The C-terminal of CASY-1/Calsyntenin regulates GABAergic synaptic transmission at the Caenorhabditis elegans neuromuscular junction
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.