Aldicarb Sensitivity Assays
To measure aldicarb sensitivity, 20–25 young adult animals were placed on agar plates containing 1 mM aldicarb (Chem Services). Worms were scored for paralysis at ten minute intervals for two hours. Each genotype was tested at least 10 times and paralysis curves were generated by averaging paralysis time courses for each plate as described previously (Dittman and Kaplan, 2008
Worm Tracking and Analysis
Young adult animals were picked to 6 cm agar plates with no bacterial lawn (40 worms per plate). Imaging began 1 hour after worms were transferred. At least 200 animals were assayed for each genotype. One minute digital videos of individual animals were captured on an ORCA-05G CCD camera (Hamamatsu) mounted on an Olympus SZX16 stereomicroscope at 5 Hz using 10X magnification. Plates were lightly tapped before tracking capture. The center of mass was recorded for each animal on each video frame using custom object tracking software in MatLab (Ramot et al., 2008
). Fewer than 10% of worms were excluded due to lack of movement for 30 seconds or longer.
Steady-state Fluorescence Imaging and Quantification
Animals were immobilized using a 30 mg/mL solution of 2,3-butanedione monoxime (J.T Baker) mounted on 2% agarose pads, and imaged on an inverted Olympus microscope (IX81), using a laser scanning confocal imaging system (Olympus Fluoview FV1000 with dual confocal scan heads) and an Olympus PlanApo 60X 1.42 NA objective. Rescuing complexin variants were C-terminally tagged with GFP separated by a 12 residue linker (GGSGGSGGSAAA), and synaptic protein levels were estimated by measuring background-subtracted fluorescence within dorsal cord varicosities. Data were analyzed with custom software in IGOR Pro (WaveMetrics, Lake Oswego, OR) (Burbea et al., 2002
; Dittman and Kaplan, 2006
). A fluorescent slide was imaged daily to monitor the laser stability and the dorsal cord fluorescence was normalized to the slide value. For the spatial correlation experiments shown in , individual line scans where computed from maximal intensity projections of image stacks in each color channel. Green (CPX-1) and red (RAB-3 or ELKS-1) line scans from the same animal were cross-correlated to compute the Pearson correlation coefficient in the paired measurement. Since some of the overlapping fluorescence intensity could have arisen due to chance, we also shuffled the green and red line scans so that green-red correlations between animals could be determined (shuffled correlation). In all cases, the shuffled correlation coefficient was nearly zero, confirming that the measured correlation is not due to chance. ΔF/F calculations in , were made as described previously (Dittman and Kaplan, 2006
Dynamic Fluorescence Imaging and Quantification
Complexin variants were C-terminally tagged with photoactivatable GFP (pGFP) separated by a 12 residue linker (GGSGGSGGSAAA) and co-expressed with mCherry::RAB-3 in dorsal cord motor neurons using a modified unc-129
promoter. pGFP was photo-activated with a one millisecond pulse from a stationary 405-nm laser beam centered on the synapse of interest based on mCherry::RAB-3 fluorescence. Photo-activated fluorescence was initially distributed over the synapse with a full-width half-maximum ranging from 500 to 800 nm. Line scans of length five to ten microns centered on the target synapse were collected every 1.5 – 2 milliseconds, depending on the line scan length. Kymographs of sequential line scans were assembled and analyzed to generate time courses of fluorescence decay averaged over a one micron region centered on the target synapse. For the CPX-pGFP experiments shown in , small regions of interest measuring 10 by 0.2 microns (400 by 8 pixels) centered on the target synapse were collected every 30 to 40 milliseconds, and each image was collapsed to a line scan using maximal intensity projection. Consecutive projected line scans were then assembled into a kymograph and analyzed as described above. Experiments were rejected if mCherry::RAB-3 fluorescence revealed large drift or movement artifacts. All analysis was performed in Igor using custom written software. The decay time courses reflect a nontrivial combination of diffusion and binding/unbinding kinetics. In the absence of a detailed reaction-diffusion model to account for the details of synapse geometry, diffusion, and membrane binding, we chose to fit the data empirically using a double exponential decay function. For each synapse, weighted decay times constants were obtained by fitting the normalized, background subtracted data with a double exponential function and computing τw
) where Afast
are the fast and slow amplitudes, while τfast
are the fast and slow time constants, respectively. These parameters allow for a quantitative comparison of decay kinetics but are not intended to correspond directly to a measure of either diffusion or unbinding kinetics. For untagged pGFP, diffusion out of the synapse could be estimated by fitting each line scan with a Gaussian centered on the target synapse and analyzing the increase in variance of the Gaussian over time using the formula:
is the initial variance of fluorescence. For simple diffusion, this rate is constant and thus variance will increase linearly with time (see ). Using this approach, a moderate range of diffusion constants was observed ranging between 5 and 15 µm2
/sec at the worm NMJ, consistent with small globular proteins diffusing within a restricted compartment.
CPX-1(WT)::pGFP (JP249), CPX-1(K/P)::pGFP (JP252), CPX-1(ΔCT)::pGFP (JP253), CPX-1(LV/EE)::pGFP (JP254), and CPX-1(Δ12)::pGFP (JP318) constructs were cloned into the pET28a vector containing a His6 tag to facilitate purification. These constructs also contain a 34 amino acid sequence at the N-terminus consisting of a T7 tag and thrombin cleavage site. BL21-DE3 E. coli were transformed and grown in rich media containing 50 µg/mL kanamycin to an optical density of 0.6. Cells were then induced with 400 µg/ml isopropyl thiogalactopyranoside (IPTG), grown for three hours at 37 °C, pelleted, resuspended in buffer (35 mM NaCl, 20 mM imidazole, 20 mM Tris-HCl, 1.5 mM BME, 2 mM DTT), lysed by sonication, and pelleted at 40,000 rpm for 40 minutes. For NMR samples, cells grown on rich media were spun down and resuspended in a minimal M9 media containing 15N-ammonium chloride with or without 13C-glucose prior to induction. The supernatant was purified on a Ni-NTA column (Qiagen, Hilden, Germany). Protein was eluted in elution buffer (350 mM NaCl, 250mM Imidazole, 20 mM Tris-HCl, 1.5 mM BME, 2 mM DTT). For assays in , concentrated fractions underwent buffer exchange (150 mM NaCl, 50 mM Tris-HCl, 1 mM CaCl, 1 mM DTT) using Sephadex G-25 Fine beads (Sigma). For NMR, pure complexin was dialyzed into 60 mM phosphate, 2 mM DTT, 0.5 mM EDTA, pH 6.1.
Lipids were obtained from Avanti Polar Lipids and stored at −20 °C. For anionic liposomes, a lipid mixture composed of 70% 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 30% 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) was dried under a stream of N2 gas, and residual solvent was removed under vacuum for 1 hour. The lipid film was then rehydrated in buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM CaCl2, 1 mM DTT for flotation, or 60 mM phosphate, 2 mM DTT, 0.5 mM EDTA, pH 6.1 for NMR) to obtain a final lipid concentration of either 4 mM (flotation and co-sedimentation) or 20 mM (NMR). The resulting liposomes underwent 10 cycles of freezing in liquid nitrogen and thawing (50 °C water bath), and were then extruded 21 times through either 400 or 200 nm then 100 nm pore-size polycarbonate films using a 10 mL Lipex Extruder from Northern Lipids (Canada) to form unilamellar vesicles.
Liposome flotation assay
Protein and lipids were incubated at room temperature for 1 hour. A sucrose gradient (10 mM HEPES, 150 mM NaCl, 1 mM CaCl) containing 500 µL of the sample mixed with 500 µL 60% sucrose (Amresco), 1mL 25% sucrose, 1mL 20% sucrose and 500 µL buffer was constructed. 100 µL fractions were obtained after ultracentrifugation in an MLS-50 rotor (Beckman) at 35,000 rpm for 2 hours. Fractions were run on a 10% SDS-PAGE gel followed by Western blotting. Protein was detected with a polyclonal rabbit GFP antibody and a horseradish peroxidase conjugated goat anti-rabbit secondary antibody (Genescript) and visualized using chemiluminescence (Thermo Scientific). For the flotation assays, the total amount of protein added was estimated using SDS-PAGE (WT 0.92 µg; K123P 1.2 µg; ΔCT 0.89 µg; LV/EE 0.74 µg for assay shown in , and WT 9.6 µg; Δ12 40.8 µg for the assay shown in Figure S1C
) with bands quantified using ImageJ. The protein:liposome ratio for these experiments ranged from 4:1 to 200:1 which is more than an order of magnitude below the expected ratio of binding sites:liposome (~4,400:1, estimated using the surface area occupied by a 30-residue helix freely rotating about its center on the surface of a 150 nm diameter liposome) (Georgieva et al., 2010
Liposome co-sedimentation assay
Full-length and Δ12 CPX-1 proteins were spun at 75,000 rpm for 30 min to eliminate potential protein aggregates and then incubated with lipids at room temperature for 1 hour. 20 µL fractions were taken from the ~70 µL supernatant and the resuspended pellet after ultracentrifugation in a TLA100.1 rotor (Beckman) at 75,000 rpm for 30 min. The fractions were run on a 10% SDS-PAGE gel and examined using ImageJ. Percent co-sedimentation was calculated as 100×P/(P + 3.5×S) where P is the pellet band intensity and S is the supernatant band intensity. The co-sedimentation was performed six times and the calculated co-sedimentation percentage was averaged. Protein:liposome ratios were about 20:1 for these assays.
Proton-nitrogen correlation (HSQC) spectra as well as a standard set of heteronuclear triple resonance three-dimensional spectra (HNCACB/CBCACONH, HNCACO/HNCO, and HNCANH) were collected on either a Varian Unity Inova 600 MHz (Weill Cornell NMR Facility) or a Bruker AVANCE 700 MHz (New York Structural Biology Center) spectrometer equipped with a cryoprobe. Backbone resonance assignments for free wild type complexin were obtained at a completeness level of 91% for amide group, Cα, and Cβ, and 87% for CO resonances, except for the N-terminal tag region and residues 40–66, the latter of which were not assigned due to reduced signal intensity and severe overlap, likely originating from the known helical conformation of this region (Chen et al., 2002
). Liposome titrations were monitored using HSQC spectra using 1024 and 2564 complex data points in the direct and indirect dimensions, respectively and spectral widths of 12,000 Hz (proton) and 1600 Hz (nitrogen). All spectra were collected at 20 °C with subsaturating protein:liposome ratios. Data were processed using NMRpipe and analyzed using NMRview (Delaglio et al., 1995
; Johnson and Blevins, 1994
). Spectra were referenced indirectly to water. Protein concentrations were between 50 and 75 micromolar, as measured using absorbance at 280 nm and an experimentally determined extinction coefficient of 1882 cm−1
Whole-cell patch-clamp recordings were performed on dissected animals as described previously (Madison et al., 2005
; McEwen et al., 2006
). Dissected worms were superfused in an extracellular solution containing 127 mM NaCl, 5 mM KCl, 26 mM NaHCO3
, 1.25 mM NaH2
, 20 mM glucose, 5 mM MgCl2
at 20 °C. Whole-cell recordings were carried out at −60 mV using an internal solution containing 105 mM CH3
SCs, 10 mM CsCl, 15 mM CsF, 4 mM MgCl2
, 5 mM EGTA, 0.25 mM CaCl2
, 10 mM HEPES and 4 mM Na2
ATP, adjusted to pH 7.2 using CsOH. Under these conditions, we only observed cholinergic EPSCs.
For data sets requiring a single pairwise comparison, we used Student's t test to compute significance. For all other comparisons, we used the Tukey-Kramer method for multiple comparisons. This test assumes independent, normal distributions with equal variance. In cases in which sample variance was significantly different, we employed the Newman-Keuls method to test significance. Significance was defined by the criterion p < 0.01.