. 2015 Mar 24;108(6):1318-1329.
Synaptic Activity Regulates the Abundance and Binding of Complexin
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Synaptic Activity Regulates the Abundance and Binding of Complexin
Free PMC article
Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity.
Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Using pGFP to determine mobility of synaptic proteins. (
A) Cartoon of an adult worm expressing pGFP in a dorsal motor neuron (DA/DB axon and neuromuscular junctions). The rectangle indicates the region imaged in this study. ( B) Experimental scheme using pGFP expressed in a small number of motor neuron axons. Nonactivated pGFP ( black) is distributed throughout the axon along with SVs marked by mCherry::RAB-3 ( red circles). Photoactivation of a single synapse with a 405 nm laser pulse ( purple) converts pGFP to a fluorescent state ( green) followed by diffusion and reequilibration with axonal pGFP. ( C) Kymographs of mCherry-Rab3 and pGFP line scans (∼10 μm of axon) collected every 2 msec for 5 s. Three en passant synapses are apparent based on localized Rab3 fluorescence ( gray arrows). About 1 s into the recording, a brief photoactivation of the middle synapse (#2, purple arrowhead) produced a transient increase in GFP fluorescence that rapidly dispersed throughout the axon. ( D) Time course of 1- μm spatial averages for each of the three synapses as indicated. ( E) The activated region was estimated to be ∼500 nm in diameter based on scanning a 170 nm fluorescent bead (see Materials and Methods). ( F) Six consecutive photoactivation trials at a single synapse show similar decay kinetics. Intertrial interval was 2 min. ( G) Single trial fits to 1D ( red, τ ) and 3D diffusion ( D blue, D ). See Materials and Methods for model details. app
Effects of synaptic bouton geometry on protein mobility. (
A) Example of bouton ( blue) and axonal ( black) decay kinetics for pGFP with fits to 1D diffusion. The cartoon inset indicates the locations of point photoactivation. ( B) Average decay time courses (±SE) for axonal ( black) and synaptic pGFP ( blue). Number of experiments indicated on graph. Inset: Average decay time constants using 1D diffusion show a sixfold decrease in apparent mobility in synaptic boutons compared to axons. Data are mean ± SE and ∗∗indicates p < 0.01 by Student’s t-test. ( C) Individual 3D synaptic boutons were modeled as 1- μm long ellipsoids in Virtual Cell as described in the Materials and Methods using the indicated sizes. Seven boutons were placed evenly along a 24 μm length of axon with 3 μm spacing. The center bouton was used for photoactivation simulations ( arrowhead). ( D) Simulated normalized decay curves for values of bouton radius ranging from 100 nm ( blue) to 500 nm ( yellow). For each bouton radius, five values of diffusion coefficients ranging from 2 to 8 μm 2/s were used (corresponding to the five curves for each radius). ( E) Each decay curve from D was fit to a 1D decay to estimate the diffusion coefficient. The ratios of the estimated and actual diffusion coefficients used in the simulation were plotted as a function of bouton radius, illustrating the underestimation of diffusion with increasing bouton size. An exponential fit exhibited a space constant of 72 nm. The region shaded in yellow represents the typical range of bouton sizes. Note that the r = 100 nm case did not produce a precise match to the true D value because of numerical error in the simulation using a voxel size of 20 nm. ( F) Simulated decay curves (normalized to initial pGFP concentration) for pGFP photoactivation in the axon ( black) and synapse ( blue) using D = 4 μm 2/s and a bouton radius of 200 nm.
Complexin mobility is restricted at synapses. (
A) Kymographs of mCherry-Rab3 and CPX-1 fused to pGFP (CPX-pG) line scans collected every 2 msec for 6 s. Three en passant synapses along a 10 μm region of the axon are identified using Rab3 fluorescence ( gray arrowheads). A brief photoactivation of the middle synapse (#2, purple arrowhead) produced a transient increase in CPX-pG fluorescence that dispersed throughout the axon and accumulated at neighboring synapses. ( B) Time course of 1- μm spatial averages for each of the three synapses as indicated. ( C) Synaptic fluorescence decay (uncorrected) for pGFP ( gray) and CPX-pG ( black) indicating the lower mobility of CPX-pG within the synapse, perhaps due to CPX binding to SV membrane as shown in the cartoon. ( D) Average fluorescence decay (±SE) for axonal ( black) and synaptic ( blue) CPX-pG. For comparison, the average axonal pGFP decay is shown in purple. Based on the slower axonal mobility of CPX-pG compared to pGFP (diffusion coefficients of 2 and 4 μm 2/s, respectively), simulations of synaptic CPX-pG generate the decay curve shown in red. Note that CPX-pG decays much more slowly than predicted ( arrow). ( E) Single trial decay curves for pGFP ( gray) and CPX-pG ( black) are overlaid with 1D diffusion fits ( blue and red, respectively) sampled once every 2 msec over a 5 s interval. ( F) Long timescale measurements made every 40 msec for pGFP ( gray) and CPX-pG ( black) together with 1D diffusion fits. ( G) Average values of the decay time constants based on 1D diffusion using fast ( black) and slow ( gray) sampling rates. The number of synapses is indicated in the bars. ( H) Schematic of an en passant synapse with immobile binding sites ( orange). CPX ( black) diffuses in and out of the bouton and binds to the synaptic sites with dissociation constant K and concentration D B . This scheme was used to develop a simple reaction-diffusion model in Virtual Cell. ( tot I) Photoactivation in a single bouton using a binding model with five species. The fluorescent species ( blue) diffuse out of the bouton, whereas nonfluorescent species ( gray) diffuse into the bouton after photoactivation at t = 0. The measured fluorescence was modeled as the sum of both fluorescent species ( green) and compared to measured decay traces to estimate best fitting parameters. Unbound species ( dotted lines) exchanged rapidly with the same time course as pGFP, whereas bound species ( solid lines) exchanged slowly and were limited by the off-rate of binding. ( J) The reaction-diffusion model adequately fits the example decay traces for both CPX-pG ( black) and pGFP ( gray) adjusting only the binding capacity ( B / tot K ) and using a common B D of 4 app μm 2/s for both pGFP and CPX-pG. See Materials and Methods and Fig. S2 for details. Data are mean ± SE.
Complexin is retained in synapses through multiple types of interactions. (
A) Schematic of six optical probes used for these experiments. CH is the central helix and CTD is the C-terminal domain. KY/AA is a double point mutant within the CH domain that impairs SNARE binding. Probes are ordered from highest to lowest binding ( top to bottom), as predicted from prior studies. ( B) Cartoon of two binding modes for complexin at the synapse. The CTD ( black) interacts with SVs, whereas the CH ( gray) interacts with SNARE complexes. ( C) Representative single trials of CPX-pG ( gray), ΔCT-pG ( blue), and pGFP ( black) synaptic decay curves. pGFP/mCherry ratios were computed for each decay trace and then normalized to the initial value. Data are fit with a 1D diffusion decay curve with characteristic decay time constants as indicated. ( D) Average τ values for each of the optical probes. ( D E) Residual fluorescence at 25 s normalized to initial fluorescence for each CPX variant. Note that pG-Gyrin is included for comparison ( magenta). ( F) Average values of the log of the dissociation constant (in micromolar) are plotted for each pGFP probe. Traces were fit to a 3D reaction diffusion model with immobile binding sites within the synaptic bouton and D of either 2 or 4 app μm 2/s. Best-fit estimates of the dissociation constant were generated for each of the CPX probes, allowing only the binding site concentration and the dissociation constant to vary, (see Materials and Methods and Fig. S3). Data are mean ± SE and number of synapses is indicated in ( E). ∗∗differs from CPX-pG with p < 0.01, ∗differs from CPX-pG with p < 0.05. n.s., not significant. # differs from all other species with p < 0.01. Significance was determined using the Tukey Kramer test. Sample numbers indicated in ( E).
Chronic activity mutants bidirectionally alter complexin mobility. (
A) Mean ± SE decay curves for synaptic CPX-pG in WT ( black), unc-64 Syntaxin mutant ( red), and tom-1 Tomosyn mutant ( blue) animals. ( B) Individual traces were fit to 1D diffusion and average τ values are shown for each genotype. Both synaptic mutants significantly differ from WT. ( D C) Mean ± SE synaptic decay curves for a C-terminal truncation of CPX ( ΔCT-pG) are shown for each genotype. ( D) Diffusion time constants for ΔCT-pG indicate that there is no significant change in mobility for unc-64 Syntaxin mutants and only a minor increase for tom-1 Tomosyn mutants. ( E) Mean ± SE decay curves for pGFP in each genotype. ( F) Average time constant for pGFP is the same in both mutant backgrounds. The number of synapses is indicated in the bars for each genotype. ∗∗ p < 0.01, < 0.05 by Student’s ∗p t-test. ( G) Cartoon of the SV cycle ( red arrows) depicting CPX ( blue) binding and unbinding from SVs. If SV fusion drives the release of bound CPX ( orange arrows), CPX mobility will be coupled to the SV cycle. ( H) Individual CPX-pG decay traces from WT ( black), unc-64 Syntaxin mutant ( red), and tom-1 Tomosyn mutant ( blue) animals are overlaid with simulated decays for three concentrations of binding sites using D = 2 app μm 2/s. Model binding site abundance is indicated on the right. Log( K ) values are −2.4, −2.7, and −2 for D WT, unc-64, and tom-1, respectively.
Dispersion of synaptic CPX-GFP during acute stimulation in cultured hippocampal neurons. (
A) Colocalization of CPX-GFP ( green) and the presynaptic marker VAMP-mCherry ( red) in cultured hippocampal neurons. Scale bar = 5 μm. ( B) CPX-GFP fluorescence at rest ( top) and during a train of 300 AP at 10 Hz ( bottom). Note the decreased fluorescence in boutons and increased fluorescence in neighboring regions of axon upon stimulation. Scale bar = 2 μm. ( C) Time course of average fluorescence intensity (mean ± SE) in synaptic boutons ( red) and axonal regions ( gray) from one neuron during a train of 300 AP at 10 Hz. ( D) Time course of average synaptic bouton fluorescence (mean ± SE) for neurons expressing the tetanus toxin light chain (TeNT). ( E) Average change in bouton fluorescence following the 10 Hz stimulus train for control ( red) and TeNT-expressing ( blue) neurons.
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