Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Sep 24:6:8392.
doi: 10.1038/ncomms9392.

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

Niclas Gimber  1 , Georgi Tadeus  1 , Tanja Maritzen  1 , Jan Schmoranzer  1   2 , Volker Haucke  1   3   2
Affiliations

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins

Niclas Gimber et al. Nat Commun. .

Abstract

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Newly exocytosed Syb2 is largely retained within presynaptic boutons.
(a) Schematic of experimental workflow to monitor newly exocytosed pHluorin-tagged SV proteins. (b) Syb2-pHluorin fluorescence (F) before and after selective photobleaching. Right, enhanced contrast shows small residual F post-bleaching. Scale bar, 5 μm. (c,d) Syb2-pHluorin F intensity changes from time-lapse images (400 ms frame−1) of stimulated (40 APs, 20 Hz) surface-eclipsed neurons. (c) Presynaptic boutons, (d) axonal areas (mean±s.e.m.; N=10 neurons).
Figure 2
Figure 2. Syb2 spread, confinement and reclustering within presynaptic boutons.
(a) Representative images of a Syb2-pHluorin expressing synapse of stimulated (40 APs, 20 Hz) surface-eclipsed neurons, either background subtracted (ΔF), Gaussian fit (Fit), FWHM (white line) or Gaussian fit residual. Scale bar, 1 μm. (b,c) FMWH2 of Gaussian fit over time. Fast diffusional spread during and after stimulation, followed by confinement (b) and reclustering (c). Mean±s.e.m.; n=5 independent experiments.
Figure 3
Figure 3. Diffusional spread, confinement, and reclustering of newly exocytosed SV proteins.
(a,b) FMWH2 of Gaussian fit over time shows spreading, confinement (a), and reclustering (b) of Syb2-, Syt1- and Syp-pHluorin in surface-eclipsed stimulated (40 AP, 20 Hz) neurons imaged at 400 ms frame−1. (c–e) Maximal spread (c), last time point (d) and half-life t1/2 of reclustering (e). (f) Measured Syb2-pHluorin F increase used to model-free diffusion of successively fusing SVs. (g) Modelled intensity distributions from successive SV fusions and sum distribution. (h) Best-fit of model and experimentally determined FMWH2. (i) Diffusion coefficients (D) for Syb2, Syt1, Syp. Mean±s.e.m.; independent experiments: Syb2 (n=5), Syt1 (n=3), Syp (n=3). Statistical analysis was done by two-tailed unpaired t-test (c-e, i) or two-way analysis of variance with repeated measures (h time points 0.8–2 s). NS: P>0.05.
Figure 4
Figure 4. Spread and confinement of Syb2 are modulated by its association with AP180 or CALM.
(a,b) FMWH2 of Gaussian fit over time shows spreading, confinement (a), and reclustering (b) of Syb2-pHluorin WT or M46A in surface-eclipsed stimulated (40 AP, 20 Hz) neurons imaged at 400 ms frame−1. (c–e) Maximal spread (c), last time point (d) and half-life t1/2 of reclustering (e). (f,g) Best-fit of model and experimentally determined FMWH2 for Syb2 WT and M46A. (h) Diffusion coefficients for Syb2 WT and M46A. Mean±s.e.m.; n=3 independent experiments. Statistical analysis was done by two-tailed paired t-test (c–e,h) or two-way analysis of variance with repeated measures (f,g) time points 0.8–2 s). **P<0.01, *P<0.05, NS: P>0.05.
Figure 5
Figure 5. AP180 and CALM modulate spread and confinement of newly exocytosed Syb2.
(a,b) FMWH2 of Gaussian fit over time shows spreading, confinement (a) and reclustering (b) of Syb2-pHluorin in surface-eclipsed hippocampal neurons lacking AP180 (KO) and depleted of CALM (knockdown (KD)) or corresponding WT control neurons treated with scrambled (Scr) shRNA (stimulation: 40 AP, 20 Hz). (c–e) Maximal spread (c), last time point (d) and half-life t1/2 of reclustering (e). Mean±s.e.m.; n=3 independent experiments). Statistical analysis was done by two-tailed paired t-test. **P<0.01, *P<0.05, NS: P>0.05.
Figure 6
Figure 6. Newly exocytosed proteins co-cluster with endocytic proteins in the periactive zone.
(a) 3D-Structured Illumination Microscopy (3D-SIM) analysis of Bassoon (Bass), AP180 and clathrin heavy chain (CHC) in stimulated (40 APs, 20 Hz) or non-stimulated hippocampal neurons. Scale bar, 1 μm; zoom, 500 nm. (b) Schematic for immunolabeling of the newly exocytosed SV protein pool (NEP). (c–e) SD-dSTORM imaging of newly exocytosed SV proteins (stimulation: 40 AP, 20 Hz) in hippocampal neurons expressing Syb2-c-myc. (c) 2-colour SD-dSTORM analysis of NEP and AP180, overlaid with wide-field image of Bassoon. Scale bar, 1 μm; zoom, 100 nm. (d) Ripley's L(r)-r function shows sub-synaptic clustering of NEP and of AP180, indicated by positive L(r)-r values with a maximum at radii of about 100 nm (NEP) and 200 nm (AP180). Mean±s.e.m.; n=31 synapses. Dotted lines represent upper envelopes of complete spatial randomness (CSR). (e) Radial intensity profiles of NEP and AP180 signals centred on the maxima of sub-synaptic NEP clusters show colocalization of both proteins (mean±s.e.m.; n=47 synapses). (f) k-Nearest neighbour analysis (k=10) of NEP and AP180 showing a maximum at 25 nm. The red line represents results after toroidal shift of one channel. Values above the red line indicate colocalization of NEP and AP180 (mean±s.e.m.; n=31 synapses).
Figure 7
Figure 7. Model of protein diffusion, confinement and reclustering.
Schematic model of SV protein confinement and reclustering and the role of the endocytic machinery: following rapid diffusion (WT, top) newly exocytosed SV proteins remain confined within the presynaptic bouton (WT, middle), aided by their association with the endocytic machinery. Confinement is followed by SV protein reclustering within the periactive zone (WT, bottom). In the absence of a functional endocytic machinery diffusion of newly exocytosed SV proteins is accelerated (bold arrows) and both confinement and the extent, but not the rate, of reclustering are compromised.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dittman J. & Ryan T. A. Molecular circuitry of endocytosis at nerve terminals. Annu. Rev. Cell Dev. Biol. 25, 133–160 (2009). - PubMed
    1. Haucke V., Neher E. & Sigrist S. J. Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat. Rev. Neurosci. 12, 127–138 (2011). - PubMed
    1. Saheki Y. & De Camilli P. Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645 (2012). - PMC - PubMed
    1. Kononenko N. L. & Haucke V. Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85, 484–496 (2015). - PubMed
    1. Royle S. J. & Lagnado L. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules. Traffic 11, 1489–1497 (2010). - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-