The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans

Abstract

Neuroactive peptides are packaged as proproteins into dense core vesicles or secretory granules, where they are cleaved at dibasic residues by copackaged proprotein convertases. We show here that the Caenorhabditis elegans egl-3 gene encodes a protein that is 57% identical to mouse proprotein convertase type 2 (PC2), and we provide evidence that this convertase regulates mechanosensory responses. Nose touch sensitivity (mediated by ASH sensory neurons) is defective in mutants lacking GLR-1 glutamate receptors (GluRs); however, mutations eliminating the egl-3 PC2 restored nose touch sensitivity to glr-1 GluR mutants. By contrast, body touch sensitivity (mediated by the touch cells) is greatly diminished in egl-3 PC2 mutants. Taken together, these results suggest that egl-3 PC2-processed peptides normally regulate the responsiveness of C. elegans to mechanical stimuli.

Fig. 1.

Mechanosensory circuits for nose and body touch responses. A, Nose touch stimuli evoke backward locomotion and are sensed by ASH neurons (black circle). ASH neurons have dendritic processes that are exposed to the external environment through the amphid pore at the tip of the worm's nose (Perkins et al., 1986). Body touch sensitivity is mediated by six touch cells (gray circles), which have sensory endings in either the anterior or posterior body regions. Touch to the anterior half of the body elicits backward locomotion and vice versa for posterior touch stimuli (Chalfie and Au, 1989). B,C, Synaptic connections underlying the nose and body touch responses are illustrated. Chemical synapses are indicated bylines with black triangles, and gap junctions by lines with bars.B, Nose touch responses are primarily mediated by the ASH neurons. Two other sensory neurons also contribute to this response but quantitatively account for <40% of the normal responsiveness (Kaplan and Horvitz, 1993). For simplicity, these minor nose touch sensory neurons are not shown here. The ASH neurons also mediate responsiveness to hyperosmolarity and volatile repellents (Bargmann et al., 1990; Troemel et al., 1995). All ASH stimuli evoke backward movement via synapses between ASH and the command neurons for locomotion (AVA, AVB, and AVD). The glr-1 GluRs are clustered at command neuron synapses (Rongo et al., 1998).C, Body touch responses are mediated by the touch cells. Touch cells form gap junctions and chemical synapses with the command neurons. The touch cell–command neuron synapses are thought to be inhibitory (Chalfie et al., 1985; Wicks and Rankin, 1995; Wicks et al., 1996) and glutamatergic (Lee et al., 1999). Adapted from Kaplan and Horvitz (1993) and Driscoll and Kaplan (1997).

Fig. 2.

Restoration of the nose touch response byegl-3 PC2 mutations. Nose and body touch responses of several strains were compared, as detailed in Materials and Methods.A, The egl-3(nu349) PC2 allele restored full nose touch responsiveness to two different glr-1GluR mutants: n2461 a premature stop codon, andky176 a deletion allele (Hart et al., 1995; Maricq et al., 1995). The egl-3(nu349) PC2 allele also restored nose touch responsiveness to lin-10 mutants, which fail to cluster glr-1 GluRs at sensory-command neuron synapses. Two other egl-3 PC2 alleles (n150ts and nr2090) also restored nose touch responsiveness to glr-1 GluR mutants. These results suggest that egl-3 PC2 mutations functionally bypass the requirement for glr-1 GluRs at ASH-command neuron synapses. B, Body touch responsiveness was reduced in homozygous egl-3 PC2 mutants. Values shown are mean ± SEs. The numbers listed below each genotype indicate the number of animals and trials, as follows: number of animals(number of trials).

Fig. 3.

The structure of the egl-3 PC2 protein (as predicted from the cDNA sequence) is 57% identical to human PC2 and contains predicted propeptide, catalytic, and P domains. Within the catalytic domain, all four amino acid residues known to be critical for protease activity are conserved. Missense mutations inegl-3 PC2 alleles are indicated. Thenr2090 allele is an in-frame deletion that removes amino acids 151–377, as indicated. The synthetic peptide (peptide 2) sequence used to raise anti-egl-3 PC2 antisera is indicated by the gray bar.

Fig. 4.

Expression of egl-3 PC2. Anti-egl-3 PC2 antibody was used to stain transgenic animals expressing KP#454, a full-length rescuing EGL-3:: GFP construct (A–C). Endogenously expressed EGL-3 was visualized by staining nontransgenic wild-type animals (D) or egl-3(nu349) mutants (E). Expression of egl-3 PC2 in the command neurons was examined by staining transgenic animals containing both KP#454 and nuIs24, a GLR-1:: GFP transgene, with anti-egl-3 PC2 and anti-glr-1 GluR antibodies (B).A, Expression of EGL-3:: GFP was found in the cell bodies of many neurons in the head and in ganglia in the tail (TG). In addition, axons in the nerve ring (NR) stained brightly with the anti-egl-3PC2 antibody. B, Expression of EGL-3:: GFP in the PVC command neurons was documented by double staining with anti-egl-3 PC2 (left) and anti-glr-1 GluR (middle) antibodies. The merged image is shown on the right. This panel shows a mosaic animal in which one PVC neuron (indicated by theasterisk) expressed both GLR-1 (green) and EGL-3 (red), whereas the second PVC neuron (indicated by the arrowhead) expressed GLR-1 but not EGL-3. Similar results were obtained documenting expression of EGL-3:: GFP in AVB and AVD (data not shown). D, E, Endogenously expressed EGL-3 was stained with anti-egl-3 PC2 antibodies. In wild-type animals (D), bright staining was observed in the nerve ring (NR) and ventral cord (data not shown) axons. Cell bodies (indicated by arrows) stained very weakly. In egl-3(nu349) mutants (E), the axons of the nerve ring (NR) and ventral cord (data not shown) were poorly stained, whereas bright staining of neuronal cell bodies was observed (arrows). Scale bars, 10 μm.

Fig. 5.

Role of egl-3 PC2 in restoration of the nose touch response. A, The nose touch responses of wild-type and of glr-1 GluR;egl-3 PC2 double mutants are greatly diminished when the ASH neurons were killed with a laser microbeam. B, Theegl-3(nu349) PC2 mutation did not restore nose touch responsiveness to eat-4 VGLUT mutants. C,egl-3 PC2 expression in the command neurons is sufficient to regulate ASH-mediated nose touch sensitivity. Wild-type EGL-3 was expressed in either the ASH neurons (using theosm-10 promoter) or in the command neurons (using theglr-1 promoter) ofglr-1(n2461)GluR;egl-3(nu349)PC2 double mutants. For each transgene, two independent transgenic strains were analyzed. Strains expressing egl-3 PC2 in the command neurons (Interneuron#1 and 2) had greatly reduced nose touch sensitivity, indicating rescue of the suppression defect. Rescue was not observed in transgenic strains expressingegl-3 PC2 in the ASH neurons (ASH#1 and2). Values shown are mean ± SEs. Thenumbers listed below each genotype indicate the number of animals and nose touch trials for each data point, as follows: number of animals(number of trials).