Fig 1.
Mutants in casy-1 are hypersensitive to Aldicarb.
(A) A schematic representation of the CASY-1 protein showing the N-terminal signal peptide (SP), two-tandem cadherin repeats, LG/LNS domain, transmembrane region (TM) and cytosolic acidic region. The domains deleted in casy-1 mutants are indicated as triangles. The tm718 and hd41 alleles are putative null alleles as deletion starts in the N-terminal region and results in a frame-shift in both cases. (B) Aldicarb-induced paralysis in casy-1 mutants was compared to wild-type (WT) animals. Both casy-1 mutant alleles (tm718 and hd41) are hypersensitive in the Aldicarb assays. Assays were done at least 6 times. (C) A schematic representing the transgenes used in the experiment. Expression of casy-1 isoforms under their endogenous promoters completely rescues the Aldicarb hypersensitivity of casy-1 mutant animals. In B and C, number of assays (~20 C. elegans/assay) is indicated for each genotype. Data are represented as mean ± S.E.M. (***p<0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test) “ns” indicates not significant in all Figures. (D) Pictorial representation of the genomic locus of three isoforms. CASY-1B and CASY-1C are expressed by alternative promoters that exist in between the 8th and 9th intron of CASY-1A isoform, which is unusually long (~ 4000bp) and carries their own SL1 leader sequences. The location of promoter sequences utilized in the study are indicated. (E) Representative confocal images of transcriptional reporters of the three casy-1 isoforms. Expression of GFP under isoform-specific promoters showed expression of casy-1a in most of the head neurons including amphid sensory neurons, in VNC, some tail neurons, in the intestine as well as in the gonadal sheath. casy-1b and casy-1c also showed expression in some head neurons, in the ventral cord motor neurons and some tail neurons but no expression in the gonadal sheath. Dotted lines indicate the position of gonadal sheath. Scale bar, 20μm.
Fig 2.
Motor neuron development is normal in casy-1 mutants.
(A) Representative fluorescent images of WT and casy-1 mutant animals expressing GFP in all GABAergic neurons (juIs76 [Punc-25:: GFP]). The number of cell bodies and axonal commissures were largely normal in casy-1 mutant animals (n-25) analyzed. Scale bar, 8μm. (B) CASY-1 functions in the mature nervous system to regulate synaptic transmission at the NMJ. Expression of CASY-1A isoform just three hours before the assay using a heat- shock promoter (hsp16.2) completely rescues the Aldicarb hypersensitivity phenotype seen in casy-1 mutant animals. The number of assays (~20 C. elegans/assay) is indicated for each genotype. (C) The C-terminal of the CASY-1A isoform is required to regulate synaptic transmission at the NMJ. Transgenic lines expressing either the CASY-1A N-terminal (ΔC) or C-terminal (ΔN) alone expressed under ins-1 promoter suggest that the CASY-1A C- terminal is sufficient to rescue the Aldicarb hypersensitivity in casy-1 mutants. Assays were done 3 times as indicated in Figures (~20 C. elegans/ assay). Data are represented as mean ± S.E.M. (***p<0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test). “ns” indicates not significant in all Figures.
Fig 3.
The casy-1 isoforms functions in GABAergic neurons to regulate synaptic transmission.
(A) Expression of CASY-1 isoforms using tissue specific promoters; ACh neurons (Punc-17), GABA neurons (Punc-25) and muscle (Pmyo-3) establishes that expressing CASY-1 in GABAergic neurons completely rescues the Aldicarb hypersensitivity in casy-1 mutants. The number of assays (~20 C. elegans/assay) is indicated for each genotype. (B) The C-terminal of the CASY-1 isoforms functions in GABAergic neurons to regulate synaptic function. The CASY-1A isoform expressed under GABAergic promoter completely rescues the Aldicarb hypersensitivity in casy-1 mutants. However, removing the entire C-terminal from the CASY-1A [CASY-1A (ΔC)] isoform does not rescue the Aldicarb hypersensitivity in casy-1 mutants. The number of assays (~20 C. elegans/assay) is indicated for each genotype. (C) The casy-1 mutants show higher sensitivity to GABA receptor antagonist PTZ than the WT animals. Representative still frame images demonstrating casy-1 mutant C. elegans with anterior convulsions. The still frame images are representative frames from movies (7 frames/second), which are available in the supporting information. Scale bar, 100μm. The graph shows the fraction of animals showing anterior ‘head bobs’ after 30 minute and 60-minute exposure to 10 mg/ml PTZ. The sensitivity to PTZ could be fully rescued by expressing all casy-1 isoforms in GABAergic neurons but not in cholinergic neurons or muscle. Assays were done (~10 C. elegans/assay) at least thrice. Data are represented as mean ± S.E.M. Values that differ significantly from WT animals are indicated (*p<0.01, ***p<0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test). Representative fluorescent images of (D) GABAergic [nuIs376 (Punc-25::SNB-1::GFP)] or (E) cholinergic [nuIs152 (Punc-129:: SNB-1::GFP)] synapses in the dorsal cord of WT or casy-1 mutant. Scale bar, 8μm. Cholinergic synapses are largely normal in casy-1 mutants, while GABAergic synapses showed a subtle but significant decrease in fluorescent intensity when compared to WT animals. Quantification of fluorescent intensity is normalized to WT values. The number of animals analyzed for each genotype is indicated at the base of the bar graph. Quantified data are displayed as mean ± S.E.M. and were analyzed by two-tailed Student’s t-test, “ns” indicates not significant in all Figures.
Fig 4.
casy-1 mutants show decreased GABAergic synaptic transmission at the NMJ.
(A) Expression of the CASY-1C specifically in DD and VD neuronal subtypes using GABAergic motor neuron specific promoter (Punc-30) establish that CASY-1 is explicitly functioning in GABAergic motor neurons to regulate synaptic transmission. Assays were done 3 times as indicated in Figures (~20 C. elegans/ assay). Data are represented as mean ± S.E.M. (***P<0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test). “ns” indicates not significant in all Figures. (B) Optogenetic stimulation of GABAergic neurons using Channelrhodopsin (ChR2) [zxIs3 (unc-47p::ChR2(H134R)::YFP)] showed that the casy-1 mutant animals relax significantly less than WT C. elegans upon blue light exposure (percent change in body length before and after optogenetic stimulation). Expressing the CASY-1C isoform under a GABAergic neuron specific promoter (Punc-25) completely rescues the relaxation defect in casy-1 mutant animals. Data is shown for two independent rescue lines. The graph shows the percentage change in body length for both +ATR and–ATR controls. The numbers of animals analyzed for each genotype are indicated. Data are represented as mean ± S.E.M. (*p<0.01 using one-way ANOVA and Bonferroni's Multiple Comparison Test). “ns” indicates not significant in all Figures. (C) mIPSCs were recorded from body wall muscles of adult animals for the indicated genotypes. Representative traces of mIPSCs and summary data for frequency and amplitude are shown. The casy-1 mutants showed a significant decrease in mIPSCs rate compared to WT C. elegans, suggesting a decreased GABAergic neurotransmission at the NMJ. The mIPSC amplitude, however, remains unaltered suggesting normal muscle responsiveness in the mutant. The decreased mIPSCs rate of casy-1 mutants can be significantly rescued by expressing CASY-1C specifically in GABA motor neurons (Punc-25). (D) Depicts traces and quantified data for the mEPSCs recorded from the C. elegans body-wall muscles. There is a subtle but significant increase in mEPSC frequency in casy-1 mutants, and this defect was not rescued by expressing CASY-1C in GABA motor neurons. For both C and D, the number of animals analyzed for each genotype is indicated. Data are represented as mean ± S.E.M. *p<0.05 with respect to WT and # p<0.05 with respect to casy-1 mutants, using the one-way ANOVA with Dunnett’s post test).
Fig 5.
CASY-1 isoforms shows differential spatial localization.
(A) Expression of GFP under isoform-specific casy-1 promoters. casy-1a transcriptional reporter does not co-localize with mCherry marked cholinergic or GABAergic motor neurons. casy-1b and casy-1c expression reporters show expression in both cholinergic and GABAergic motor neurons. Anterior is to the left in all panels. Scale bar, 8μm. (B) Representative fluorescent images of Pcasy-1c::CASY-1C::mCherry translational reporter showing co-localization with the GABAergic nuIs376 [Punc-25::SNB-1::GFP] pre-synaptic markers suggesting the presence of CASY-1C in the GABAergic NMJ pre-synaptic termini. Scale bar, 10μm. (C) Representative fluorescent images of Pcasy-1c::CASY-1C::mCherry translational reporter showing co-localization with the cholinergic nuIs152 [Punc-2129::SNB-1::GFP] pre-synaptic markers suggesting the presence of CASY-1C in the cholinergic synapses. Scale bar, 10μm.
Fig 6.
casy-1 isoforms are required for normal GABA release at NMJ.
(A) GABA SV release is compromised in casy-1 mutants. Images show the DNC of adult hermaphrodites expressing pH-sensitive GFP reporter (superecliptic pHluorin) tagged to the luminal domain of synaptobrevin. pHluorin fluorescent intensity is significantly reduced in the casy-1 mutants suggesting fewer GABA vesicles functional at the synapse. Quantification of fluorescent intensity is normalized to WT values. The number of animals analyzed for each genotype is indicated at the base of the bar graph. Quantified data are displayed as mean ± S.E.M. and were analyzed by two-tailed Student’s t-test. Scale bar, 8μm. (B) FRAP analysis of SNB-1::GFP levels in GABAergic motor neurons reveals that the dynamics of SV mobility is reduced in casy-1 mutants. Representative confocal images of pGABAergic::SNB-1::GFP levels compared between WT, casy-1 and casy-1; pGABAergic::CASY-1C rescue shows images before photo-bleaching (pre-bleach), immediately after photo-bleaching (post-bleach) and 360 sec after photo-bleaching (recovery). Scale bar, 2μm. At time 0, a single puncta of SNB-1::GFP was photo-bleached. Recovery of SNB-1::GFP levels were subsequently monitored at the photo-bleached and a neighboring control puncta. The fractional recovery of fluorescence 360 sec after photo-bleaching is shown. Recovery was measured with the pre-bleach fluorescence intensity being 100% and the post-bleach intensity at time 0 being 0%. The fluorescence intensity of control unbleached puncta did not change significantly during the period of recovery. (C) FRAP analysis of SNB-1::GFP levels in cholinergic motor neurons illustrated that mobility dynamics of SNB-1::GFP is normal in cholinergic motor neurons in casy-1 mutant animals. Representative confocal images of pCholinergic::SNB-1::GFP levels images prior to photo-bleaching (pre-bleach), immediately after photo-bleaching (post-bleach) and 400 sec after photo-bleaching (recovery) Scale bar, 2μm. The fractional recovery of fluorescence 400 sec after photo-bleaching is shown. The number of animals analyzed are indicated for each genotype. Data are represented as mean ± S.E.M. (**p<0.001 using one-way ANOVA and Bonferroni's Multiple Comparison Test).
Fig 7.
CASY-1 is required for transport of SV precursors in GABAergic motor neurons.
(A) Representative GABAergic::SNB-1::GFP transport kymographs in WT, casy-1 and casy-1; pGABAergic::CASY-1C rescue. For all kymographs, ventral cell body is to the right. Anterograde movement is from right to left. All kymographs are clipped to 10 μm (distance) x 1 min (time) (77x180 pixels). Scale bar, 3 μm. (B-C) Quantification of anterograde and retrograde SV flux in young adult animals. (B) Comparison of mean anterograde and retrograde flux (normalized to a distance of 10 μm and a time of 1 min) between WT, casy-1 mutant and casy-1; pGABAergic::CASY-1C rescue animals. (C) Comparison of mean anterograde and retrograde velocities between WT, casy-1 mutant and casy-1; pGABAergic::CASY-1C rescue animals are shown. casy-1 mutants show significantly reduced anterograde vesicular flux compared to WT animals. Reduced GABAergic anterograde flux was completely rescued by expressing CASY-1C specifically in GABAergic motor neurons. (D) Representative Cholinergic::SNB-1::GFP trafficking kymographs in WT and casy-1 mutant animals. For all kymographs, ventral cell body is to the right. All kymographs are clipped to 10 μm (distance) x 1 min (time). Scale bar, 3 μm. (E-F) Quantification of anterograde and retrograde SV transport in young adult animals (E) Average flux and (F) Average velocity are shown. Flux is defined as the number of moving particles per 10 μm per min. n represents number of particles analyzed for the analysis. Data are represented as mean ± S.E.M. (***p<0.0001 using two-way ANOVA and Bonferroni's Multiple Comparison Test, “ns” indicates not significant in all Figures).
Fig 8.
CASY-1 interacts with the kinesin motor UNC-104/KIF1A to regulate the trafficking of GABA vesicles.
(A) FRAP analysis of SNB-1::GFP levels in GABAergic motor neurons reveals that the dynamics of SV mobility is reduced after unc-104 RNAi. Representative confocal images of pGABAergic::SNB-1::GFP levels compared between WT, casy-1 mutant, unc-104 RNAi and casy-1; unc-104 RNAi shows images before photo-bleaching (pre-bleach), immediately after photo-bleaching (post-bleach) and 240 sec after photo-bleaching (recovery). Scale bar, 2μm. The fractional recovery of fluorescence 240 sec after photo-bleaching is shown. (B) The upper panel shows the domain organization of CASY-1C illustrating the two conserved WDDS motifs surrounding an acidic region containing stretches of glutamic acid residues. The CASY-1C (ΔKIF) represents a deletion in the region harboring the two WDDS motifs and the acidic region (amino acids 70–148) in the C- terminal region of CASY-1C. The lower panel shows a GST pull down assay showing that CASY-1 C-terminal can interact with the tail region of UNC-104. HA-tagged UNC-104 expressed in bacterial cell extract was incubated with bead-bound GST fusion proteins CASY-1C GST and CASY-1C (ΔKIF) GST. The blot was probed with anti-HA antibody. Significant pull down of 110 KDa HA-tagged UNC-104 was observed with CASY-1C GST. A deletion in the KIF-binding domain in CASY-1C eliminates the pull down of HA-tagged UNC-104 from the bacterial lysate. 10 μl of bacterial cell lysate was loaded as control. (C) Representative fluorescence micrographs summarizing the results of BiFC assay. BiFC signals from UNC-104 VN and UNC-104 VC interactions along the VNC (positive control). No BiFC signals were observed from UNC-104 VN and Empty VC (negative control). BiFC signals of UNC-104 VN and CASY-1C VC interactions observed in the GABA ventral cord motor neurons. Intensity of BiFC signals were significantly reduced in UNC-104 VN and CASY-1C (ΔKIF) VC BiFC pair. Scale bar, 10μm. (D) Intracellular localization of Pcasy-1c::CASY-1C::GFP in VNC cell bodies in unc-104 mutant background. In this mutant background, CASY-1C::GFP is significantly sequestered in cell bodies compared to WT control animals. A circular ROI was quantified in each image for two cell bodies and then averaged for each image intensity values. (E) In parallel, the fluorescence intensity of CASY-1C::GFP was significantly decreased at the DNC synapses in unc-104 mutants. Quantification of fluorescent intensity is normalized to WT values. The number of animals analyzed for each genotype is indicated at the base of the bar graph. Quantified data are displayed as mean ± S.E.M. and were analyzed by two-tailed Student’s t-test. Scale bar, 10μm.
Fig 9.
Proposed model for CASY-1 functioning at the NMJ.
CASY-1B and CASY-1C, the shorter isoforms are present on the SV precursors. The conserved C-terminal of CASY-1 act as an adaptor to mediate interaction of GABA-specific SVs with the tail region of UNC-104/KIF1A motor protein that mediates fast anterograde axonal transport of synaptic cargo. Mobility dynamics of SV precursors in turn regulates the release kinetics of GABA at NMJ. However in the absence of casy-1, this cargo-adaptor-motor bridge is lost resulting in aberrant anterograde flux of the GABA-specific SV cargo along the axonal pathway. This function of CASY-1B/C shows some similarity to how the mammalian CLSTN1 is thought to function. CLSTN1 has been shown to be required for fast anterograde axonal transport and loss of interaction with kinesin motor results in decreased anterograde trafficking and an increase in retrograde transport of vesicular cargo [18].