Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision

Abstract

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

Figure 1.

Experimental configuration for imaging single molecules and synaptic vesicles using TIRF microscopy. a, Schematic of the system in which samples were imaged in microfluidic channels. Fluorescent samples were flowed into the channels and excited by green (488 nm) and red (633 nm) lasers simultaneously. Images were focused using a CRIFF (continuous reflective-interface feedback focus system; depicted as a light blue line). Emitted light was split with a dual-view imaging module and collected by a cooled, high-sensitivity CCD camera. OBJ, Objective; DM, dichoric mirror; SM, scanning mirror; PSD, position-sensitive diode; L, lens; BP, bandpass filter; CCD, camera. b, Diagram of the TIRF-imaging environment. Minimally labeled vesicles (left), single antibody complexes (middle), and fully labeled vesicles (right). The decay of laser intensity is illustrated in the plot to the left of the drawing, which we measured to be ∼300 nm (1/e). c, High signal-to-noise ratio obtained with antibody-labeled synaptic vesicles. Labeled vesicles were adsorbed onto the floor of a microchannel and imaged (left). The line scan plots the intensity of each pixel along the indicated line. d, Schematic of the two-color labeling scheme in which synaptic vesicles were labeled with antibodies directed against two different synaptic vesicle proteins, each of which was detected with a different colored fluorescent secondary antibody. e, Verification of vesicle labeling. 1, 2, Sample image of synaptic vesicles labeled with anti-SV2 and goat-anti-mouse Alexa-488 (green, 1) and anti-synaptotagmin 1 and goat-anti-rabbit Alexa-635 (red, 2). 3, Two-color overlay in which colocalization of the two probes appears yellow. 4, Sample in which primary antibodies were excluded from the labeling protocol. 5, Sample in which vesicles were excluded from the labeling protocol. Scale bars, 1 μm.

Figure 2.

Determining the concentration of antibody required to produce fully and minimally labeled vesicles. Vesicles were labeled with the indicated antibody dilutions (from a stock concentration of ∼2 mg/ml). Vesicles were judged to be fully labeled at antibody concentrations that produced no further increase in measured intensity per fluorescent spot (i.e., vesicle, green arrows), and to be minimally labeled at antibody concentrations that produced no further decrease in measured intensity per fluorescent spot (i.e., vesicle, red arrows).

Figure 3.

Verification of single vesicle samples. AFM imaging of labeled synaptic vesicles and illustrative example of microscope resolution. a, AFM imaging of labeled synaptic vesicles. Shown are AFM images of antibody-labeled vesicles in solution on a mica surface. Scale bars, 100 nm. b, Histogram showing the diameter of the labeled vesicles as measured with contact-mode AFM. c, Filtering of synaptic vesicles to test for vesicle aggregation. To ensure we were not imaging aggregates of vesicles, we filtered a vesicle sample through a 0.11 μm pore-diameter polycarbonate filter and measured the mean intensity of the synaptic vesicles before and after filtration. Aggregates of two or more vesicles labeled with antibodies are unlikely to pass though the filter. The mean intensity did not change between the filtered and unfiltered samples. Error bars represent SE between data sets. d, Left, Three-dimensional Gaussian plots of a spot at the resolution of our setup (2.3 pixels); right, plot of a spot much above our measured resolution (10 pixels).

Figure 4.

Procedure used to generate calibration distributions. a, Intensity distributions of single-antibody complexes (red) compared with minimally labeled synaptic vesicles (blue). b, Intensity distribution of single-antibody complexes from (a) after it has been scaled to the same mean intensity as the minimally labeled vesicles. The goodness of fit is indicated by a reduced χ² value of 1.10. The residual of the fit (the difference between the scaled antibody and the minimally labeled vesicles) is plotted as a black dotted line. c, The scaled distribution of the single-antibody complexes was multiplied by integers to generate theoretical (calibration) distributions. Ab1 denotes the scaled distribution of the single-antibody complexes; Ab2 through Ab5 were obtained by multiplying the Ab1 distribution by the corresponding integers 2 through 5. d, Calibration distributions displayed as semilog cumulative probability plots. This panel illustrates that multiplication of the calibration distribution does not change the multiplicative standard deviation or shape of the distribution.

Figure 5.

Quantification of monodispersed synaptic vesicle proteins. Plots of representative fits for each protein is shown on the left, and histograms showing the percentage of vesicles containing n number of the indicated vesicle protein averaged across ∼10,000–20,000 vesicles (see Table 1) is shown on the right. Error bars, SE between different datasets.

Figure 6.

Quantification of polydispersed synaptic vesicle proteins. Plots of representative fits for each protein is shown on the left, and histograms showing the percentage of vesicles containing n number of the indicated vesicle protein averaged across ∼10,000–20,000 vesicles (see Table 1) is shown on the right. Error bars, SE between different datasets.