Fig. 5.
Dominant-negative effect of α1BC-terminal fragment on synaptic function. A, The experimental protocol for live labeling of active synapses with synaptotagmin luminal antibodies (STI) was adapted from Matteoli et al. (1992). The hippocampal neuronal cultures were preincubated with STI antibodies for 3 min in the presence of 45 mm KCl and 2 mm Ca2+. After stimulation, STI antibodies were washed out, and neurons were fixed, permeabilized, and stained with rhodamine-conjugated anti-mouse secondary antibody to visualize STI antibody localization. The intraluminal epitope of synaptotagmin is shown as a black box. B, Live labeling for STI (red) followed by fixation and labeling for MAP2 (green). Note that all STI-positive clusters are apposed to cell body and dendrites (MAP2-positive). Scale bar, 20 μm. C, Live staining for STI (left panel, red in merged image) followed by fixation and staining for synapsin (center panel, green on merged image) shows colocalization of STI clusters with presynaptic sites. On average, 93% of STI and synapsin clusters were colocalized (n = 200). Scale bar, 10 μm. D, Live staining for STI (red) of GFP-NC3 (green)-transfected neurons followed by fixation and staining for MAP2 (blue). Axons of transfected cells (green, MAP2-negative) are indicated bylarge arrows. The boxed portion of the image is enlarged in the D′ inset. Note that the intensity of individual STI clusters associated with axons of transfected neurons is reduced when compared with control clusters formed by GFP-NC3-negative axons. Small arrows inD′ indicate synapses of transfected cells. Scale bars:D, 20 μm; D′, 10 μm.E, Live staining for STI (red) of neurons expressing mGFP (green). Note that the intensity of individual STI clusters associated with axons of mGFP-transfected neurons is not affected. Small arrows in Eindicate synapses of transfected cells. Scale bar, 10 μm.F, Quantitative analysis of the intensity of individual clusters of STI (left panel), synapsin (center panel), and PSD-95 (right panel) associated with control (nontransfected) cells and cells transfected with mGFP or GFP-NC3 as indicated. For each transfection, the fluorescence intensity of individual STI, synapsin, and PSD-95 clusters was normalized to the average fluorescence intensity of corresponding clusters observed for control (nontransfected) cells in the same field. The normalized intensities are shown as mean ± SEM (n = number of clusters analyzed). For STI plot: control, n = 309; mGFP, n = 55; GFP-NC3, n = 90 (*p < 0.001 when GFP-NC3 is compared with control and mGFP). For synapsin and PSD-95, n ≥ 75 for controls and n ≥ 25 for mGFP and GFP-NC3.G, Cumulative distribution of intensities of individual STI clusters associated with axons of nontransfected neurons (control, ■) or axons of neurons expressing mGFP (○) and GFP-NC3 (●).H, Effect of GFP-NC3 expression on STI uptake by neurons preincubated with 5 μm ω-GVIA or 5 μmω-MVIIC (n ≥ 25 for each group).