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. 2006 Jul 25;103(30):11399-404.
doi: 10.1073/pnas.0600784103. Epub 2006 Jul 14.

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

Jeremy S Dittman  1 , Joshua M Kaplan
Affiliations

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

Jeremy S Dittman et al. Proc Natl Acad Sci U S A. .

Abstract

After synaptic vesicle fusion, vesicle proteins must be segregated from plasma membrane proteins and recycled to maintain a functional vesicle pool. We monitored the distribution of synaptobrevin, a vesicle protein required for exocytosis, in Caenorhabditis elegans motor neurons by using a pH-sensitive synaptobrevin GFP fusion protein, synaptopHluorin. We estimated that 30% of synaptobrevin was present in the plasma membrane. By using a panel of endocytosis and exocytosis mutants, we found that the majority of surface synaptobrevin derives from fusion of synaptic vesicles and that, in steady state, synaptobrevin equilibrates throughout the axon. The surface synaptobrevin was enriched near active zones, and its spatial extent was regulated by the clathrin adaptin AP180. These results suggest that there is a plasma membrane reservoir of synaptobrevin that is supplied by the synaptic vesicle cycle and available for retrieval throughout the axon. The size of the reservoir is set by the relative rates of exo- and endocytosis.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Measuring surface and vesicular SpH in cholinergic motor neurons. (A) A representative experiment in a wild-type animal. Photodiode current measurements from the fluorescent spot were made every 15 sec while the extracellular solution was exchanged as indicated (see Methods). Note that there is a large contribution of background light pooled onto the photodetector (see Supporting Methods for details). (B) Summary of SpH surface ratios in a panel of synaptic mutants: wild-type nuIs122, ric-4 SNAP-25, unc-64 syntaxin, unc-13 Munc13, unc-18 Munc18, dpy-23 μ2 AP2, dyn-1 dynamin, unc-57 endophilin A, and unc-11 AP180. See Methods for the calculation of surface ratios. Data are shown as mean ± SEM. ∗∗, P < 0.01 by Student’s t test, compared with wild type.
Fig. 2.
Fig. 2.
Imaging synaptobrevin in intact animals. (Aa) The N-terminal tag places GFP (NGFP-SNB) in the cytoplasm. (Ab) Motor neurons expressing a C-terminal pHluorin-tagged synaptobrevin (SpH) that places the fluorophore in the vesicle lumen. (Scale bars, 5 μm.) (Ba) Expression of SpH in the ventral cord. Neuromuscular junctions are located on the bottom cord (∗). (Bb) Expression of UNC-10 RIM1::RFP in the ventral cord. (Bc) Colocalization of SpH (green) and UNC-10 (red). (C) A line scan of SpH in a wild-type animal. Peaks (red arrowheads) and baseline (dashed line) were located by using automated software, as described in Supporting Methods.
Fig. 3.
Fig. 3.
Effects of synaptic mutants on synaptobrevin distribution in vivo. (A) Representative image of the dorsal cord in wild-type (a), unc-13 Munc13 (b), and unc-11 AP180 (c) mutant animals. (Left) NGFP-SNB. (Right) SpH. (B) Peak absolute SpH fluorescence across the nine strains: nuIs122, ric-4, unc-64, unc-13, unc-18, dpy-23, dyn-1, unc-57, and unc-11. (C) Axon absolute fluorescence. Individual images are normalized to fluorescent bead standards (see Methods). (D) Plot of percentage change in peak fluorescence vs. percentage change in axon fluorescence for each of the nine strains measured in B and C. A linear regression of the data gave a slope of 0.9, y intercept of 8.2%, and Pearson’s R2 of 0.99 (P < 0.001). All data are normalized to wild type; bars are mean ± SEM. ∗∗, P < 0.01 by Student’s t test, compared with wild type. Note that some error bars are too small to be visible on this scale.
Fig. 4.
Fig. 4.
Loss of tomosyn increases surface synaptobrevin abundance. (A) Representative images of the dorsal cord from the wild-type strain (Upper) and from the tomosyn mutant tomo-1 (Lower). (Scale bar, 5 μm.) (Ba) Absolute peak fluorescence (relative to a fluorescent bead standard, see Methods) for wild type and tomo-1. (Bb) Absolute axonal fluorescence. Data are normalized to wild type; bars are mean ± SEM. ∗∗, P < 0.001 by Student’s t test, compared with wild type.
Fig. 5.
Fig. 5.
Effects of endocytosis mutants on surface synaptobrevin puncta width. (A) Summary of endocytosis mutant effects on SpH punctal width for the following strains (number of animals): wild type, dpy-23 μ2 AP2, dyn-1 dynamin, unc-57 endophilin A, and unc-11 AP180. (B) Assortment of representative puncta from wild-type (Upper) and unc-11 AP180 mutant (Lower) animals. (Scale bar, 1 μm.) (C) Histogram of punctal widths (measured as full width at half maximum, see Methods) for wild-type (black) and unc-11 (red) animals. Widths are binned in 0.2-μm intervals and normalized to the wild-type peak. Data are from 1,672 puncta (90 animals) for wild type and 414 puncta (35 animals) for unc-11. All data are normalized to wild type; bars are mean ± SEM. ∗∗, P < 0.01 by Student’s t test, compared with wild type.
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