to the Golgi occurs normally in unc-108 mutants. Finally, we also examined anterograde transport of the plasma membrane SNARE Syntaxin-1 (encoded by unc-64). In both wild type and unc-108 mutants, GFP-tagged UNC-64 expressed in PVC interneurons was found both in intracellular membranes, and at the periphery of the cell body, which likely corresponds to the plasma membrane. We found that the extent of colocalization of GFP::UNC-64 with ssTomato::KDEL was not significantly different in unc-108 mutants compared with wild-type controls (Figure 5, F–H), suggesting that UNC-64 was not retained in the ER in these mutants. Collectively, these results suggest that unc-108 mutants do not have a significant defect in anterograde transport.
GLR-1::GFP Accumulates in Endosomal Structures in unc-108/Rab2 Mutants
To further address the nature of the trafficking defect observed in unc-108 mutants, we analyzed the distribution of GLR-1::GFP in neuronal cell bodies. We found that GLR-1::GFP also had an aberrant distribution in interneuron cell bodies of unc-108(nu415) and unc-108(n501dm) mutants (Figure 6, A–C). In both unc-108 mutants, GLR-1::GFP accumulated in large tubulovesicular structures that were observed infrequently in wild-type neurons. The tubulovesicular structures had very bright GLR-1::GFP pixel intensities (corresponding to the brightest 
5% of pixels in each cell) (Supplemental Figure S4, B, D, and E). By contrast, wild-type neurons lacked these tubulovesicular structures, and they had fewer pixels with high GLR-1::GFP intensities (Supplemental Figure S4, A, C, and E). Thus, in unc-108 mutant neurons, there was an accumulation of GLR-1::GFP in these aberrant structures.
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65% of GLR-1::GFP is retained in the ER based on Endo H sensitivity assays (Grunwald and Kaplan, 2003) (Supplemental Figure S5, G–I). A panel of other RFP-tagged intracellular compartment markers were examined for colocalization with GLR-1::GFP in unc-108(nu415) mutant neurons, including Rab14, Rab11, Rab10, GOS-28, and Syntaxin-16, but none were significantly colocalized with GLR-1::GFP (Supplemental Figure S3). Together, these results suggest that GLR-1::GFP accumulates in abnormal endosomal derivatives in unc-108 mutants. In addition to the cell body, we also expect to find endosomal structures in neuronal processes. Therefore, we analyzed the distribution of an endosomal marker (GFP::Syntaxin-13) in the ventral cord, after transgenic expression in the ventral cord interneurons or in the cholinergic DA motor neurons (using the glr-1 or unc-129 promoter, respectively). In both cases, we found that the intensity of GFP::Syntaxin-13 puncta in the ventral cord significantly increased in unc-108(nu415) mutants compared with wild-type controls (Figure 7, A–D; data not shown). This further supports the conclusion that there is an endosomal trafficking defect in unc-108(nu415) mutant neurons.
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). Postendocytic trafficking of TR-BSA in coelomocytes was monitored at sequential time points (Figure 8). In wild-type animals, internalized TR-BSA was observed in primarily RME-8::GFP– positive vesicles 10 min after injection, whereas after 30 min, TR-BSA was observed increasingly in RME-8::GFP-negative vesicles, which presumably correspond to late endosomes and lysosomes (Zhang et al., 2001). If in unc-108(nu415) mutant animals, endocytosis occurs normally, but a postendocytic trafficking step is disrupted, you would expect to see changes in the size or distribution of endosomal compartments in the coelomocytes of these animals. In fact, in unc-108(nu415) mutants, there was a significant reduction in the number of RME-8::GFP–positive intracellular vesicles, but each vesicle was significantly larger than those observed in wild-type controls. In addition to this change in the morphology of the RME-8–containing vesicles, TR-BSA trafficking into RME-8::GFP–negative vesicles was significantly delayed in unc-108(nu415) mutants. 60 min after injection, TR-BSA was visible only in RME-8::GFP–positive vesicles of unc-108(nu415) mutant coelomocytes, eventually becoming visible in RME-8::GFP negative vesicles at 120 min (Figure 8B). Trafficking defects seen in unc-108(nu415) mutants were rescued by expression of FLAG-tagged UNC-108 by using the coelomocyte-specific unc-122 promoter (Loria et al., 2004) (Figure 8C). These results demonstrate that UNC-108/Rab2 is required for postendocytic trafficking in coelomocytes, and specifically for endosome-to-lysosome trafficking. A similar coelomocyte post-endocytic trafficking defect was recently described in mutants carrying a dominant unc-108 allele, and after knockdown of unc-108 by RNAi (Lu et al., 2008). These results are also consistent with the observed trafficking defects in ventral cord interneurons: accumulation of GLR-1::GFP in an endosomal compartment in cell bodies, and altered distribution of the endosomal marker Syntaxin-13 in the ventral cord.
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). Overexpression of Myc-tagged ubiquitin (MUb) in the ventral cord interneurons results in the formation of MUb–GLR-1 conjugates and decreased density of GLR-1::GFP puncta in the ventral cord (Burbea et al., 2002). The effect of MUb on GLR-1::GFP puncta density was prevented by mutations that block endocytosis (e.g., unc-11 AP180 mutations) (Burbea et al., 2002). In other systems, ubiquitin-promoted degradation of membrane proteins is mediated by targeting ubiquitin-conjugates to the internalized vesicles of multivesicular bodies (MVBs) (Katzmann et al., 2001; Bilodeau et al., 2003). To further characterize MUb-mediated degradation of GLR-1, we tested the role of the MVB sorting pathway in this process. The VPS-4 AAA ATPase is required for internalizing cargo from the limiting membrane of endosomes to the internalized vesicles of MVBs (Babst et al., 2002; Yeo et al., 2003). As expected, expressing a dominant-negative VPS-4 mutant, vps-4(DN), significantly decreased the effects of MUb on the density of GLR-1::GFP puncta (Figure 9, A–E). These results indicate that GLR-1, like other membrane proteins, undergoes post-endocytic degradation by ubiquitin-mediated sorting into the MVB pathway.
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; Yeo et al., 2003). Consistent with this prediction, GLR-1::GFP accumulated in the limiting membrane of large vesicular structures in PVC cell bodies of vps-4(DN) mutants (Figure 10B). By contrast, in unc-108(nu415) mutants, GLR-1::GFP accumulated in tubulovesicular structures in PVC interneuron cell bodies (Figures 6, B and C, and 10C). Thus, VPS-4 and UNC-108 had distinct effects on the distribution of GLR-1::GFP in cell bodies. Together, these results suggest that UNC-108/Rab2 is not required for ubiquitin-mediated sorting of GLR-1::GFP into the MVB pathway.
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; Lin et al., 2000). If this were the case for GLR-1, then one might expect that GLR-1 could be subject to endocytosis by both the ubiquitin/MVB pathway, and by additional endocytic pathways which may require UNC-108/Rab2. To test this idea, we examined the distribution of GLR-1::GFP in vps-4(DN); unc-108(nu415) double mutants. If UNC-108/Rab2 and the MVB pathway define two alternative recycling mechanisms regulating GLR-1, then we would expect that these mutations would have additive effects. The distribution of GLR-1::GFP in PVC interneuron cell bodies in vps-4(DN); unc-108(nu415) double mutants seemed different from that of either single mutant (Figure 10, B–D) suggesting possible additivity. In the ventral cord, GLR-1::GFP puncta fluorescence in vps-4(DN); unc-108(nu415) double mutants was significantly greater than that observed in either single mutant (Figure 10, F–J), demonstrating that these mutations had additive effects on GLR-1::GFP. These results suggest that UNC-108/Rab2 and VPS-4 define two parallel pathways that regulate postendocytic trafficking of GLR-1. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Grosshans et al., 2006). UNC-108 is more similar to RAB-2 (87% identical to mouse Rab2) than any other C. elegans Rab protein. Thus, this study represents the first analysis of a Rab2 loss-of-function phenotype in a metazoan.
A New Function for Rab2: UNC-108 Promotes Postendocytic Trafficking
We showed that UNC-108/Rab2 is required for postendocytic trafficking in both neurons and coelomocytes. In coelomocytes, uptake of Texas Red-BSA was normal, but there was a decreased rate of trafficking from RME-8::GFP–positive endosomes to later endocytic compartments. The decreased rate in endosome to lysosome traffic leads to the enlargement of the RME-8::GFP–positive endosomes. Similar coelomocyte trafficking defects were recently described in mutants carrying a dominant unc-108 allele, and after knockdown of unc-108 by RNA interference (Lu et al., 2008). In unc- 108 mutant interneurons, there is an accumulation of GLR-1::GFP in tubulovesicular structures that colocalize with markers characteristic of early and recycling endosomes, as well as increased abundance of GLR-1::GFP and of GFP::Syntaxin-13 in the ventral nerve cord. Two very recent papers described a defect in the degradation of engulfed cell death corpses in mutants carrying dominant unc-108 alleles (Lu et al., 2008; Mangahas et al., 2008). These cell death defects were apparently caused by failure of the phagosomes to mature into lysosomes. A direction function for UNC-108 in endosomal trafficking is also supported by colocalization of UNC-108 with endosomal structures. In coelomocytes, GFP::UNC-108 is localized to early endosomes (Lu et al., 2008). In hypodermal cells, UNC-108 was localized to phagosomes containing engulfed cell death corpses (Lu et al., 2008; Mangahas et al., 2008). Collectively, these results strongly suggest that RAB-2 regulates an aspect of endosomal trafficking in C. elegans.
Rab2 belongs to a subfamily of Rabs that includes Rab4 and Rab14. These Rab proteins share a high level of homology within their GTPase domains and hypervariable domains, but they vary near their carboxy-terminal tails. Both Rab4 and Rab14 have been shown to localize to and function at the early endosome (Daro et al., 1996; Junutula et al., 2004). It has been shown that Rabs sharing a high level of similarity tend to function at similar sites within the cell. For example, Rab7 and Rab9 function at the late endosome (Soldati et al., 1995), whereas Rab11 and Rab25 function at the recycling endosome (Ullrich et al., 1996; Casanova et al., 1999). Based on homology, one might predict that Rab2 could function at endosomes, as we have shown here for UNC-108.
Is UNC-108 Required for COPI-mediated Transport?
Previous studies suggested that Rab2 functions in the COPI-mediated retrograde trafficking of proteins between the Golgi apparatus and the ER, based on overexpression of mutant Rab2 proteins in cell lines (Tisdale and Balch, 1996; Tisdale and Jackson, 1998). Therefore, we analyzed unc-108 mutants for defects in the COPI pathway. Mutants disrupting retrograde transport secrete KDEL-containing ER resident proteins, e.g., Kar2/BiP (Semenza et al., 1990; Andag et al., 2001). However, we found that ssVenus::KDEL was retained in the ER, and it was not secreted in unc-108 mutants. COPI mutants also often have alterations in the morphology of the ERGIC; however, we found that ERGIC morphology (visualized with RFP::BET-1) was unaltered in unc-108 mutants. In addition to their retrograde transport defects, some COPI mutants also have anterograde transport defects for proteins, e.g., VSV-G. Therefore, we also examined anterograde transport in unc-108 mutants. Delivery of GLR-1::GFP to the Golgi (assayed as endoglycosidase H resistance), and transport of GFP::UNC-64 Syntaxin out of the ER (assayed by colocalization with ssVenus::KDEL) were both unaffected in unc-108 mutants. Consequently, it is very unlikely that the GLR-1::GFP trafficking defects observed in unc-108 mutants are a secondary consequence of a primary defect in COPI-mediated retrograde transport. Our results do not exclude the possibility that UNC-108 also plays a role in retrograde transport in other tissues, or in other systems.
In addition to its well-characterized function in Golgi-to-ER retrograde transport, several articles have documented a requirement for COPI function in endosomal trafficking. In particular, in a mutant Chinese hamster ovary cell line deficient for the epsilon subunit of COPI, defects were observed in the recycling of transferrin receptors, infection by Semliki forest virus and vesicular stomatitis virus viruses, trafficking of internalized horseradish peroxidase to lysosomes, and an absence of multivesicular endosomes (Daro et al., 1997; Gu et al., 1997). Similar endosomal trafficking defects were also observed in a subset of yeast COPI mutants (Gabriely et al., 2007). These defects are likely to reflect direct functions of COPI on endosomes, because a subset of COPI subunits are found on endosomal membranes in both mammalian cells and in yeast (Whitney et al., 1995; Gabriely et al., 2007). Given these precedents for COPI function in endosomal trafficking, we speculate that UNC-108 (and perhaps Rab2 generally) will play a role in recruiting COPI to endosomal membranes. Consistent with this idea, several yeast COPI mutants have aberrant endosomal structures characteristic of the class E Vps mutants (Gabriely et al., 2007), which are similar to the aberrant endosomal structures we describe in unc-108 mutant interneurons and coelomocytes.
Postendocytic Trafficking of GLR-1
In the ventral cord interneurons, UNC-108/Rab2 was required for the proper distribution of the AMPA-type glutamate receptor GLR-1. Our results suggest that after internalization from the plasma membrane, GLR-1::GFP is sorted into either of two postendocytic pathways. Degradation of GLR-1::GFP is mediated by ubiquitin-mediated sorting into the MVB/lysosome pathway, whereas UNC-108 mediates trafficking of GLR-1::GFP from Syntaxin-13–positive endosomes. Several results support this model. First, in the cell bodies of unc-108 mutant interneurons, GLR-1::GFP accumulates in tubulovesicular structures that colocalize with endosomal markers. By contrast, GLR-1::GFP accumulates in the limiting membrane of intracellular organelles in vps-4(DN) mutants, which are defective for MVB trafficking. Second, the endosomal marker GFP::Syntaxin-13 accumulates in the ventral cord processes of unc-108 mutants. Third, ubiquitin-mediated degradation of GLR-1::GFP occurs normally in unc-108 mutants, but was significantly reduced in vps-4(DN) mutants. Fourth, the unc-108(nu415) and vps-4(DN) mutations had additive effects on GLR-1::GFP accumulation. Collectively, these results demonstrate that post-endocytic trafficking of GLR-1::GFP is mediated by two parallel pathways, the ubiquitin/MVB pathway and the RAB-2/Syntaxin-13 pathway.
Tubulovesicular endosomes similar to those observed in the cell bodies of unc-108 mutant interneurons are most commonly associated with recycling endosomes (Prekeris et al., 1999). Tubulovesicular recycling endosomes are typically located within neuronal cell bodies, in a perinuclear location (Hopkins et al., 1994). These results suggest that UNC-108/Rab2 may function in recycling endosomes. Consistent with this idea, in unc-108 mutants, GLR-1::GFP accumulates in structures containing the recycling endosome marker Rab8. Thus, we propose that UNC-108/Rab2 regulates sorting of GLR-1::GFP from early or recycling endosomes; however, the target membrane for this pathway remains unclear. Our results do not indicate whether UNC-108/Rab2 promotes recycling of GLR-1 from endosomes to the plasma membrane, or to another intracellular compartment.
Our results are consistent with prior studies of mammalian AMPA receptors. Mammalian AMPA receptors have multiple, alternative postendocytic fates. After internalization, the AMPA receptor GluR1 is sorted into both a recycling pathway, from which it can subsequently be returned to the cell surface, and into a lysosomal degradation pathway (Ehlers, 2000). Similarly, we find that GLR-1 undergoes two postendocytic trafficking pathways, which function in parallel. Very little is known about the cellular mechanisms that control differential sorting of GluRs between these postendocytic pathways. Different signaling pathways selectively bias GluR1 into either of these two postendocytic pathways. For example, insulin signaling and proteasome activity have both been implicated in postendocytic degradation of GluR1 (Ehlers, 2000; Patrick et al., 2003). Our results suggest that Rab2 activity may selectively promote trafficking of GluRs through the recycling endosome pathway.
Implications for Synaptic Plasticity: UNC-108/Rab2 as a Regulator of GluR Recycling
Recycling endosomes are thought to play a pivotal role in activity-dependent remodeling of synapses, e.g., those occurring during LTP. After potentiation, AMPA-type GluRs are inserted into spine membranes, and these newly inserted receptors are thought to be derived from Syntaxin-13–positive recycling endosomes (Lee et al., 2001; Park et al., 2004). Similarly, potentiated synapses are typically found at larger dendritic spines, and recycling endosomes are proposed to provide the new membrane inserted into potentiated spines (Park et al., 2004). Thus, regulation of the recycling endosome pathway is likely to play a role in expressing activity-dependent forms of plasticity. We speculate that the regulation of Rab2 activity may play an important role in this process.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work. 
Present address: Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA 90095.
Address correspondence to: Joshua M. Kaplan (kaplan{at}molbio.mgh.harvard.edu)
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