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Mol Cells
2024 Oct 16;:100124. doi: 10.1016/j.mocell.2024.100124.
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A FMRFamide-like neuropeptide FLP-12 signaling regulates head locomotive behaviors in C. elegans.
Kim DY
,
Moon KM
,
Heo W
,
Du EJ
,
Park CG
,
Cho J
,
Hahm JH
,
Suh BC
,
Kang K
,
Kim K
.
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Neuropeptides play a critical role in regulating behaviors across organisms, but the precise mechanisms by which neuropeptides orchestrate complex behavioral programs are not fully understood. Here, we show that the FMRFamide-like neuropeptide FLP-12 signaling from the SMB head motor neurons, modulates head locomotive behaviors, including stomatal oscillation in C. elegans. lim-4 mutants, in which the SMB neurons are not properly specified, exhibited various head and body locomotive defects, including stomatal oscillation. Chronic activation or inhibition of neuropeptidergic signaling in the SMB motor neurons resulted in a decrease or increase in stomatal oscillation, respectively. The flp-12 neuropeptide gene is expressed and acts in the SMB neurons to regulate head and body locomotion, including stomatal oscillation. Moreover, the frpr-8 GPCR and gpa-7 Gα genes are expressed in the AVD command interneurons to relay the FLP-12 signal to mediate stomatal oscillation. Finally, heterologous expression of FRPR-8 either Xenopus oocytes or HEK293T cells conferred FLP-12 induced responses. Taken together, these results indicate that the C. elegans FMRFamide neuropeptide FLP-12 acts as a modulator of stomatal oscillation via the FRPR-8 GPCR and the GPA-7 G-protein.
Fig. 1. The SMB neurons regulate head and body locomotion. (A) Scheme of the SMB sensory/inter/motor neurons. (B) Wavelength and wave width of wild-type and lim-4 mutant. n = 30. (C) Scheme of head lift, stomatal oscillation, reversal, and omega turn (left) and time lapse images during head lift and stomatal oscillation (right). (D) The frequency of stomatal oscillation, head lift, reversal, and omega turn behavior of wild-type and lim-4 mutant for 3 minutes. n = 30 ~ 86. (E) Scheme of the optogenetic activation of the SMB neurons. (F) The frequency of stomatal oscillation, head lift, reversal, and omega turn behavior of wild-type animals upon optogenetic activation of the SMB neurons. Light stimulation was performed in the absence or presence of all-trans-retinal (ATR) for 3 minutes. n = 20. Error bars represent the SEM. *, **, and *** indicate a significant difference from the control at p<0.05, 0.01, and 0.001 by a student t-test, respectively.
Fig. 2. FLP-12 neuropeptide expressed in the SMB neurons regulates head and body locomotion. (A) Schematic of UNC-31 function in neurons. (B) Wavelength and wave width of wild-type and transgenic animals expressing SMB(flp-12)p::unc-31 dsRNAi. n = 30. (C) The frequency of head and body locomotive behaviors of wild-type and transgenic animals expressing SMBp::unc-31 dsRNAi for 3 minutes. n = 30. (D) The genomic structure of the flp-12 gene. (E) Expression pattern of flp-12p::gfp. The anterior is to the left. Scale bar: 20 μm. (F) Wavelength and wave width of wild-type and flp-12 mutant animals. n = 30. Error bars represent the SEM. *, **, and *** indicate a significant difference from the control at p<0.05, 0.01, and 0.001 by a student t-test, respectively. (G) The frequency of head and body locomotive behaviors of wild-type, flp-12 mutant, and flp-12 mutant expressing flp-12 cDNA under the control of SMB-specific (flp-12) promoter for 3 minutes. n = 30 ~ 86. Error bars are the SEM. *, **, and *** indicate significantly different from the wild-type at p<0.05, 0.01, and 0.001 by a one-way ANOVA with a Tukey post hoc test, respectively.
Fig. 3. FRPR-8 GPCR is expressed in and acts in the AVD neurons to mediate FLP-12 mediated stomatal oscillation. (A) The frequency of normalized stomatal oscillation of flp-12 mutants and RNAi-treated worms against neuropeptide candidate receptors for 3 minutes. n = 30 ~ 32. Error bars are the SEM. *, **, and *** indicate significantly different from the wild-type at p<0.05, 0.01, and 0.001 by a one-way ANOVA with a Dunnett post hoc test, respectively. (B) The genomic structure of the frpr-8 gene. (C) The frequency of stomatal oscillation, head lift, reversal, and omega turn behavior of wild-type, flp-12, frpr-8, and flp-12 frpr-8 double mutants for 3 minutes. n = 30 ~ 86. (D) Representative image of wild-type expressing the frpr-8p::mCherry transgene in the AVD neurons. Anterior is to the left. Scale bar: 20 μm. (E) Schematic drawing of the AVD neurons in C. elegans. (F) Representative images of NeuroPAL otIs670 worm expressing frpr-8p::frpr-8 cDNA::gfp. Left and right images represent GFP-tagged frpr-8 cDNA and NeuroPAL images, respectively. Images are derived from z-stacks of confocal microscopy images. The anterior is to the left. Scale bar: 20 μm. (G) The frequency of stomatal oscillation, head lift, reversal, and omega turn behavior of wild-type, frpr-8, and transgenic animals expressing frpr-8 cDNA under the control of frpr-8 or nmr-1 promoters for 3 minutes. n = 30 ~ 86. Error bars are the SEM. *, **, and *** indicate significantly different from the wild-type at p<0.05, 0.01, and 0.001 by a one-way ANOVA with a Tukey post hoc test, respectively.
Fig. 5. FLP-12 is a cognate ligand of FRPR-8. (A) Schematic images of two distinct in vitro systems, Xenopus oocyte (left) and HEK293T cell (right). (B) A representative current trace (Left) at +60 mV from Xenopus oocytes microinjected with either FRPR-8 cRNA (green) or water (control, black). The experiments were then plotted to present FLP-12 dose dependence of the cells (Right). (C) Time-series confocal images of HEK293T cells (left), which are expressed without or with FRPR-8, were taken before (left), during 1 μM FLP-12 treatment (middle), and after washout (right). All cells expressed Gα and calcium indicator Fluo-4, which was used to detect the calcium elevation. Scale bar: 10 μm. Representative time courses of cytosolic fluorescence intensity change in HEK293T cells in response to FLP-12 treatment. Lines indicate the activity of the cells without (black) or with (green) FRPR-8. Fluorescence intensity changes (right). Gray bars indicate before treatment of FLP-12, and red bars indicate after treatment of FLP-12 neuropeptide. n ≥ 5. Error bars are the SEM. (D) Time-series images and fluorescence intensity change of FRPR-8 expressed cells with FLP-5 or FLP-12 treatment. Time-series confocal images of HEK293T cells expressing FRPR-8 were taken before (control), during FLP-5 (1 μM) treatment, washout, and FLP-12 (1 μM) treatment (left). Representative time courses of cytosolic fluorescence intensity changes in cells in response to FLP-5 and FLP-12. Fluorescence intensity changes (right). Gray bars indicate before the treatment of ligands, and colored bars indicate after the treatment of ligands. n≥ 5. Error bars indicate SEM.
Fig. 6. Model of FLP-12 and FRPR-8 regulating stomatal oscillation.
Fig. 4. Gα subunit protein gpa-7 is coupled to frpr-8 GPCR to regulate stomatal oscillation. (A) Timescale images of optogenetics activation of the AVDneuron (left panel). The frequency of stomatal oscillation (right upper) and head lift (right lower). n = 30. Error bars represent the SEM. *, **, and *** indicate a significant difference from the control at p<0.05, 0.01, and 0.001 by a student t-test, respectively. (B) Expression pattern of transgenic animals expressing gpa-7p::gfp (left) and frpr-8p::mCherry transgene (middle). Merged images are in the right panel. Images are derived from z-stacks of confocal microscopy images. The anterior is to the left. Scale bar: 20 μm. (C) The frequency of stomatal oscillation, head lift, reversal, and omega turn behavior of wild-type, frpr-8, gpa-7, and gpa-7; frpr-8 double mutants for 3 minutes. n = 30 ~ 86. Error bars are the SEM. *, **, and *** indicate significant differences from the wild-type at p<0.05, 0.01, and 0.001 by a one-way ANOVA with a Tukey post hoc test, respectively.
