Explore the vast range of titles available.

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.

Coordinating the balance between development and stress responses is critical for organismal survival. However, the cellular signaling controlling this mechanism is not well understood. In Caenorhabditis elegans, it has been hypothesized that a genetic network regulated by NIPI-3/Tibbles may control the balance between animal development and immune response. Using a nipi-3(0) lethality suppressor screen in C. elegans , we reveal a novel role for N-terminal acetyltransferase C complex natc-1/2/3 and histone deacetylase hda-4 , in the control of animal development. These signaling proteins act, at least in part, through a PMK-1 p38 MAP kinase pathway (TIR-1–NSY-1–SEK-1–PMK-1), which plays a critical role in the innate immunity against infection. Additionally, using a transcriptional reporter of SEK-1, a signaling molecule within this p38 MAP kinase system that acts directly downstream of C/EBP bZip transcription factor CEBP-1, we find unexpected positive control of sek-1 transcription by SEK-1 along with several other p38 MAP kinase pathway components. Together, these data demonstrate a role for NIPI-3 regulators in animal development, operating, at least in part through a PMK-1 p38 MAPK pathway. Because the C. elegans p38 MAP kinase pathway is well known for its role in cellular stress responses, the novel biological components and mechanisms pertaining to development identified here may also contribute to the balance between stress response and development.

The conserved MAP3K12/Dual Leucine Zipper Kinase (DLK) plays versatile roles in neuronal development, axon injury and stress responses, and neurodegeneration, depending on cell-type and cellular contexts. Emerging evidence implicates abnormal DLK signaling in several neurodegenerative diseases. However, our understanding of the DLK-dependent gene network in the central nervous system remains limited. Here, we investigated the roles of DLK in hippocampal glutamatergic neurons using conditional knockout and induced overexpression mice. We found that dorsal CA1 and dentate gyrus neurons are vulnerable to elevated expression of DLK, while CA3 neurons appear less vulnerable. We identified the DLK-dependent translatome that includes conserved molecular signatures and displays cell-type specificity. Increasing DLK signaling is associated with disruptions to microtubules, potentially involving STMN4. Additionally, primary cultured hippocampal neurons expressing different levels of DLK show altered neurite outgrowth, axon specification, and synapse formation. The identification of translational targets of DLK in hippocampal glutamatergic neurons has relevance to our understanding of selective neuron vulnerability under stress and pathological conditions.

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron’s oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron’s intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of the C. elegans RNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron-type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5′UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5′UTR-dependent manner. Our study reveals an in vivo mechanism for eIF3 in governing neuronal protein levels to control neuronal activity states and offers insights into how eIF3 dysregulation contributes to neurological disorders.

We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce \"miniSOG\" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.

The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca2+ entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca2+ entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone. Neurons are connected to each other by junctions called synapses. When an electrical signal travelling along a neuron arrives at a synapse, it causes the release of bubble-like structures called synaptic vesicles that contain chemicals called neurotransmitters. When released by the vesicles these neurotransmitters bind to receptors on a second neuron and allow the signal to continue on its way through the nervous system. The release of synaptic vesicles from the neuron depends largely on the number of calcium ions that enter this neuron via structures called ion channels, and also on the rate at which they enter. Vesicles are released in one of three ways: they can be released quickly (within a few milliseconds) in response to the influx of calcium ions; they can be released slowly (over a period of tens or hundreds of milliseconds) in response to the influx; or they can be released at random times that are not related to the influx. It is known that the sensitivity of certain calcium sensors near the synapse influences the release of the vesicles. It had been thought that the distance between the “active zone” where the calcium ions enter the neuron and the region where the vesicles reside might also influence rate of release, but the molecular mechanism underlying this hypothesis is poorly understood. Zhou et al. have now shed new light on this question by performing a series of experiments that involved manipulating a protein called UNC-13 – which is known to be involved in the release of vesicles – in neurons from C. elegans, a nematode worm. First it was shown that the precise position of UNC-13 in the active zone depended on a domain within the protein called the C2A domain. Next it was shown that the distance between the UNC-13 protein and the calcium ion channels strongly influences the quick mode of vesicle release. Finally, Zhou et al. showed that the C2A domain also had a significant influence on the spontaneous release of vesicles, which suggests that a common fleet of vesicles might be used for both the quick and the spontaneous modes of vesicle release. Zhou et al. also generated mutant worms that mimicked a neurological disease, epileptic seizure, and showed that eliminating the C2A domain can relieve some of the symptoms associated with the disease. Many neurological diseases are caused by signals not being transmitted properly at synapses, so in addition to providing insights into the basic mechanism underlying synaptic action, these results could also assist with the development of new strategies for managing neurological diseases.

Apical extracellular matrices (aECMs) are complex extracellular compartments that form important interfaces between animals and their environment. In the adult C. elegans cuticle, layers are connected by regularly spaced columnar structures known as struts. Defects in struts result in swelling of the fluid-filled medial cuticle layer (‘blistering’, Bli). Here we show that three cuticle collagens BLI-1, BLI-2, and BLI-6, play key roles in struts. BLI-1 and BLI-2 are essential for strut formation whereas activating mutations in BLI-6 disrupt strut formation. BLI-1, BLI-2, and BLI-6 precisely colocalize to arrays of puncta in the adult cuticle, corresponding to struts, initially deposited in diffuse stripes adjacent to cuticle furrows. They eventually exhibit tube-like morphology, with the basal ends of BLI-containing struts contact regularly spaced holes in the cuticle. Genetic interaction studies indicate that BLI strut patterning involves interactions with other cuticle components. Our results reveal strut formation as a tractable example of precise aECM patterning at the nanoscale. The extracellular matrix (ECM) is critical for animal development and intricately patterned at multiple scales. Here Adams and Pooranachithra et al . show how specific collagens are precisely localized to struts, highly patterned sub-micron structures in the nematode cuticle ECM.