Identifying individual scaffolding molecules in the postsynaptic density.
Identifying individual scaffolding molecules in the postsynaptic density.
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识别突触后密度中的单个支架分子。
DOI：
10.1017/s1431927608085449
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Reese,Ts
中科院分区：
文献类型：
--
作者：
Chen,X;Winters,C;Azzam,R;Crocker,V;Li,X;Galbraith,J;Leapman,Rd;Reese,Ts
突触后密度（PSD）是锚定在神经细胞突触后膜上的大分子信号传导组件。它包含受体、支架和信号分子以及细胞骨架元件。分离的 PSD 的质谱分析揭示了数百种蛋白质 [1]。突触后信号通路在控制突触强度方面发挥着重要作用，并最终可能成为学习和记忆机制的基础。最近通过 EM 断层扫描解析了培养海马棘中 PSD 的基本分子组织。三维重建显示水平支架分子的正交组装与垂直定向的细丝相关，而垂直定向的细丝又与主要的跨膜结构相关[2]。现在核心单个蛋白质可以通过断层扫描可视化，因此需要方法来识别它们。结构可以与特定细胞区室中蛋白质的预测大小和形状相匹配，但直接识别标签会让人放心。在这里，我们通过将电子显微镜断层扫描与免疫标记相结合，对通过高压冷冻、冷冻替代和冷冻包埋制备的培养的大鼠海马神经元进行标记，标记了突触后密度中的关键支架分子 PSD-95 [3]。在 3 mm 金室中的星形胶质细胞上生长三周的解离培养神经元最初固定在 4% 多聚甲醛中，并与 PSD-95 的一抗一起孵育，然后与 Nanogold 偶联的二抗一起孵育。经过短暂（6-8 分钟）的银增强后，通过高压冷冻制备细胞，并正面切割厚度 100-200 nm 的 Lowicryl HM-20 切片，拍照用于双轴断层扫描。我们发现 PSD-95 分子可以在断层扫描重建中单独标记。我们的方法可以广泛应用于识别神经元细胞中大型蛋白质复合物中的单个分子。
The postsynaptic density (PSD) is a macromolecular signaling assembly anchored in the postsynaptic membrane of nerve cells. It contains receptors, scaffold and signaling molecules, and cytoskeletal elements. Mass spectrometry of isolated PSDs reveals several hundred protein species [1]. The postsynaptic signal pathways have a major role in controlling synaptic strength, and ultimately may underlie the mechanisms involved in learning and memory. The fundamental molecular organization of the PSD in cultured hippocampal spines has recently been resolved by EM tomography. Three-dimensional reconstructions show an orthogonal assembly of horizontal scaffolding molecules associated with the vertically oriented filaments that in turn associate with major transmembrane structures [2]. Now that core individual proteins can be visualized with tomography, methods are needed to identify them. Structures can be matched with predicted sizes and shapes of proteins at specific cellular compartment, but a direct identifying label would be reassuring. Here, we label a key scaffolding molecule in the postsynaptic density, PSD-95 [3], by combining electron microscopic tomography with immunolabeling on cultured rat hippocampal neurons prepared by high pressure freezing, freeze substitution and cryoembedding. Dissociated cultured neurons grown on astrocytes in a 3 mm gold chamber for three weeks were initial fixed in 4% paraformaldehyde and incubated with primary antibody to PSD-95, followed by a Nanogold conjugated secondary antibody. After a short (6-8 min) silver enhancement, the cells underwent preparation by high pressure freezing, and Lowicryl HM-20 sections of thickness 100-200 nm were cut en face, photographed for dual axis tomography. We find that PSD-95 molecules can be individually labeled in the tomographic reconstructions. Our methods could have broad applications for identifying individual molecules in large protein complexes in neuronal cells.