Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling
Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling
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DOI：
10.1002/cne.20633
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发表时间：
2005-08-22
影响因子：
2.5
通讯作者：
Shigemoto, R
中科院分区：
文献类型：
--
作者：
Hagiwara, A;Fukazawa, Y;Shigemoto, R
突触小泡释放发生在称为突触前活性区 (AZ) 的特殊膜区域。几种膜蛋白参与囊泡释放过程，例如对接、引发和胞吐融合。活性区细胞基质 (CAZ) 蛋白是 AZ 的结构成分，并且高度集中在其中。然而，其他释放相关蛋白的定位，包括目标可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (t-SNARE) 蛋白，在 AZ 中尚未得到很好的证实。在这里，我们使用十二烷基硫酸钠消化的冷冻断裂复制标记（SDS-FRL）定量分析海马 CA3 区域中 CAZ 和 t-SNARE 蛋白的分布。复制膜中的 AZ 通过 CAZ 蛋白（CAZ 相关结构蛋白 [CAST] 和巴松管）的免疫标记来鉴定。在苔藓纤维和缔合/连合 (A/C) 纤维突触前末端的 P 面上发现了这些蛋白质的免疫金颗粒簇。与 CAST 共同标记揭示了 AZ 以及 AZ 周围突触外膜 (SZ) 中 t-SNARE 蛋白突触蛋白和 25 kDa 突触体相关蛋白 (SNAP-25) 的分布。定量分析表明，AZ 中 CAST 的免疫颗粒密度比 SZ 中高 100 倍以上，而 A/C 和苔藓纤维末端中 AZ 和 SZ 之间突触蛋白和 SNAP-25 的免疫颗粒密度没有显着差异。这些结果支持 t-SNARE 蛋白参与 AZ 的胞吐融合以及 CAST 在 AZ 膜结构域特化中的作用。 (C) 2005 Wiley-Liss, Inc.
Synaptic vesicle release occurs at a specialized membrane domain known as the presynaptic active zone (AZ). Several membrane proteins are involved in the vesicle release processes such as docking, priming, and exocytotic fusion. Cytomatrix at the active zone (CAZ) proteins are structural components of the AZ and are highly concentrated in it. Localization of other release-related proteins including target soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (t-SNARE) proteins, however, has not been well demonstrated in the AZ. Here, we used sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) to analyze quantitatively the distribution of CAZ and t-SNARE proteins in the hippocampal CA3 area. The AZ in replicated membrane was identified by immunolabeling for CAZ proteins (CAZ-associated structural protein [CAST] and Bassoon). Clusters of immunogold particles for these proteins were found on the P-face of presynaptic terminals of the mossy fiber and associational/commissural (A/C) fiber. Co-labeling with CAST revealed distribution of the t-SNARE proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) in the AZ as well as in the extrasynaptic membrane surrounding the AZ (SZ). Quantitative analysis demonstrated that the density of immunoparticles for CAST in the AZ was more than 100 times higher than in the SZ, whereas that for syntaxin and SNAP-25 was not significantly different between the AZ and SZ in both the A/C and mossy fiber terminals. These results support the involvement of the t-SNARE proteins in exocytotic fusion in the AZ and the role of CAST in specialization of the membrane domain for the AZ. (C) 2005 Wiley-Liss, Inc.