C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release

Significance Caenorhabditis elegans is a round worm that has been successfully used to study synaptic transmission, the process by which neurons communicate with targets cells in the nervous system through the release of chemical neurotransmitters. Many synaptic proteins involved in this release process are highly conserved with some species-specific variations in individual components. Here, we examined how the C. elegans protein, clarinet (CLA-1), an elusive member of the Piccolo, Fife and Rab3-interactingmolecule (Rim) protein family, fits into the organization and function of synaptic release sites in the worm. Using CRISPR/Cas-9 to endogenously tag key synaptic players, we determined the functional hierarchy of these C. elegans synaptic proteins and provide insights into conserved design principles that govern nervous system function.


RT-PCR Validation of cla-1(S) expression
RNA was isolated from N2 and cla-1(S) mutants by grinding in liquid nitrogen using a mortar and pestle, followed by homogenization using Qiashredder Columns (Qiagen), and then finally purified with the RNeasy Plus Mini Kit (Qiagen). RNA was reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). Primer sets were designed to amplify a 5′ region of the cla-1a long isoform, across the junction between cla-1a exons 32 and 33 (the location of the intron deletion), and the cla-1 3′ PDZ domain. PCR of these sites from cDNA was performed using a fixed series of cycles (22, 26, and 29 cycles) and cla-1 long and medium isoform mRNA was expressed in the cla-1(S) allele at similar levels as wildtype N2 animals, with amplicon bands appearing at the same cycle number as the appearance of N2 bands.

Electron Microscopy
Twenty to thirty young adult worms were placed in specimen chambers filled with E. coli and frozen at −180°C, using liquid nitrogen under high pressure (Leica HPM 100, Oberkochen, Germany). Samples then underwent freeze substitution (Reichert AFS, Leica) using the following program: −90°C for 107 hours with 0.1% tannic acid followed by 2% OsO4 in anhydrous acetone, incrementally warmed at a rate of 5°C/hour to −20°C, and kept at −20°C for 14 hours before increasing temperature by 10°C/hour to 20°C; samples were then infiltrated with 50% Epon/acetone for 4 hours, 90% Epon/acetone for 18 hours, and 100% Epon for 5 hours; finally, samples were embedded in Epon and incubated for 48 hours at 65°C (5). Ultrathin (40 nm) serial sections were acquired using an Ultracut 6 (Leica) and collected on formvarcovered, carbon-coated copper grids (EMS, FCF2010-Cu). Sections were post-stained with 2.5% aqueous uranyl acetate for 4 minutes, followed by Reynolds lead citrate for 2 minutes (5).
Images were obtained using either a JEOL JEM1220 or JEM-1400F transmission electron microscope, operating at 80 kV. Micrographs were acquired using one of the following cameras: Gatan Es1000W 11MP CCD, AMT NanoSprint1200-S CMOS or BioSprint 12M-B CCD Camera with AMT software (Version 7.01). Cholinergic synapses at the NMJ of the ventral nerve cord were identified based on established synaptic morphology (6). Sections containing a DP, as well as two flanking sections on either side of the DP, were analyzed blinded to genotype using NIH FIJI/ImageJ software. SVs were counted as docked when the SV membrane was fully contacting the plasma membrane of the neuron terminal (distance = 0 nm), SVs that were within 1-5 nm of the plasma membrane that exhibited small tethers were not scored as docked. The distribution of docked SVs from the DP was calculated for each section containing a DP, as well as one section on either side, using the ROI data from FIJI with Matlab scripts written by the Watanabe and Jorgensen labs (7). Values were imported to Prism (GraphPad) for statistical analysis using One-way ANOVA with Tukey post hoc analysis, or Kruskal-Wallis with Dunn's test, for multiple comparisons. An unpaired t-test was used when comparing only two genotypes.

Fluorescence Microscopy
Image acquisition and quantification for endogenously tagged UNC-2 and AZ proteins.
Fluorescent microscopy was performed as described previously (1 Images were obtained on a 63x/1.4 numerical aperture on a Zeiss Axio-Observer Z1 microscope. Images were captured using a Zyla 4.2 PLUS (Andor) with Spectra X solid-state light engine (Lumencor) as light source. For a given GFP transgenic animal, the same light intensity and exposure time setting were used. While not all animals were imaged simultaneously, each strain was imaged with its respective control to ensure consistency among image quality and quantification. Variations in intensity or peak number within the same genotype between different imaging sessions were negligible. Images were obtained as horizontal slices or Z-stacks, and maximal projection was applied and used for quantification.
With the exception of UNC-13 data, a line-scanning method (Metamorph) was utilized to quantify the maximal projection images to produce the average peak fluorescent intensity. Pixel intensity of 282 pixel length or 30 microns were measured in each image. To obtain the true peak intensity of each puncta, the average background intensity from an adjacent area was also measured and subtracted. Peaks above a threshold value were counted as a true peaks. The threshold was arbitrarily set to exclude small background fluctuations, and this same threshold value was applied to all the images of a given transgenic line. Since UNC-13 images are comprised of discrete puncta and surrounding diffuse signals adaptive thresholding was used on selected 282 x 47 pixel areas from maximum projection images to obtain a series of puncta from which maximal puncta intensity values and puncta numbers were using Integrative Morphometry Analysis (Metamorph). For clear separation of these two methods we used peak number/average intensity for the linescan method and puncta number/max intensity for adaptive thresholding.

Confocal acquisition of UNC-13 isoforms under the pUNC-129 promoter
Young adult worms (~10) were placed in a drop of M9 on 2% agarose pads (in M9 buffer containing 10 mM Sodium Azide (NaN3)) on a glass slide under a coverslip. Images of dorsal nerve cords were collected on an Olympus Fluoview FV10i inverted laser scanning confocal microscope with the 60X (NA 1.35) oil immersion lens and optical zooming to a total magnification of 120X. The same imaging parameters were used for each genetic background of the same fluorescence marker. Control strains were always imaged on the same day as mutant strains. Fluorescent analysis was conducted using NIH FIJI/ImageJ software in which max projections were created from obtained z-stacks, nerve cords were straightened, and all images were subjected to background subtraction with a rolling ball radius of 50. Fluorescent levels were extracted from a 40µm line along the nerve cord and peaks were identified from the Plot Profile data using peak finder in Matlab with an arbitrary threshold set to account for background fluctuations which was applied to all images for a given fluorescent marker.
Statistical analysis was conducted in Prism (GraphPad) using one-way ANOVA with Dunnett's multiple comparison test.