Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters
Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters
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DOI：
10.1074/jbc.m112.353250
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发表时间：
2012-08-03
影响因子：
4.8
通讯作者：
Ashery, Uri
中科院分区：
文献类型：
--
作者：
Bar-On, Dana;Wolter, Steve;Ashery, Uri
来自可溶性 SNARE（N-乙基马来酰亚胺敏感因子附着蛋白受体）家族的关键突触蛋白等，在细胞质膜上组织成包含数十到数百个蛋白质拷贝的簇。然而，由于成像和分析工具的限制，蛋白质在簇或单分子中的确切膜分布、簇内分子的组织以及簇机制尚不清楚。重点关注 Syntaxin 1 和 SNAP-25，我们实施了直接随机光学重建显微镜与定量聚类算法，以展示一种以单分子精度探索 PC12 细胞膜上聚集和非聚集分子分布的新方法。直接随机光学重建显微镜图像首次揭示了孤立的突触蛋白/SNAP-25 分子和小簇以及较大的簇。非簇突触蛋白或 SNAP-25 分子主要集中在与其自身簇相邻的区域。在团簇中，分子的密度从致密的团簇核心向外围逐渐减小。我们进一步检测到包含多个密度梯度的大簇。这表明一些簇是由几个簇统一形成的，这些簇保留了它们的原始组织或重新组织成一个单元。尽管 Syntaxin 和 SNAP-25 具有一些共同的分布特征，但它们的簇彼此明显不同。 SNAP-25 星团明显更大、更椭圆且密度更低。最后，本研究建立了用于分析基于单分子的超分辨率成像数据的方法工具，并为揭示膜蛋白组织的新水平铺平了道路。
Key synaptic proteins from the soluble SNARE (N-ethylma-leimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.