Voltage-gated calcium channels direct neuronal migration in Caenorhabditis elegans

Calcium signaling is known to be important for regulating the guidance of migrating neurons, yet the molecular mechanisms underlying this process are not well understood. We have found that two different voltage-gated calcium channels are important for the accurate guidance of postembryonic neuronal migrations in the nematode Caenorhabditis elegans. In mutants carrying loss-of-function alleles of the calcium channel gene unc-2, the touch receptor neuron AVM and the interneuron SDQR often migrated inappropriately, leading to misplacement of their cell bodies. However, the AVM neurons in unc-2 mutant animals extended axons in a wild-type pattern, suggesting that the UNC-2 calcium channel specifically directs migration of the neuronal cell body and is not required for axonal pathfinding. In contrast, mutations in egl-19, which affect a different voltage-gated calcium channel, affected the migration of the AVM and SDQR bodies, as well as the guidance of the AVM axon. Thus, cell migration and axonal pathfinding in the AVM neurons appear to involve distinct calcium channel subtypes. Mutants defective in the unc-43/CaM kinase gene showed a defect in SDQR and AVM positioning that resembled that of unc-2 mutants; thus, CaM kinase may function as an effector of the UNC-2-mediated calcium influx in guiding cell migration.     

The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates

A stochastic cell fate decision mediated by axon contact and calcium signaling causes one of the two bilaterally symmetric AWC neurons, either AWCL or AWCR, to express the candidate olfactory receptor str-2. nsy-1 mutants express str-2 in both neurons, disrupting AWC asymmetry. nsy-1 encodes a homolog of the human MAP kinase kinase kinase (MAPKKK) ASK1, an activator of JNK and p38 kinases. Based on genetic epistasis analysis, nsy-1 appears to act downstream of the CaMKII unc-43, and NSY-1 associates with UNC-43, suggesting that UNC-43/CaMKII activates the NSY-1 MAP kinase cassette. Mosaic analysis demonstrates that UNC-43 and NSY-1 act primarily in a cell-autonomous execution step that represses str-2 expression in one AWC cell, downstream of the initial lateral signaling pathway that coordinates the fates of the two cells.     

UNC-73/trio RhoGEF-2 activity modulates Caenorhabditis elegans motility through changes in neurotransmitter signaling upstream of the GSA-1/Galphas pathway

Rho-family GTPases play regulatory roles in many fundamental cellular processes. Caenorhabditis elegans UNC-73 RhoGEF isoforms function in axon guidance, cell migration, muscle arm extension, phagocytosis, and neurotransmission by activating either Rac or Rho GTPase subfamilies. Multiple differentially expressed UNC-73 isoforms contain a Rac-specific RhoGEF-1 domain, a Rho-specific RhoGEF-2 domain, or both domains. The UNC-73E RhoGEF-2 isoform is activated by the G-protein subunit Gαq and is required for normal rates of locomotion; however, mechanisms of UNC-73 and Rho pathway regulation of locomotion are not clear. To better define UNC-73 function in the regulation of motility we used cell-specific and inducible promoters to examine the temporal and spatial requirements of UNC-73 RhoGEF-2 isoform function in mutant rescue experiments. We found that UNC-73E acts within peptidergic neurons of mature animals to regulate locomotion rate. Although unc-73 RhoGEF-2 mutants have grossly normal synaptic morphology and weak resistance to the acetylcholinesterase inhibitor aldicarb, they are significantly hypersensitive to the acetylcholine receptor agonist levamisole, indicating alterations in acetylcholine neurotransmitter signaling. Consistent with peptidergic neuron function, unc-73 RhoGEF-2 mutants exhibit a decreased level of neuropeptide release from motor neuron dense core vesicles (DCVs). The unc-73 locomotory phenotype is similar to those of rab-2 and unc-31, genes with distinct roles in the DCV-mediated secretory pathway. We observed that constitutively active Gαs pathway mutations, which compensate for DCV-mediated signaling defects, rescue unc-73 RhoGEF-2 and rab-2 lethargic movement phenotypes. Together, these data suggest UNC-73 RhoGEF-2 isoforms are required for proper neurotransmitter signaling and may function in the DCV-mediated neuromodulatory regulation of locomotion rate.     

UNC-52/perlecan affects gonadal leader cell migrations in C. elegans hermaphrodites through alterations in growth factor signaling

The unc-52 gene of Claenorhabditis elegans encodes a homologue of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. We propose that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways.     

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 netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and,in parallel,Rac and UNC-115/AbLIM

Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, we generated a gain-of-function UNC-40 molecule, MYR::UNC-40. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. Our results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton.     

MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion

The netrin UNC-6 repels motor axons by activating the UNC-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the UNC-5 receptor signaling pathway.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

Sulfated signal from ASJ sensory neurons modulates stomatin-dependent coordination in Caenorhabditis elegans

The neuronal stomatin-like proteins UNC-1 and UNC-24 play important roles in the nervous system of Caenorhabditis elegans. These neuronal stomatin-like proteins are putative chaperone proteins that can modify volatile anesthetic sensitivity and disrupt coordinated locomotion. A suppressor of unc-1 and unc-24, named ssu-1(fc73) (for suppressor of stomatin uncoordination), suppresses three phenotypes of neuronal stomatin-like protein deficiency as follows: volatile anesthetic sensitivity, uncoordinated locomotion, and a constitutive alternative developmental phenotype known as dauer. Here we provide the first phenotypic characterization of ssu-1, predicted to be the only C. elegans cytosolic alcohol sulfotransferase, a family of enzymes that catalyze a sulfate linkage with the alcohol group of small molecules for the purposes of detoxification or modification of signaling. In vitro enzyme analysis of bacterially expressed SSU-1 demonstrates sulfotransferase activity and thus confirms the function predicted by protein sequence similarities. Whereas unc-1 is expressed in the majority of neurons of C. elegans, expression of SSU-1 protein in only the two ASJ amphid interneurons is sufficient to restore the wild type phenotype. This work demonstrates that SSU-1 is a functional sulfotransferase that likely modifies endocrine signaling in C. elegans. The expression of SSU-1 in the ASJ neurons refines the understanding of the function of these cells and supports their classification as endocrine tissue. The relationship of unc-1, unc-24, and ssu-1 is the first association of neuronal stomatin-like proteins sharing regulatory roles with a sulfotransferase enzyme.     

Multiple signaling mechanisms of the UNC-6/netrin receptors UNC-5 and UNC-40/DCC in vivo.

Cell and growth cone migrations along the dorsoventral axis of Caenorhabditis elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, we have examined genetic interactions between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. We report that cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. We show that hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, we observed that unc-5 hypomorphs exhibit dominant negative effects. We propose that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes.     

UNC-108/Rab2 regulates postendocytic trafficking in Caenorhabditis elegans

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.     

Presynaptic UNC-31 (CAPS) is required to activate the G alpha(s) pathway of the Caenorhabditis elegans synaptic signaling network

C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal G alpha(s) pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal G alpha(s) are different parts of the same pathway and that the UNC-31/G alpha(s) pathway is functionally distinct from the presynaptic G alpha(q) pathway with which it interacts. UNC-31 acts upstream of G alpha(s) because mutations that activate the G alpha(s) pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G alpha(s) pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal G alpha(s) pathway near synaptic active zones.     

The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C. elegans

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has previously been implicated in axon pathfinding. We have discovered the role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding. We show that unc-115 double mutants with ced-10 Rac, mig-2 Rac or unc-73 GEF but not with rac-2/3 Rac displayed synthetic axon pathfinding defects, and that loss of unc-115 function suppressed the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, we show that UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding.     

UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ～50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the μ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as μ2 adaptin.     

Noncell- and cell-autonomous G-protein-signaling converges with Ca2+/mitogen-activated protein kinase signaling to regulate str-2 receptor gene expression in Caenorhabditis elegans

In the sensory system of C. elegans, the candidate odorant receptor gene str-2 is strongly expressed in one of the two AWC neurons and weakly in both ASI neurons. Asymmetric AWC expression results from suppression of str-2 expression by a Ca2+/MAPK signaling pathway in one of the AWC neurons early in development. Here we show that the same Ca2+/MAPK pathway promotes str-2 expression in the AWC and ASI neurons together with multiple cell-autonomous and noncell-autonomous G-protein-signaling pathways. In first-stage larvae and adult animals, signals mediated by the Galpha subunits ODR-3, GPA-2, GPA-5, and GPA-6 and a Ca2+/MAPK pathway involving the Ca2+ channel subunit UNC-36, the CaMKII UNC-43, and the MAPKK kinase NSY-1 induce strong str-2 expression. Cell-specific rescue experiments suggest that ODR-3 and the Ca2+/MAPK genes function in the AWC neurons, but that GPA-5 and GPA-6 function in the AWA and ADL neurons, respectively. In Dauer larvae, the same network of genes promotes strong str-2 expression in the ASI neurons, but ODR-3 functions in AWB and ASH and GPA-6 in AWB. Our results reveal a complex signaling network, encompassing signals from multiple cells, that controls the level of receptor gene expression at different developmental stages.     

Protein interaction surface of the POU transcription factor UNC-86 selectively used in touch neurons

The Caenorhabditis elegans POU protein UNC-86 specifies the HSN motor neurons, which are required for egg-laying, and six mechanosensory neurons. To investigate how UNC-86 controls neuronal specification, we characterized two unc-86 mutants that do not respond to touch but show wild-type egg-laying behavior. Residues P145 and L195, which are altered by these mutations, are located in the POU-specific domain and abolish the physical interaction of UNC-86 with the LIM homeodomain protein, MEC-3. This results in a failure to maintain mec-3 expression and in loss of expression of the mechanosensory neuron-specific gene, mec-2. unc-86-dependent expression of genes in other neurons is not impaired. We conclude that distinct residues in the POU domain of UNC-86 are involved in modulating UNC-86 activity during its specification of different neurons. A structural model of the UNC-86 POU domain, including base pairs and amino acid residues required for MEC-3 interaction, revealed that P145 and L195 are part of a hydrophobic pocket which is similar to the OCA-B-binding domain of the mammalian POU protein, Oct-1.