Adult <i>C</i>. <i>elegans</i> rhythmic swimming video.

Representative 10 second video of adult C. elegans swimming aligned with kymograph and eigenworm amplitude probability density estimates. (MOV)</p

Proposed model for CASY-1 functioning at the NMJ.

<p>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 <i>casy-1</i>, 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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007263#pgen.1007263.ref018" target="_blank">18</a>].</p

<i>casy-1</i> mutants show decreased GABAergic synaptic transmission at the NMJ.

<p>(A) Expression of the CASY-1C specifically in DD and VD neuronal subtypes using GABAergic motor neuron specific promoter (P<i>unc-30</i>) 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 <i>C</i>. <i>elegans</i>/ 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) [<i><a href="http://www.wormbase.org/species/all/transgene/WBTransgene00005248" target="_blank">zxIs3</a></i> (<i>unc-47</i>p::ChR2(H134R)::YFP)] showed that the <i>casy-1</i> mutant animals relax significantly less than WT <i>C</i>. <i>elegans</i> 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 (P<i>unc-25</i>) completely rescues the relaxation defect in <i>casy-1</i> 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. (*<i>p</i><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 <i>casy-1</i> mutants showed a significant decrease in mIPSCs rate compared to WT <i>C</i>. <i>elegans</i>, 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 <i>casy-1</i> mutants can be significantly rescued by expressing CASY-1C specifically in GABA motor neurons (P<i>unc-25</i>). (D) Depicts traces and quantified data for the mEPSCs recorded from the <i>C</i>. <i>elegans</i> body-wall muscles. There is a subtle but significant increase in mEPSC frequency in <i>casy-1</i> 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. *<i>p</i><0.05 with respect to WT and # <i>p</i><0.05 with respect to <i>casy-1</i> mutants, using the one-way ANOVA with Dunnett’s post test).</p

CASY-1 isoforms shows differential spatial localization.

<p>(A) Expression of GFP under isoform-specific <i>casy-1</i> promoters. <i>casy-1a</i> transcriptional reporter does not co-localize with mCherry marked cholinergic or GABAergic motor neurons. <i>casy-1b</i> and <i>casy-1c</i> 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 P<i>casy-1c</i>::CASY-1C::mCherry translational reporter showing co-localization with the GABAergic <i>nuIs376</i> [P<i>unc-25</i>::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 P<i>casy-1c</i>::CASY-1C::mCherry translational reporter showing co-localization with the cholinergic <i>nuIs152</i> [P<i>unc-2129</i>::SNB-1::GFP] pre-synaptic markers suggesting the presence of CASY-1C in the cholinergic synapses. Scale bar, 10μm.</p

List of strains.

(DOCX)</p

<i>casy-1</i> isoforms are required for normal GABA release at NMJ.

<p>(A) GABA SV release is compromised in <i>casy-1</i> mutants. Images show the DNC of adult hermaphrodites expressing pH-sensitive GFP reporter (<i>superecliptic pHluorin</i>) tagged to the luminal domain of synaptobrevin. pHluorin fluorescent intensity is significantly reduced in the <i>casy-1</i> 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 <i>t</i>-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 <i>casy-1</i> mutants. Representative confocal images of pGABAergic::SNB-1::GFP levels compared between WT, <i>casy-1</i> and <i>casy-1</i>; pGABAergic::CASY-1C rescue shows images before photo-bleaching <i>(pre-bleach)</i>, immediately after photo-bleaching <i>(post-bleach)</i> and 360 sec after photo-bleaching <i>(recovery)</i>. 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 <i>casy-1</i> mutant animals. Representative confocal images of pCholinergic::SNB-1::GFP levels images prior to photo-bleaching <i>(pre-bleach)</i>, immediately after photo-bleaching <i>(post-bleach)</i> and 400 sec after photo-bleaching <i>(recovery)</i> 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. (**<i>p</i><0.001 using one-way ANOVA and Bonferroni's Multiple Comparison Test).</p

The <i>casy-1</i> isoforms functions in GABAergic neurons to regulate synaptic transmission.

<p>(A) Expression of CASY-1 isoforms using tissue specific promoters; ACh neurons (P<i>unc-17</i>), GABA neurons (P<i>unc-25</i>) and muscle (P<i>myo-3</i>) establishes that expressing CASY-1 in GABAergic neurons completely rescues the Aldicarb hypersensitivity in <i>casy-1</i> mutants. The number of assays (~20 <i>C</i>. <i>elegans</i>/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 <i>casy-1</i> mutants. However, removing the entire C-terminal from the CASY-1A [CASY-1A (ΔC)] isoform does not rescue the Aldicarb hypersensitivity in <i>casy-1</i> mutants. The number of assays (~20 <i>C</i>. <i>elegans</i>/assay) is indicated for each genotype. (C) The <i>casy-1</i> mutants show higher sensitivity to GABA receptor antagonist PTZ than the WT animals. Representative still frame images demonstrating <i>casy-1</i> mutant <i>C</i>. <i>elegans</i> 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 <i>casy-1</i> isoforms in GABAergic neurons but not in cholinergic neurons or muscle. Assays were done (~10 <i>C</i>. <i>elegans</i>/assay) at least thrice. Data are represented as mean ± S.E.M. Values that differ significantly from WT animals are indicated (*<i>p</i><0.01, ***<i>p</i><0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test). Representative fluorescent images of (D) GABAergic [<i>nuIs376</i> (P<i>unc-25</i>::SNB-1::GFP)] or (E) cholinergic [<i>nuIs152</i> (P<i>unc-129</i>:: SNB-1::GFP)] synapses in the dorsal cord of WT or <i>casy-1</i> mutant. Scale bar, 8μm. Cholinergic synapses are largely normal in <i>casy-1</i> 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 <i>t</i>-test, “ns” indicates not significant in all Figures.</p

<i>ocr-2(ak47)</i>, <i>ocr-2(ok1711</i>,<i>)</i> and <i>kin-29(oy38)</i> mutants do not have exploration defects.

(A) ocr-2(ak47), ocr-2(ok1717) (strains BJH2763 and BJH2762, respectively) and kin-29(oy38) mutants do not have exploration defects. Shown is the number of squares (out of 50) single animals covered in ~20 h. (ns, P>0.05. One way ANOVA followed by a Bonferroni test. Error bars = SEM; n = 20–24). (B) kin-29(oy38) mutant animals do not have an exploration defect and the double mutant grk-2(gk268); kin-29(oy38) has an exploration defect similar to grk-2(gk268). (ns, P>0.05. One way ANOVA followed by a Bonferroni test. Error bars = SEM; n = 20–21). (PDF)</p

Motor neuron development is normal in <i>casy-1 mutants</i>.

<p>(A) Representative fluorescent images of WT and <i>casy-1</i> mutant animals expressing GFP in all GABAergic neurons (<i>juIs76</i> [P<i>unc-25</i>:: GFP]). The number of cell bodies and axonal commissures were largely normal in <i>casy-1</i> 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 (<i>hsp16</i>.<i>2</i>) completely rescues the Aldicarb hypersensitivity phenotype seen in <i>casy-1</i> mutant animals. The number of assays (~20 <i>C</i>. <i>elegans</i>/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 <i>ins-1</i> promoter suggest that the CASY-1A C- terminal is sufficient to rescue the Aldicarb hypersensitivity in <i>casy-1</i> mutants. Assays were done 3 times as indicated in Figures (~20 <i>C</i>. <i>elegans</i>/ assay). Data are represented as mean ± S.E.M. (***<i>p</i><0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test). “ns” indicates not significant in all Figures.</p

<i>grk-2</i> mutant animals have dispersal and exploration defects.

(A) Schematic representation of the assay used to measure dispersal (see Methods). (B) grk-2(gk268) mutant animals have a dispersal defect in the presence of food and dop-3(vs106) does not rescue this phenotype. Shown is the distance (cm) animals traveled from the center of the plate in the presence of food in 1 h. (****, P0.05. One way ANOVA followed by a Bonferroni test. Error bars = SEM; n = 30–53). (C) grk-2(gk268) mutant animals have a dispersal defect in the absence of food and dop-3(vs106) does not rescue this phenotype. Shown is the distance (cm) animals traveled from the center of the plate without food in 20 min. (****, P0.05. One way ANOVA followed by a Bonferroni test. Error bars = SEM; n = 47–60). (D) Schematic representation of the assay used to measure exploration (see Methods). (E) grk-2(gk268) mutant hermaphrodites have an exploration defect and dop-3(vs106) does not rescue this defect. Shown is the number of squares (out of 50) single animals covered in approximately 20 h. (****, P0.05. One way ANOVA followed by a Bonferroni test. Error bars = SEM; n = 30–32). (F) grk-2(gk268) mutant males have an exploration defect. (****, P<0.0001. Student’s t-test. Error bars = SEM; n = 30).</p