CDK-5 regulates the abundance of GLR-1 glutamate receptors in the ventral cord of Caenorhabditis elegans

The proline-directed kinase Cdk5 plays a role in several aspects of neuronal development. Here, we show that CDK-5 activity regulates the abundance of the glutamate receptor GLR-1 in the ventral cord of Caenorhabditis elegans and that it produces corresponding changes in GLR-1-dependent behaviors. Loss of CDK-5 activity results in decreased abundance of GLR-1 in the ventral cord, accompanied by accumulation of GLR-1 in neuronal cell bodies. Genetic analysis of cdk-5 and the clathrin adaptin unc-11 AP180 suggests that CDK-5 functions prior to endocytosis at the synapse. The scaffolding protein LIN-10/Mint-1 also regulates GLR-1 abundance in the nerve cord. CDK-5 phosphorylates LIN-10/Mint-1 in vitro and bidirectionally regulates the abundance of LIN-10/Mint-1 in the ventral cord. We propose that CDK-5 promotes the anterograde trafficking of GLR-1 and that phosphorylation of LIN-10 may play a role in this process.     

Distinct LIN-10 domains are required for its neuronal function, its epithelial function, and its synaptic localization

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) mediate excitatory neurotransmission at neuronal synapses, and their regulated localization plays a role in synaptic plasticity. In Caenorhabditis elegans, the PDZ and PTB domain-containing protein LIN-10 is required both for the synaptic localization of the AMPAR subunit GLR-1 and for vulval fate induction in epithelia. Here, we examine the role that different LIN-10 domains play in GLR-1 localization. We find that an amino-terminal region of LIN-10 directs LIN-10 protein localization to the Golgi and to synaptic clusters. In addition, mutations in the carboxyl-terminal PDZ domains prevent LIN-10 from regulating GLR-1 localization in neurons but do not prevent LIN-10 from functioning in the vulval epithelia. A mutation in the amino terminus prevents the protein from functioning in the vulval epithelia but does not prevent it from functioning to regulate GLR-1 localization in neurons. Finally, we show that human Mint2 can substitute for LIN-10 to facilitate GLR-1 localization in neurons and that the Mint2 amino terminus is critical for this function. Together, our data suggest that LIN-10 uses distinct modular domains for its functions in neurons and epithelial cells and that during evolution its vertebrate ortholog Mint2 has retained the ability to direct AMPAR localization in neurons.     

RAB-6.2 and the retromer regulate glutamate receptor recycling through a retrograde pathway

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

RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent endocytosis pathway

Regulated endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, the specific combination of clathrin-dependent and -independent mechanisms that mediate AMPAR trafficking in vivo have not been fully characterized. Here, we examine the trafficking of the AMPAR subunit GLR-1 in Caenorhabditis elegans. GLR-1 is localized on synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit. Animals lacking RAB-10, a small GTPase required for endocytic recycling of intestinal cargo, are similar in phenotype to animals lacking LIN-10, a postsynaptic density 95/disc-large/zona occludens-domain containing protein: GLR-1 accumulates in large accretions and animals display a decreased frequency of reversals. Mutations in unc-11 (AP180) or itsn-1 (Intersectin 1), which reduce clathrin-dependent endocytosis, suppress the lin-10 but not rab-10 mutant phenotype, suggesting that LIN-10 functions after clathrin-mediated endocytosis. By contrast, cholesterol depletion, which impairs lipid raft formation and clathrin-independent endocytosis, suppresses the rab-10 but not the lin-10 phenotype, suggesting that RAB-10 functions after clathrin-independent endocytosis. Animals lacking both genes display additive GLR-1 trafficking defects. We propose that RAB-10 and LIN-10 recycle AMPARs from intracellular endosomal compartments to synapses along distinct pathways, each with distinct sensitivities to cholesterol and the clathrin-mediated endocytosis machinery.     

The T-box gene tbx-2, the homeobox gene egl-5 and the asymmetric cell division gene ham-1 specify neural fate in the HSN/PHB lineage

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, we have been studying a Caenorhabditis elegans lineage that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. We have previously shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. In this study we examine tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, we identified the T-box protein TBX-2 as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage.     

C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.     

Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons.     

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.     

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.     

Caenorhabditis elegans Galphaq regulates egg-laying behavior via a PLCbeta-independent and serotonin-dependent signaling pathway and likely functions both in the nervous system and in muscle

egl-30 encodes the single C. elegans ortholog of vertebrate Galphaq family members. We analyzed the expression pattern of EGL-30 and found that it is broadly expressed, with highest expression in the nervous system and in pharyngeal muscle. We isolated dominant, gain-of-function alleles of egl-30 as intragenic revertants of an egl-30 reduction-of-function mutation. Using these gain-of-function mutants and existing reduction-of-function mutants, we examined the site and mode of action of EGL-30. On the basis of pharmacological analysis, it has been determined that egl-30 functions both in the nervous system and in the vulval muscles for egg-laying behavior. Genetic epistasis over mutations that eliminate detectable levels of serotonin reveals that egl-30 requires serotonin to regulate egg laying. Furthermore, pharmacological response assays strongly suggest that EGL-30 may directly couple to a serotonin receptor to mediate egg laying. We also examined genetic interactions with mutations in the gene that encodes the single C. elegans homolog of PLCbeta and mutations in genes that encode signaling molecules downstream of PLCbeta. We conclude that PLCbeta functions in parallel with egl-30 with respect to egg laying or is not the major effector of EGL-30. In contrast, PLCbeta-mediated signaling is likely downstream of EGL-30 with respect to pharyngeal-pumping behavior. Our data indicate that there are multiple signaling pathways downstream of EGL-30 and that different pathways could predominate with respect to the regulation of different behaviors.     

CYSL-1 interacts with the O2-sensing hydroxylase EGL-9 to promote H2S-modulated hypoxia-induced behavioral plasticity in C. elegans

The C. elegans HIF-1 proline hydroxylase EGL-9 functions as an O(2) sensor in an evolutionarily conserved pathway for adaptation to hypoxia. H(2)S accumulates during hypoxia and promotes HIF-1 activity, but how H(2)S signals are perceived and transmitted to modulate HIF-1 and animal behavior is unknown. We report that the experience of hypoxia modifies a C. elegans locomotive behavioral response to O(2) through the EGL-9 pathway. From genetic screens to identify novel regulators of EGL-9-mediated behavioral plasticity, we isolated mutations of the gene cysl-1, which encodes a C. elegans homolog of sulfhydrylases/cysteine synthases. Hypoxia-dependent behavioral modulation and H(2)S-induced HIF-1 activation require the direct physical interaction of CYSL-1 with the EGL-9 C terminus. Sequestration of EGL-9 by CYSL-1 and inhibition of EGL-9-mediated hydroxylation by hypoxia together promote neuronal HIF-1 activation to modulate behavior. These findings demonstrate that CYSL-1 acts to transduce signals from H(2)S to EGL-9 to regulate O(2)-dependent behavioral plasticity in C. elegans.     

FGF signaling functions in the hypodermis to regulate fluid balance in C. elegans

Signaling by the Caenorhabditis elegans fibroblast growth factor receptor EGL-15 is activated by LET-756, a fibroblast growth factor, and attenuated by CLR-1, a receptor tyrosine phosphatase. Hyperactive EGL-15 signaling results in a dramatic Clr phenotype characterized by the accumulation of clear fluid within the pseudocoelomic space, suggesting that regulated EGL-15 signaling is essential for fluid homeostasis in C. elegans. To determine the cellular focus of EGL-15 signaling, we identified an enhancer element (e15) within the egl-15 promoter, which is both necessary for the promoter activity and sufficient when duplicated to drive either egl-15 or clr-1 rescue activity. This enhancer drives GFP expression in hypodermal cells. Consistent with this finding, immunofluorescence studies of EGL-15 indicate that EGL-15 is expressed in hypodermal cells, and hypodermal promoters can drive full clr-1 and egl-15 rescue activity. Moreover, a mosaic analysis of mpk-1, which acts downstream of egl-15, suggests that its suppression of Clr (Soc) function is required in the hypodermis. These results suggest that EGL-15 and CLR-1 act in the hypodermis to regulate fluid homeostasis in worms.     

The kinesin-3 family motor KLP-4 regulates anterograde trafficking of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

The transport of glutamate receptors from the cell body to synapses is essential during neuronal development and may contribute to the regulation of synaptic strength in the mature nervous system. We previously showed that cyclin-dependent kinase-5 (CDK-5) positively regulates the abundance of GLR-1 glutamate receptors at synapses in the ventral nerve cord (VNC) of Caenorhabditis elegans. Here we identify a kinesin-3 family motor klp-4/KIF13 in a cdk-5 suppressor screen for genes that regulate GLR-1 trafficking. klp-4 mutants have decreased abundance of GLR-1 in the VNC. Genetic analysis of klp-4 and the clathrin adaptin unc-11/AP180 suggests that klp-4 functions before endocytosis in the ventral cord. Time-lapse microscopy indicates that klp-4 mutants exhibit decreased anterograde flux of GLR-1. Genetic analysis of cdk-5 and klp-4 suggests that they function in the same pathway to regulate GLR-1 in the VNC. Interestingly, GLR-1 accumulates in cell bodies of cdk-5 but not klp-4 mutants. However, GLR-1 does accumulate in klp-4-mutant cell bodies if receptor degradation in the multivesicular body/lysosome pathway is blocked. This study identifies kinesin KLP-4 as a novel regulator of anterograde glutamate receptor trafficking and reveals a cellular control mechanism by which receptor cargo is targeted for degradation in the absence of its motor.     

Regulation of anchor cell invasion and uterine cell fates by the egl-43 Evi-1 proto-oncogene in Caenorhabditis elegans

Cell invasion is a tightly controlled process occurring during development and tumor progression. The nematode Caenorhabditis elegans serves as a genetic model to study cell invasion during normal development. In the third larval stage, the anchor cell in the somatic gonad first induces and then invades the adjacent epidermal vulval precursor cells. The homolog of the Evi-1 oncogene, egl-43, is necessary for basement membrane destruction and anchor cell invasion. egl-43 is part of a regulatory network mediating cell invasion downstream of the fos-1 proto-oncogene. In addition, EGL-43 is required to specify the cell fates of ventral uterus cells downstream of or in parallel with LIN-12 NOTCH. Comparison with mammalian Evi-1 suggests a conserved pathway controlling cell invasion and cell fate specification.