Synaptic function defects in hid-1 mutants. (A) hid-1 mutants are moderately resistant to aldicarb. Wild-type (open circle), hid-1(sa722) (closed circle), hid-1(sa691) (open box), hid-1(js828) (open triangle), and aex-3(js815) (closed box) animals were exposed to 1 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM. (B) hid-1 mutants are hypersensitive to levamisole. Wild-type (open circle), hid-1(sa722) (closed circle), and rab-3(js49) (open box) animals were exposed to 100 μm of levamisole and scored for the percentage of animals still responding to gentle touch. Error bars represent SEM. (C) UNC-29 distribution is not altered in hid-1 mutants: Wild-type (top) and hid-1(sa722) (bottom) immunostaining in the anterior regions of the ventral nerve cord. UNC-17 (red) and UNC-10 (blue) mark cholinergic synapses and active zones, respectively. UNC-29 (green) is usually concentrated at or near the UNC-10 puncta in both strains. No significant differences were observed in the UNC-29 pattern between wild-type and hid-1 mutant animals. Bar, 10 μm.

Sites of HID-1 function. (A) HID-1 is broadly expressed in secretory tissues. Top left panel: A young adult hid-1(sa722) animal expressing HID-1-GFP driven by the hid-1 promoter. Expression is seen in neuronal and intestinal cells. In the inset a solid arrowhead indicates expression in the nerve ring, an open arrowhead indicates expression in the anterior intestinal cells, and an open arrow indicates expression in the pharynx. Bar, 50 μm. Bottom left panel: Expression of HID-1-GFP driven by the rab3 promoter in the nerve ring, the dorsal nerve cord, and the ventral nerve cord of a young adult hid-1(sa722) animal. Bar, 50 μm. Top right panel: Punctate pattern of neuronal HID-1-GFP in the dorsal (DC) and ventral nerve cords (VC) of a young adult hid-1(sa722) animal. Bar, 40 μm. Middle right panel: Neuronal HID-1-GFP is shown concentrated in small spots (arrowheads) in cell bodies of motor neurons. Bar, 5 μm. Bottom right panel: Neuronal HID-1-GFP expressed in nerve ring and cell bodies of the head neurons. The pharynx is outlined as an anatomical guide. Bar, 10 μm. (B) The aldicarb resistance of hid-1(sa722) is rescued by expression of GFP-tagged HID-1 in neurons. Wild-type (open circle), hid-1(sa722) mutants (closed circle), and hid-1(sa722) mutants carrying the transgenes driving the expression of wild-type HID-1-GFP under the hid-1 promoter (open box), the panneuronal rab-3 promoter (closed box), and intestinal ges-1 promoter (asterisk) were exposed to 0.5 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM. (C) The increased sensitivity to levamisole of hid-1(sa722) is rescued by expression of GFP-tagged HID-1 in neurons. Wild-type (open circle), hid-1(sa722) mutants (closed circle), and hid-1(sa722) mutants carrying a transgene driving the expression of HID-1-GFP in neurons under the rab-3 promoter (closed box) were exposed to 100 μm of levamisole and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM. (D) Rescue of the defecation defects of hid-1(sa722) mutants by tissue-specific expression plasmids and replication of the defecation phenotype by hid-1 RNAi. The percentage of enteric muscle contractions (EMC) in 10 defecation cycles is shown on the y-axis. hid-1; HID-1, hid-1; neuronal_p HID-1, or hid-1; intestinal_p HID-1 indicates hid-1(sa722) animals carrying HID-1-GFP under a native, neuronal, or intestinal promoter, respectively. WT (RNAi vector) and WT (RNAi hid-1) indicate wild-type animals exposed to bacteria transformed with an empty RNAi vector as a control and an RNAi vector targeting hid-1, respectively. Ten animals were observed for each strain. Error bars represent SEM.

HID-1 functions in parallel with aex-6 RAB-27 and RAB-3 in neurosecretion. (A) hid-1 mutant animals partially mislocalize a GFP-tagged version of the RAB-27 effector RBF-1 rabphilin. Top panel: Image of an integrated GFP-tagged RBF-1 transgenic animal (jsIs423) showing primarily nerve ring staining. Bottom panel: RBF-1-GFP is partially mislocalized to neuronal cell bodies in hid-1(js828) mutant animals. The same defect was observed in hid-1(sa722) and hid-1(sa691) mutants. Bar, 20 μm. (B) The combination of hid-1 and aex-6 RAB-27 mutations causes a severe resistance to aldicarb. Wild-type (open circle), hid-1(sa722) (closed circle), aex-6(sa24) RAB-27 (open triangle), and aex-6(sa24) RAB-27; hid-1(sa722) double mutants (closed box) were exposed to 1 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM. (C) The combination of hid-1 and rab-3 mutations causes a severe resistance to aldicarb. Wild-type (open circle), hid-1(sa722) (closed circle), rab-3(js49) (open box), and rab-3(js49); hid-1(sa722) double mutants (closed box) were exposed to 1 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM.

HID-1 is a potentially myristoylated protein associated with membranes. (A) HID-1-GFP localization is UNC-104 dependent. Left panel: Image of GFP-tagged HID-1 localized in the synaptic-rich nerve ring of a hid-1(sa722) mutant animal. Right panel: HID-1-GFP is mislocalized to the ganglia of neuronal cell bodies surrounding the nerve in unc-104(e1265) mutant animals. An arrow points to a representative cell body. Bar, 10 μm. (B) HID-1(G2N)-GFP mutant protein is missorted in the nervous system. Left panel: Image of the ventral nerve cord of a hid-1(sa722) animal showing the punctate pattern of wild-type HID-1-GFP expressed in neurons. Right panel: The expression of neuronal HID-1(G2N)-GFP (mutated in its potential myristoylated site) in the ventral nerve cord of hid-1(sa722) animals shows a diffuse localization pattern and a redistribution to neuronal cell bodies. An arrow points to a representative cell body. Bar, 10 μm. (C) Neuronal HID-1-GFP is associated with membrane fractions. C. elegans extracts were sonicated and separated by ultracentrifugation. Comparable amounts of the supernatant (S) and pellet (P) were analyzed by immunoblotting with anti-GFP and anti-SNB-1. Neuronal HID-1-GFP was exclusively membrane associated, while a portion of neuronal HID-1(G2N)-GFP was recovered in the soluble fraction. The vesicular marker SNB-1 synaptobrevin was used as a control for proteins retained in the pellet. The data shown are representative of four independent experiments with similar results. (D) Murine HID-1 is a potentially myristoylated protein associated with membranes. PC12 cells were transfected with a mHID-1-GFP construct and 24 hr later the distribution of GFP fluorescence was analyzed. Left: Wild-type murine HID-1 protein associates with internal membrane structures. Tubules extending from these murine HID-1-labeled vesicular structures were frequently observed. Insets show tubular projections attached to a vesicle. Right: The distribution of the murine HID-1(G2N)-GFP mutant is diffused. Bar, 5 μm.

HID-1 acts in a neuropeptide processing pathway. (A) Double mutants containing mutations in hid-1 and egl-3 PC2 show similar aldicarb resistance to each single mutant. Wild-type (open circle), hid-1(sa722) (closed circle), egl-3(n150) (open box), and egl-3(n150); hid-1(sa722) double mutants (closed box) were exposed to 1 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM. (B) Double mutants containing mutations in hid-1 and egl-21 CPE show similar aldicarb resistance to each single mutant. Wild-type (open circle), hid-1(sa722) (closed circle), egl-21(n476) (open box), and egl-21(n476); hid-1(sa722) double mutants (closed box) were exposed to 1 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM.

hid-1 mutants alter intestinal secretion patterns. (A) Representative DIC (top) and fluorescent (bottom) images of the AEX-5-VENUS-derived fluorescence in coelomocytes of wild-type, aex-6, hid-1(sa722), and aex-6; hid-1 mutant animals. Coelomocytes are outlined in the DIC image with a dashed open line. Bar, 5 μm. (B) Quantification of fluorescence measured from coelomocytes of young adult worms expressing intestinal-expressed AEX-5-VENUS. The number of worms analyzed are as follows: WT = 13, aex-6=16, hid-1 = 15, aex-6; hid-1 = 15. Asterisks indicate significant difference among the pairs at P < 0.001 (Student's t-test).

Relationship between HID-1 and other regulators of DCVs signaling. (A) The combination of hid-1 and ida-1 mutations increases the sensitivity to aldicarb compared to each single mutation. Wild-type (open circle), hid-1(sa722) (closed circle), ida-1(ok409) (open box), and ida-1(409); hid-1(sa722) double mutants (closed box) were exposed to 0.5 mm of aldicarb and scored periodically for the percentage of animals still responding to gentle touch. Error bars represent SEM. (B) The combination of hid-1 and unc-108 mutations causes an increase in the locomotion defect compared to each single mutation. Spontaneous (unstimulated) locomotion rates of wild-type compared to hid-1(sa722), unc-108(nu415) and unc-108(nu415); hid-1(sa722) mutant animals. Error bars are SEM of 10 animals per genotype. Triple asterisks indicate that the difference relative to the wild type is significant with a P value of <0.002. P-values are from the unpaired t-tests with Welch corrections. (C) Representative images of Venus-tagged NLP-21 neuropeptides in DA/DB motor neuron axons in wild-type, unc-108(nu415), hid-1(sa722), and unc-108(nu415); hid-1(sa722) mutant animals. Images are scaled identically for brightness. Bar, 1 μm. (D) Quantification of NLP-21-Venus fluorescence in the dorsal axons of the indicated genotypes. Graph shows the total integrated fluorescence per micrometer of dorsal axon length. Data are means and SEMs from images acquired from 11–13 animals per genotype. Triple asterisks indicate that the difference relative to the wild type is significant with a P-value of <0.001. P-values are from the unpaired t-test with Welch correction.

Neuronal HID-1-GFP accumulates in unc-13 and unc-31 mutants. (A) neuronal HID-1-GFP distribution in the dorsal cord of lin-15(n765), unc-31 CAPS; lin-15(n765) and unc-13(s69); lin-15(n765) mutant animals. Bar, 10 μm. (B) Quantification of neuronal HID-1-GFP puncta fluorescence. The number of worms analyzed are as follows: lin-15(n765) = 19, unc-31 CAPS; lin-15(n765) = 13, and unc-13(s69); lin-15(n765) = 8. Asterisks indicate a significant difference from the lin-15 control for P < 0.001 (Student's t-test). AU, arbitrary units. Error bars represent SEM.

hid-1 mutants exhibit reduced levels of ANF-GFP fluorescence in the dorsal nerve cord and coelomocytes. (A) Representative images of the ANF-GFP-derived fluorescence in the dorsal nerve cord of wild-type, unc-31(e928), hid-1(sa722), and unc-31(e928); hid-1 mutant animals. Bar, 5 μm. (B) Quantification of fluorescence normalized to wild type in the dorsal nerve cord. The number of worms analyzed are as follows: WT = 20, hid-1(sa722) = 17, unc-31(e928); hid-1 = 15, and unc-31(e928) = 10. Asterisks indicate significant difference from the wild-type control for P < 0.001 using a Student's t-test. Error bars represent SEM. (C) Representative images of the ANF-GFP-derived fluorescence in posterior coelomocytes of wild-type, unc-31 (e928), hid-1(sa722), and unc-31(e928); hid-1 mutant animals. Bar, 5 μm. (D) Quantification of fluorescence, normalized to the wild type, measured from posterior coelomocytes of young adult worms expressing neuronal ANF-GFP. The number of worms analyzed are as follows: WT = 50, hid-1(sa722) = 48, unc-31(e928); hid-1 = 15, and unc-31(e928) = 10. Asterisks indicate significant difference from the wild-type control for P < 0.001 (Student's t-test). Error bars represent SEM. (E) Representative images of the ANF-GFP-derived fluorescence in the dorsal nerve cord of wild-type, hid-1(sa722), egl-3(ok979), and egl-3(ok979); hid-1(sa722) mutant animals. Bar, 5 μm. (F) Quantification of fluorescence in the dorsal nerve cord normalized to the wild type. The number of worms analyzed are as follows: WT = 25, hid-1(sa722) = 23, egl-3(ok979) = 21, and egl-3(ok979); hid-1 = 22. Asterisks indicate significant difference from the wild-type control for P < 0.001 using a Student's t-test. Error bars represent SEM.

hid-1 mutants exhibit reduced levels of FMRFamide neuropeptides in the dorsal nerve cord. (A) Representative images of anti-FMRFamide immunostaining in the dorsal nerve cord of wild-type and hid-1(sa722) mutant animals. Bar, 5 μm. (B) Quantification of fluorescence in dorsal nerve cord normalized to wild type. The number of worms analyzed are as follows: WT = 30, hid-1(sa722) = 15. Asterisks indicate significant difference from the wild-type control for P < 0.001 using a Student's t-test. Error bars represent SEM. (C) Images of anti-FMRFamide immunostaining in the cell bodies of neurons located in the left lateral posterior ganglia of wild-type and hid-1(sa722) mutant animals. Arrows indicate the increased accumulation of anti-FMRFamide immunostaining observed in hid-1 mutants compared to wild type. Bar, 5 μm.