The Ubiquitin Ligase RPM-1 and the p38 MAPK PMK-3 Regulate AMPA Receptor Trafficking

Ubiquitination occurs at synapses, yet its role remains unclear. Previous studies demonstrated that the RPM-1 ubiquitin ligase organizes presynaptic boutons at neuromuscular junctions in C. elegans motorneurons. Here we find that RPM-1 has a novel postsynaptic role in interneurons, where it regulates the trafficking of the AMPA-type glutamate receptor GLR-1 from synapses into endosomes. Mutations in rpm-1 cause the aberrant accumulation of GLR-1 in neurites. Moreover, rpm-1 mutations enhance the endosomal accumulation of GLR-1 observed in mutants for lin-10, a Mint2 ortholog that promotes GLR-1 recycling from Syntaxin-13 containing endosomes. As in motorneurons, RPM-1 negatively regulates the pmk-3/p38 MAPK pathway in interneurons by repressing the protein levels of the MAPKKK DLK-1. This regulation of PMK-3 signaling is critical for RPM-1 function with respect to GLR-1 trafficking, as pmk-3 mutations suppress both lin-10 and rpm-1 mutations. Positive or negative changes in endocytosis mimic the effects of rpm-1 or pmk-3 mutations, respectively, on GLR-1 trafficking. Specifically, RAB-5(GDP), an inactive mutant of RAB-5 that reduces endocytosis, mimics the effect of pmk-3 mutations when introduced into wild-type animals, and occludes the effect of pmk-3 mutations when introduced into pmk-3 mutants. By contrast, RAB-5(GTP), which increases endocytosis, suppresses the effect of pmk-3 mutations, mimics the effect of rpm-1 mutations, and occludes the effect of rpm-1 mutations. Our findings indicate a novel specialized role for RPM-1 and PMK-3/p38 MAPK in regulating the endosomal trafficking of AMPARs at central synapses

Hypoxia regulates glutamate receptor trafficking through an HIF‐independent mechanism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102065/1/embj2011499-reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102065/2/embj2011499-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102065/3/embj2011499.pd

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (α α-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans. D uring animal development, specialized junctional domains are crucial for the function of epithelial cell sheets. In both vertebrates and invertebrates, adherens junctions are thought to regulate cell-cell adhesion and dynamic changes in cell morphology Here we show that the novel coiled-coil protein AJM-1 (for 'apical junction molecule') is required for the integrity of epithelial junctions of C. elegans and that it localizes to an apical junctional domain. (AJM-1 was originally called JAM-1 (refs 13, 14) but has been renamed to avoid confusion with the vertebrate transmembrane tight junction protein, JAM-1.) This domain is basal to the HMR-HMP(cadherin-catenin) complex; on the basis of the localization of the Discs large homologue DLG-1 to the same domain, it might be required for maintaining a tight apical seal between epithelial cells at apical junctions. Furthermore, we show that AJM-1 directly binds DLG-1, which is required for the proper distribution of AJM-1 around the junctional belt but not for general cell polarity. In addition, we show that in embryos lacking LET-413 the patterns of both DLG-1 and AJM-1 are equally disrupted, including a delay in concentration of these proteins at a narrow apical domain. Almost complete loss of junctional AJM-1 is observed in the absence of both LET-413 and DLG-1, whereas HMP-1 (α-catenin) localization is reduced but junctional. We propose a model in which LET-413 and DLG-1 control the integrity of a distinct apical subdomain by cooperatively regulating the localization of AJM-1. Results AJM-1 encodes a novel coiled-coil protein localizing to C. elegans apical junctions. As an initial step in understanding the molecular composition of apical junctions in C. elegans, we characterized the antigen recognized by the MH27 antibody. The antibody had been previously shown to stain apical borders of C. elegans epitheli

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (α α-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans. D uring animal development, specialized junctional domains are crucial for the function of epithelial cell sheets. In both vertebrates and invertebrates, adherens junctions are thought to regulate cell-cell adhesion and dynamic changes in cell morphology Here we show that the novel coiled-coil protein AJM-1 (for 'apical junction molecule') is required for the integrity of epithelial junctions of C. elegans and that it localizes to an apical junctional domain. (AJM-1 was originally called JAM-1 (refs 13, 14) but has been renamed to avoid confusion with the vertebrate transmembrane tight junction protein, JAM-1.) This domain is basal to the HMR-HMP(cadherin-catenin) complex; on the basis of the localization of the Discs large homologue DLG-1 to the same domain, it might be required for maintaining a tight apical seal between epithelial cells at apical junctions. Furthermore, we show that AJM-1 directly binds DLG-1, which is required for the proper distribution of AJM-1 around the junctional belt but not for general cell polarity. In addition, we show that in embryos lacking LET-413 the patterns of both DLG-1 and AJM-1 are equally disrupted, including a delay in concentration of these proteins at a narrow apical domain. Almost complete loss of junctional AJM-1 is observed in the absence of both LET-413 and DLG-1, whereas HMP-1 (α-catenin) localization is reduced but junctional. We propose a model in which LET-413 and DLG-1 control the integrity of a distinct apical subdomain by cooperatively regulating the localization of AJM-1. Results AJM-1 encodes a novel coiled-coil protein localizing to C. elegans apical junctions. As an initial step in understanding the molecular composition of apical junctions in C. elegans, we characterized the antigen recognized by the MH27 antibody. The antibody had been previously shown to stain apical borders of C. elegans epitheli

MAGI-1 Modulates AMPA Receptor Synaptic Localization and Behavioral Plasticity in Response to Prior Experience

It is well established that the efficacy of synaptic connections can be rapidly modified by neural activity, yet how the environment and prior experience modulate such synaptic and behavioral plasticity is only beginning to be understood. Here we show in C. elegans that the broadly conserved scaffolding molecule MAGI-1 is required for the plasticity observed in a glutamatergic circuit. This mechanosensory circuit mediates reversals in locomotion in response to touch stimulation, and the AMPA-type receptor (AMPAR) subunits GLR-1 and GLR-2, which are required for reversal behavior, are localized to ventral cord synapses in this circuit. We find that animals modulate GLR-1 and GLR-2 localization in response to prior mechanosensory stimulation; a specific isoform of MAGI-1 (MAGI-1L) is critical for this modulation. We show that MAGI-1L interacts with AMPARs through the intracellular domain of the GLR-2 subunit, which is required for the modulation of AMPAR synaptic localization by mechanical stimulation. In addition, mutations that prevent the ubiquitination of GLR-1 prevent the decrease in AMPAR localization observed in previously stimulated magi-1 mutants. Finally, we find that previously-stimulated animals later habituate to subsequent mechanostimulation more rapidly compared to animals initially reared without mechanical stimulation; MAGI-1L, GLR-1, and GLR-2 are required for this change in habituation kinetics. Our findings demonstrate that prior experience can cause long-term alterations in both behavioral plasticity and AMPAR localization at synapses in an intact animal, and indicate a new, direct role for MAGI/S-SCAM proteins in modulating AMPAR localization and function in the wake of variable sensory experience

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

Going Mobile: AMPA Receptors Move Synapse to Synapse In Vivo

Plasticity models invoke the synaptic delivery of AMPARs, yet we know little about how receptors move in vivo. In this issue of Neuron, Hoerndli et al. (2013) show that lateral diffusion and kinesin-mediated transport move AMPARs between synapses in vivo

The role of RNA localization and translational regulation in Drosophila germ cell determination

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1996.Includes bibliographical references (leaves 165-180).by Christopher Gabriel Rongo.Ph.D