Dynamic behavior of the proteins involved in the synaptic vesicle cycle

当动作电位到达神经末梢时，Ca^<2+>-通过开放的电压敏感Ca通道内流触发突触囊泡胞外分泌，从而释放神经递质分子。最近的研究表明突触SNARE蛋白（syntaxin， SNAP-25和VAMP/synaptobrevin）是突触囊泡与突触前膜融合的最小机制。胞质蛋白包括NSF、SNAPs、nSec1/Munc18-1和突触蛋白/络合蛋白调节融合。我们利用果蝇幼虫的神经肌肉连接处来研究这些参与突触囊泡周期及其调控的蛋白质的定位。固定后用免疫荧光法测定这些蛋白的定位。静息状态下，syntaxin沿神经末梢内表面分布，部分区域染色密集。突触的定位与syntaxin非常相似。高K^+处理诱导胞外分泌后，syntaxin定位与静息状态无显著差异。然而，突触分布在细胞质上变得弥漫性，表明其在胞吐过程中或胞吐后的动态运动。Sibire^<ts>突变体在限制性温度下也表现出突触蛋白的动态运动，突触囊泡的内吞作用完全被抑制，而不影响胞吐作用。因此突触蛋白的动态运动与突触囊泡胞外分泌有关，而与胞吞作用无关。酪蛋白激酶II （CKII）抑制剂（5,6-二氯苯并咪唑核糖体和4,5,6,7-四溴三唑）可阻断突触蛋白的运动，但Ca^<2+ b> /CaM激酶II、a激酶或c激酶抑制剂不能阻断突触蛋白的运动，这表明CKII参与了突触蛋白的磷酸化。

When an action potential reaches the nerve terminal, Ca^<2+>-influx through open voltage-sensitive Ca channels triggers the exocytosis of synaptic vesicles, thereby releasing neurotransmitter molecules. Recent studies indicate that synaptic SNARE proteins (syntaxin, SNAP-25 and VAMP/synaptobrevin) are the minimal machinery for the synaptic vesicle fusion with the presynaptic membrane. Cytosolic proteins including NSF,SNAPs,nSec1/Munc18-1 and synaphin/complexin regulate the fusion. We have used the neuromuscular junction of Drosophila larvae to examine the localization of these proteins involved in the synaptic vesicle cycle and its regulation. Localization of these proteins was determined by immunofluorescence after fixation. In resting conditions, syntaxin was distributed along the internal surface of the nerve terminal with some densely stained regions. Synaphin localization was very similar to that of syntaxin. After exocytosis induced by high K^+-treatment, syntaxin localization did not significantly differ from the resting state. However, synaphin distribution became diffuse over the cytoplasm, suggesting its dynamic movement during or after exocytosis. Sibire^<ts> mutant also showed the dynamic movement of synaphin at a restrictive temperature where endocytosis of synaptic vesicles was completely suppressed without affecting exocytosis. Thus the dynamic movement of synaphin is related to synaptic vesicle exocytosis but not to endocytosis. The movement was blocked by the casein kinase II (CKII) inhibitors (5,6-dichlorobenzimidazole riboside and 4,5,6,7-tetrabromotriazole) but not by inhibitors for Ca^<2+>/CaM kinase II, A-kinase or C-kinase, suggesting the involvement of protein phosphorylation by CKII in the movement of synaphin.