PPM-1, a PP2Cα/β phosphatase, Regulates Axon Termination and Synapse Formation in Caenorhabditis elegans

The PHR (Pam/Highwire/RPM-1) proteins are evolutionarily conserved ubiquitin ligases that regulate axon guidance and synapse formation in Caenorhabditis elegans, Drosophila, zebrafish, and mice. In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) functions in synapse formation, axon guidance, axon termination, and postsynaptic GLR-1 trafficking. Acting as an E3 ubiquitin ligase, RPM-1 negatively regulates a MAP kinase pathway that includes: dlk-1, mkk-4, and the p38 MAPK, pmk-3. Here we provide evidence that ppm-1, a serine/threonine phosphatase homologous to human PP2Cα(PPM1A) and PP2Cβ(PPM1B) acts as a second negative regulatory mechanism to control the dlk-1 pathway. We show that ppm-1 functions through its phosphatase activity in a parallel genetic pathway with glo-4 and fsn-1 to regulate both synapse formation in the GABAergic motorneurons and axon termination in the mechanosensory neurons. Our transgenic analysis shows that ppm-1 acts downstream of rpm-1 to negatively regulate the DLK-1 pathway, with PPM-1 most likely acting at the level of pmk-3. Our study provides insight into the negative regulatory mechanisms that control the dlk-1 pathway in neurons and demonstrates a new role for the PP2C/PPM phosphatases as regulators of neuronal development

N-terminal nesprin-2 variants regulate β-catenin signalling

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity

The Caenorhabditis elegans microtubule minus-end binding homolog PTRN-1 stabilizes synapses and neurites

Microtubule dynamics facilitate neurite growth and establish morphology, but the role of minus-end binding proteins in these processes is largely unexplored. CAMSAP homologs associate with microtubule minus-ends, and are important for the stability of epithelial cell adhesions. In this study, we report morphological defects in neurons and neuromuscular defects in mutants of the C. elegans CAMSAP, ptrn-1. Mechanosensory neurons initially extend wild-type neurites, and subsequently remodel by overextending neurites and retracting synaptic branches and presynaptic varicosities. This neuronal remodeling can be activated by mutations known to alter microtubules, and depends on a functioning DLK-1 MAP kinase pathway. We found that PTRN-1 localizes to both neurites and synapses, and our results suggest that alterations of microtubule structures caused by loss of PTRN-1 function activates a remodeling program leading to changes in neurite morphology. We propose a model whereby minus-end microtubule stabilization mediated by a functional PTRN-1 is necessary for morphological maintenance of neurons. DOI: http://dx.doi.org/10.7554/eLife.01637.00

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development

The Nesprin Family Member ANC-1 Regulates Synapse Formation and Axon Termination by Functioning in a Pathway with RPM-1 and β-Catenin

<div><p>Mutations in Nesprin-1 and 2 (also called Syne-1 and 2) are associated with numerous diseases including autism, cerebellar ataxia, cancer, and Emery-Dreifuss muscular dystrophy. Nesprin-1 and 2 have conserved orthologs in flies and worms called MSP-300 and abnormal nuclear <u>Anc</u>horage 1 (ANC-1), respectively. The Nesprin protein family mediates nuclear and organelle anchorage and positioning. In the nervous system, the only known function of Nesprin-1 and 2 is in regulation of neurogenesis and neural migration. It remains unclear if Nesprin-1 and 2 regulate other functions in neurons. Using a proteomic approach in <i>C. elegans</i>, we have found that ANC-1 binds to the <u>R</u>egulator of <u>P</u>resynaptic <u>M</u>orphology 1 (RPM-1). RPM-1 is part of a conserved family of signaling molecules called <u>P</u>am/<u>H</u>ighwire/<u>R</u>PM-1 (PHR) proteins that are important regulators of neuronal development. We have found that ANC-1, like RPM-1, regulates axon termination and synapse formation. Our genetic analysis indicates that ANC-1 functions via the β-catenin BAR-1, and the ANC-1/BAR-1 pathway functions cell autonomously, downstream of RPM-1 to regulate neuronal development. Further, ANC-1 binding to the nucleus is required for its function in axon termination and synapse formation. We identify variable roles for four different Wnts (LIN-44, EGL-20, CWN-1 and CWN-2) that function through BAR-1 to regulate axon termination. Our study highlights an emerging, broad role for ANC-1 in neuronal development, and unveils a new and unexpected mechanism by which RPM-1 functions.</p></div

<i>anc-1</i> and <i>unc-84</i> function in the same genetic pathway to regulate axon termination.

<p>Quantitation of axon termination defects (hook) in PLM neurons for the indicated genotypes using <i>muIs32</i>. (A) <i>unc-84</i> mutant analysis. (B) <i>emr-1</i> mutant analysis. Analysis was done on young adults grown at 23°C. Significance was determined using an unpaired Student's <i>t</i> test; error bars represent the standard error of the mean. *<i>P</i><0.05, ***<i>P</i><0.001, ns = not significant.</p

<i>anc-1</i> functions cell autonomously to regulate axon termination in the PLM mechanosensory neurons.

<p>(A) Upper panel diagrams the mechanosensory neurons of <i>C. elegans</i> (inspired by Worm Atlas). PLM neurons were visualized using <i>muIs32</i> [P_mec7GFP]. The black box indicates the region of the animal that is visualized by epifluorescent microscopy and shown on the right. Shown for the <i>rpm-1</i> mutant is the PLM axon termination phenotype that we refer to as a hook defect (arrowhead). Scale bar is 10 µm. (B) Quantitation of axon termination (hook) defects in PLM neurons for the indicated genotypes. (C) An ANC-1 dominant negative construct (ANC-1 DN) was expressed using a pan-neuronal promoter (P<i>rgef-1</i>) or a mechanosensory neuron specific promoter (P<i>mec-3</i>) with the indicated genotypes. A full length ANC-1 rescue construct (P<i>mec-7</i>::ANC-1) was expressed in <i>anc-1; fsn-1</i> double mutants. The data shown is an average of 5 or more transgenic lines for each genotype. Analysis was done on young adults grown at 23°C. Significance was determined using an unpaired Student's <i>t</i> test; error bars represent the standard error of the mean. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ns = not significant.</p

ANC-1 binds to RPM-1.

<p>(A) Schematic of ANC-1 protein structure which consists of two calponin homology (CH) domains that bind F-actin (dashed boxes), 6 repeat regions (grey), and a KASH domain (black) that mediates binding to the nucleus. Also shown is the C-terminal domain of ANC-1 that functions as a dominant negative. (B) CoIP of endogenous ANC-1 with RPM-1::GFP. CoIPs were performed from whole worm lysates prepared from transgenic animals (<i>juIs58</i>) or non-transgenic animals (N2). (C) Epifluorescent microscopy was used to visualize SUR-5::GFP in the multinucleated hypodermal cells of <i>C. elegans</i>. In wild-type animals, nuclei are anchored to the actin cytoskeleton and evenly spaced throughout the syncytium (arrowheads). In <i>anc-1</i> mutants, impaired nuclear anchorage leads to aggregation of nuclei (arrows). Scale bar is 20 µm.</p