Alexandr Goncharov

Regulation of a DLK-1 and p38 MAP Kinase Pathway by the Ubiquitin Ligase RPM-1 Is Required for Presynaptic Development

Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.

Distinct Innate Immune Responses to Infection and Wounding in the C. elegans Epidermis

BACKGROUND In many animals, the epidermis is in permanent contact with the environment and represents a first line of defense against pathogens and injury. Infection of the nematode Caenorhabditis elegans by the natural fungal pathogen Drechmeria coniospora induces the expression in the epidermis of antimicrobial peptide (AMP) genes such as nlp-29. Here, we tested the hypothesis that injury might also alter AMP gene expression and sought to characterize the mechanisms that regulate the innate immune response. RESULTS Injury induces a wound-healing response in C. elegans that includes induction of nlp-29 in the epidermis. We find that a conserved p38-MAP kinase cascade is required in the epidermis for the response to both infection and wounding. Through a forward genetic screen, we isolated mutants that failed to induce nlp-29 expression after D. coniospora infection. We identify a kinase, NIPI-3, related to human Tribbles homolog 1, that is likely to act upstream of the MAPKK SEK-1. We find NIPI-3 is required only for nlp-29 induction after infection and not after wounding. CONCLUSIONS Our results show that the C. elegans epidermis actively responds to wounding and infection via distinct pathways that converge on a conserved signaling cassette that controls the expression of the AMP gene nlp-29. A comparison between these results and MAP kinase signaling in yeast gives insights into the possible origin and evolution of innate immunity.

Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase

Axons of adult Caenorhabditis elegans neurons undergo robust regenerative growth after laser axotomy. Here we show that axotomy of PLM sensory neurons triggers axonal calcium waves whose amplitude correlates with the extent of regeneration. Genetic elevation of Ca2+ or cAMP accelerates formation of a growth cone from the injured axon. Elevated Ca2+ or cAMP also facilitates apparent fusion of axonal fragments and promotes branching to postsynaptic targets. Conversely, inhibition of voltage-gated calcium channels or calcium release from internal stores reduces regenerative growth. We identify the fusogen EFF-1 as critical for axon fragment fusion and the basic leucine zipper domain (bZip) protein CREB (cAMP response element-binding protein) as a key effector for branching. The effects of elevated Ca2+ or cAMP on regrowth require the MAPKKK (mitogen-activated protein kinase kinase kinase) DLK-1. Increased cAMP signaling can partly bypass the requirement for the bZip protein CEBP-1, a downstream factor of the DLK-1 kinase cascade. These findings reveal the relationship between Ca2+/cAMP signaling and the DLK-1 MAPK (mitogen-activated protein kinase) cascade in regeneration.

Kinesin-13 and Tubulin Posttranslational Modifications Regulate Microtubule Growth in Axon Regeneration

The microtubule (MT) cytoskeleton of a mature axon is maintained in a stabilized steady state, yet after axonal injury it can be transformed into a dynamic structure capable of supporting axon regrowth. Using Caenorhabditis elegans mechanosensory axons and in vivo imaging, we find that, in mature axons, the growth of MTs is restricted in the steady state by the depolymerizing kinesin-13 family member KLP-7. After axon injury, we observe a two-phase process of MT growth upregulation. First, the number of growing MTs increases at the injury site, concomitant with local downregulation of KLP-7. A second phase of persistent MT growth requires the cytosolic carboxypeptidase CCPP-6, which promotes Δ2 modification of α-tubulin. Both phases of MT growth are coordinated by the DLK-1 MAP kinase cascade. Our results define how the stable MT cytoskeleton of a mature neuron is converted into the dynamically growing MT cytoskeleton of a regrowing axon.

The C. elegans peroxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration

Peroxidasins form a highly conserved family of extracellular peroxidases of unknown cellular function. We identified the C. elegans peroxidasin PXN-2 in screens for mutants defective in embryonic morphogenesis. We find that PXN-2 is essential for specific stages of embryonic morphogenesis and muscle-epidermal attachment, and is also required postembryonically for basement membrane integrity. The peroxidase catalytic activity of PXN-2 is necessary for these developmental roles. pxn-2 mutants display aberrant ultrastructure of the extracellular matrix, suggesting a role in basement membrane consolidation. PXN-2 affects specific axon guidance choice points in the developing nervous system but is dispensable for maintenance of process positions. In adults, loss of pxn-2 function promotes regrowth of axons after injury, providing the first evidence that C. elegans extracellular matrix can play an inhibitory role in axon regeneration. Loss of function in the closely related C. elegans peroxidasin pxn-1 does not cause overt developmental defects. Unexpectedly, pxn-2 mutant phenotypes are suppressed by loss of function in pxn-1 and exacerbated by overexpression of wild-type pxn-1, indicating that PXN-1 and PXN-2 have antagonistic functions. These results demonstrate that peroxidasins play crucial roles in development and reveal a new role for peroxidasins as extracellular inhibitors of axonal regeneration.

C. elegans ankyrin repeat protein VAB-19 is a component of epidermal attachment structures and is essential for epidermal morphogenesis

Elongation of the epidermis of the nematode Caenorhabditis elegans involves both actomyosin-mediated changes in lateral epidermal cell shape and body muscle attachment to dorsal and ventral epidermal cells via intermediate-filament/hemidesmosome structures. vab-19 mutants are defective in epidermal elongation and muscle attachment to the epidermis. VAB-19 is a member of a conserved family of ankyrin repeat-containing proteins that includes the human tumor suppressor Kank. In epidermal cells, VAB-19::GFP localizes with components of epidermal attachment structures. In vab-19 mutants, epidermal attachment structures form normally but do not remain localized to muscle-adjacent regions of the epidermis. VAB-19 localization requires function of the transmembrane attachment structure component Myotactin. vab-19 mutants also display aberrant actin organization in the epidermis. Loss of function in the spectrin SMA-1 partly bypasses the requirement for VAB-19 in elongation, suggesting that VAB-19 and SMA-1/spectrin might play antagonistic roles in regulation of the actin cytoskeleton.

Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase

Wounding of epidermal layers triggers multiple coordinated responses to damage. We show here that the Caenorhabditis elegans ortholog of the tumor suppressor death-associated protein kinase, dapk-1, acts as a previously undescribed negative regulator of barrier repair and innate immune responses to wounding. Loss of DAPK-1 function results in constitutive formation of scar-like structures in the cuticle, and up-regulation of innate immune responses to damage. Overexpression of DAPK-1 represses innate immune responses to needle wounding. Up-regulation of innate immune responses in dapk-1 requires the TIR-1/p38 signal transduction pathway; loss of function in this pathway synergizes with dapk-1 to drastically reduce adult lifespan. Our results reveal a previously undescribed function for the DAPK tumor suppressor family in regulation of epithelial damage responses.

Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics

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. DOI: http://dx.doi.org/10.7554/eLife.01180.001

Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans

Liprin-α/SYD-2 activity promotes the polymerization of electron-dense projections in the presynaptic active zone through increased recruitment of ELKS-1/ELKS.

Caenorhabditis elegans Flamingo Cadherin fmi-1 Regulates GABAergic Neuronal Development

In a genetic screen for regulators of synaptic morphology, we identified the single Caenorhabditis elegans flamingo-like cadherin fmi-1. The fmi-1 mutants exhibit defective axon pathfinding, reduced synapse number, aberrant synapse size and morphology, as well as an abnormal accumulation of synaptic vesicles at nonsynaptic regions. Although FMI-1 is primarily expressed in the nervous system, it is not expressed in the ventral D-type (VD) GABAergic motorneurons, which are defective in fmi-1 mutants. The axon and synaptic defects of VD neurons could be rescued when fmi-1 was expressed exclusively in non-VD neighboring neurons, suggesting a cell nonautonomous action of FMI-1. FMI-1 protein that lacked its intracellular domain still retained its ability to rescue the vesicle accumulation defects of GABAergic motorneurons, indicating that the extracellular domain was sufficient for this function of FMI-1 in GABAergic neuromuscular junction development. Mutations in cdh-4, a Fat-like cadherin, cause similar defects in GABAergic motorneurons. The cdh-4 is expressed by the VD neurons and seems to function in the same genetic pathway as fmi-1 to regulate GABAergic neuron development. Thus, fmi-1 and cdh-4 cadherins might act together to regulate synapse development and axon pathfinding.