nu444 is a novel allele of pkc-1 in C. elegans

Here, we report nu444 as a novel allele of the gene pkc-1 that encodes the protein kinase C-1 in C. elegans. The nu444 allele was originally isolated from a forward genetic screen for mutants that suppressed the “Hic” (Hypersensitivity to Inhibitors of Cholinesterase) phenotype of dgk-1(nu62) mutants, which had increased acetylcholine release at the neuromuscular junction (Sieburth et al., 2007). In this screen, several genes that are important for neuropeptide secretion were recovered, including pkc-1(nu448) (Sieburth et al., 2007) and ric-7(nu447) (Hao et al., 2012). Sanger sequencing of the exons and exon-intron junctions of the pkc-1 locus revealed that nu444 had a nonsense mutation (C to T, in the coding strand of pkc-1, with left flanking sequence: 5’-GGATGAATATCATATAGGAAAGACG-3’ and right flanking sequence: 5’- AAGTTCGGCCCAAGACTAATGAACC-3’) in an early exon that is only present in pkc1a and pkc-1c isoforms (Fig.1). Thus, pkc-1(nu444) allele is probably a null allele for both pkc-1a (Q53stop) and pkc-1c (Q109stop), but presumably does not affect pkc-1b.

RIC-7 Promotes Neuropeptide Secretion

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling

Sphingosine Kinase Activates the Mitochondrial Unfolded Protein Response and Is Targeted to Mitochondria by Stress

Summary: The mitochondrial unfolded protein response (UPRmt) is critical for maintaining mitochondrial protein homeostasis in response to mitochondrial stress, but early steps in UPRmt activation are not well understood. Here, we report a function for SPHK-1 sphingosine kinase in activating the UPRmt in C. elegans. Genetic deficiency of sphk-1 in the intestine inhibits UPRmt activation, whereas selective SPHK-1 intestinal overexpression is sufficient to activate the UPRmt. Acute mitochondrial stress leads to rapid, reversible localization of SPHK-1::GFP fusion proteins with mitochondrial membranes before UPRmt activation. SPHK-1 variants lacking kinase activity or mitochondrial targeting fail to rescue the stress-induced UPRmt activation defects of sphk-1 mutants. Activation of the UPRmt by the nervous system requires sphk-1 and elicits SPHK-1 mitochondrial association in the intestine. We propose that stress-regulated mitochondrial recruitment of SPHK-1 and subsequent S1P production are critical early events for both cell autonomous and cell non-autonomous UPRmt activation. : The mitochondrial unfolded protein response (UPRmt) maintains mitochondrial protein homeostasis in response to stress. Kim and Sieburth identify SPHK-1/sphingosine kinase as a positive regulator of the UPRmt that promotes UPRmt activation in response to a variety of mitochondrial stressors. SPHK-1 associates with mitochondria and SPHK-1 mitochondrial association is stress dependent, reversible, and necessary for the UPRmt, indicating that SPHK-1 mitochondrial targeting is an early step in UPRmt activation. Keywords: sphingosine kinase, mitochondrial unfolded protein response, mitochondria, cell non-autonomous, nervous system, neuropeptide, ATFS-

PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

<div><p>Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in <i>C. elegans</i>, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the <i>C. elegans</i> PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.</p></div

Constitutively active PKA in GABAergic neurons partially bypasses the requirement of AEX-2/GPCR.

<p>(A) Diagram showing the construction of the constitutively active PKA (PKA[CA]). “R” and “C” indicate the PKA regulatory and catalytic subunit, respectively. The two asterisks (*) represent the two mutations (H96Q, W205R) in the PKA catalytic subunit KIN-1a, which presumably disrupt its association with the regulatory subunit but do not affect its enzymatic activity. (B) Quantification of the Exp step of young adults with the indicated genotypes. PKA[CA] denotes PKA constitutively active transgenic worms (<i>vjIs102</i> and <i>vjIs103</i>) expressing the mutated catalytic subunit <i>kin-1a</i>(H96Q, W205R) in the GABAergic neurons using the <i>unc-47</i> full length promoter. <i>vjIs103</i> and <i>vjIs77</i> were used for the PKA[CA];PKA[DN] strain. (C) Representative ethograms of ten consecutive defecation cycles of young adult worms with the indicated genotypes. Each dot represents 1 s. “p” stands for the pBoc step and “x” indicates the Exp step. Ectopic Exp steps are indicated by “<u>x</u>”. Means and standard errors are shown. Asterisks indicate significant differences between indicated groups: ** P<0.01, *** P<0.005 in Student's t-test.</p

Other non voltage-gated calcium channels are required from calcium influx in the GABAergic neurons.

<p>(A) and (B) Quantification of the Exp step of young adult worms with the indicated genotype types. (C) Classification of different patterns of pBoc, fluorescent spikes in DVB and Exp in worms with the indicated genotypes. <i>unc-2</i>: 40 cycles in 21 animals, <i>egl-19; unc-2</i>: 19 cycles in 11 animals, <i>PKA[CA]; egl-19; unc-2</i>: 24 cycles in 12 animals, PKA[CA]: 23 cycles in 12 animals. (D) and (E) Quantification of the duration and amplitude of regular DVB calcium spikes in worms with indicated genotypes. PKA[CA] represents transgenic worms with constitutively active PKA specifically expressed in GABAergic neurons (<i>vjIs103</i> in (B) and <i>vjIs102</i> in (C, D)). Note that in (C), (D) and (E), <i>unc-2</i> and PKA[CA] strains, but not the <i>egl-19; unc-2</i> and the PKA[CA]; <i>egl-19; unc-2</i> strains contain the <i>unc-13(s69)</i> mutation for immobilization, as <i>egl-19;unc-2</i> alone were almost completely paralyzed. <i>vjIs58</i>, the transgenic strain with GCaMP3 expressed in the DVB neuron, was used for the <i>unc-2</i> strain; while <i>vjIs64</i> was used for calcium imaging in other genotypes. Means and standard errors are shown. Asterisks indicate significant difference between indicated group and significant difference from wild type in (A), PKA[CA] in (B) and <i>egl-19; unc-2 mutants</i> in (D) and (E): * p<0.05; ***P<0.005 in Student's t-test. “n.s.” indicates no significant difference between indicated groups.</p

PKA is necessary for calcium influx in DVB neurons.

<p>(A) Expression of the genetically-encoded calcium indicator, GCaMP3 in DVB neurons (<i>vjIs58</i>). Top: diagram of the DVB neuron in the tail region. Synapse means the neuromuscular junction where the DVB neuron innervates enteric muscles. Middle and Bottom: two snapshots from a real-time imaging video of wild type animals showing an increase in fluorescence in synaptic region of DVB neurons, as indicated by the white arrow, right before the Exp step. (B) A representative trace of the GCaMP3 fluorescence in the synaptic region of DVB neurons in wild type animals showing DVB neurons are rhythmically activated during three consecutive defecation cycles. Note that the cycle length is longer than 50 seconds, likely due to <i>unc-13(s69)</i> mutation, which was used to immobilize the worms for calcium imaging (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003831#s4" target="_blank">Materials and Methods</a> for details). (C) Representative traces of the GCaMP3 fluorescence in the synaptic region of DVB neurons in worms with the indicated genotypes. The observed pBoc step and Exp step are indicated by arrows and arrowheads, respectively. (D) Classification of the different patterns of pBoc, fluorescent spikes in DVB and Exp in worms with the indicated genotypes. <i>vjIs76</i> was used for the PKA[DN] strain. In (D), wild type: 23 cycles in 11 animals; <i>unc-25</i>: 44 cycles in 11 animals; <i>aex-2</i>: 44 cycles in 11 animals; PKA[DN]: 30 cycles in 12 animals.</p

PKA functions in GABAergic neurons to regulate the Exp step.

<p>(A) Diagram showing the construction of dominant negative PKA (PKA[DN]). “R” and “C” indicate the PKA regulatory and catalytic subunit, respectively. “x” represents the substitution (G310D) in the site B of the regulatory subunit KIN-2a, which presumably blocks cAMP binding and prevents its dissociation with PKA catalytic subunit. (B) and (C) Quantification of the Exp step of young adults with the indicated genotypes. PKA[DN] denotes PKA dominant negative transgenic worms (<i>vjIs76</i> and <i>vjIs77</i>) in which the mutated regulatory subunit <i>kin-2a</i>(G310D) was expressed specifically in GABAergic neurons using <i>unc-47</i> full length promoter. <i>gsa-1(gf)</i> is a gain-of-function allele (<i>ce81)</i> of <i>gsa-1</i>/Gαs. (D) Representative ethograms of ten consecutive defecation cycles of young adult worms with the indicated genotypes. <i>vjIs77</i> is used for PKA[DN] in (D). Each dot represents 1 s. “p” stands for the pBoc step and “x” indicates the Exp step. aBoc is omitted. Means and standard errors are shown. Asterisks (***) indicate significant difference from wild type in (B) and <i>gas-1(gf)/Gas</i> in (C): p<0.005 in Student's t-test. “n.s.” indicates no significant difference between indicated groups.</p