Synaptophysin is required for synaptobrevin retrieval during synaptic vesicle endocytosis

Abstract

The integral synaptic vesicle (SV) protein synaptophysin forms ∼10% of total SV protein content, but has no known function in SV physiology. Synaptobrevin (sybII) is another abundant integral SV protein with an essential role in SV exocytosis. Synaptophysin and sybII form a complex in nerve terminals, suggesting this interaction may have a key role in presynaptic function. To determine how synaptophysin controls sybII traffic in nerve terminals, we used a combination of optical imaging techniques in cultures derived from synaptophysin knock-out mice. We show that synaptophysin is specifically required for the retrieval of the pH-sensitive fluorescent reporter sybII-pHluorin from the plasma membrane during endocytosis. The retrieval of other SV protein cargo reporters still occurred; however, their recapture proceeded with slower kinetics. This slowing of SV retrieval kinetics in the absence of synaptophysin did not impact on global SV turnover. These results identify a specific and selective requirement for synaptophysin in the retrieval of sybII during SV endocytosis and suggest that their interaction may act as an adjustable regulator of SV retrieval efficiency.

Figure 1.

SybII-pHluorin is stranded on the cell surface in synaptophysin knock-out neurons. Representative images show the differential expression of sybII-pHluorin in either wild-type (WT, A) or synaptophysin KO (B) cultures. Scale bar represents 15 μm. C, Bar graph displays the coefficient of variation of sybII-pHluorin fluorescence along axons of WT, KO, and rescued neurons in alkaline buffer. Data are presented as ±SEM, n = 6 for WT, n = 5 for KO, n = 4 for rescue, ***p < 0.001 one-way ANOVA for KO against both WT and rescue. D, Representative image of the distribution of endogenous sybII using immunofluorescence in KO cultures transfected with synaptophysin-mCerulean. SybII distribution in the rescued neuron is highlighted by arrowheads (all other neurons in field of view are KO). False coloring indicates the intensity of sybII immunolabeling (calibration bar displayed). Scale bar represents 10 μm. E, F, Representative traces displaying the proportion of sybII-pHluorin expressed on the cell surface in either WT (blue circles, E) or KO (red circles, F) neurons. SybII-pHluorin fluorescence is normalized to 100% in alkaline buffer (green bars) and to 0% in acidic buffer (yellow bars). Arrows indicate either surface fraction (neutral pH − acidic pH, SF) or total sybII-pHluorin (alkaline pH − acidic pH, Tot).

Figure 2.

Synaptophysin is required for sybII-pHluorin retrieval. SybII-pHluorin transfected wild-type (WT, blue circles), synaptophysin KO (red circles), or KO neurons expressing synaptophysin-mCerulean (Rescue, purple circles) were stimulated with a train of 200 action potentials (10 Hz, indicated by bar). Averaged traces for either WT and KO (A) or KO and Rescue (B) are displayed ±SEM, n = 10 for WT, n = 8 for KO, n = 9 for rescue, ***p < 0.001 two-way ANOVA for KO against both WT and rescue.

Figure 3.

Synaptophysin is not required for retrieval of other SV protein cargo. Wild-type (WT, blue circles) and synaptophysin KO (red circles) cultures were transfected with either vGLUT-pHluorin (A) or syt-pHluorin (B). Cultures were stimulated with a train of 200 action potentials (10 Hz, indicated by bar). Averaged traces are ±SEM, n = 8 for WT vGLUT-pHluorin, n = 4 for all other conditions, ***p < 0.001 two-way ANOVA for WT against KO for both vGLUT-pHluorin and syt-pHluorin.

Figure 4.

Global SV turnover is normal in synaptophysin knock-out neurons. A, Synaptophysin KO cultures were transfected with synaptophysin-mCerulean (Rescue) and loaded with FM2-10 (100 μm) using a train of 900 action potentials (10 Hz). FM2-10 was applied either during and after stimulation to load the total SV recycling pool or only after stimulation (to load the poststimulus recycling SV pool). B, Representative images display synaptophysin-mCerulean transfected neuron (left panel), FM2-10 loading (middle panel) and merged image (right panel). Loading in KO neurons indicated by white arrowheads and in rescued neurons by green arrowheads. Scale bar represents 20 μm. C, D, Representative traces display dye unloading in KO (red circles) and rescued (purple circles) neurons evoked by 900 action potentials (10 Hz, indicated by bar) in the same field of view for either the total recycling SV pool (C) or the poststimulus SV recycling pool (D).