Ribeye a-mCherry fusion protein: A novel tool for labeling synaptic ribbons of the hair cell
Ribeye a-mCherry fusion protein: A novel tool for labeling synaptic ribbons of the hair cell
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DOI：
10.1016/j.jneumeth.2010.11.011
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发表时间：
2011-04-30
影响因子：
3
通讯作者：
McDermott, Brian M., Jr.
中科院分区：
文献类型：
--
作者：
West, Megan C.;McDermott, Brian M., Jr.
突触带是突触前细胞，是有效地将听觉信息从毛细胞传输到中枢神经系统所必需的。在毛细胞中，每个电子致密带通过细丝拴住许多突触小泡。带状物质通常与活性区域质膜并列。缺乏适当的工具来可视化带状突触，限制了我们对其发展的了解。在这里，我们介绍了一种方法的设计和实现，以可视化的突触带在毛细胞。这个方案使用了蛋白质Ribeye a的标记版本，它是带状蛋白特有的。我们构建了DNA载体Tg(pvalb3b：ribeye a-mCherry)，在斑马鱼毛细胞中转基因表达融合蛋白Ribeye a-mCherry。融合蛋白定位于毛细胞的基底外侧表面，其图案类似于标记有识别Ribeye蛋白的抗血清的毛细胞。此外，使用该抗血清标记表达Ribeyeα-mCherry的转基因生物，我们证明融合蛋白和抗体相关的荧光信号重叠。此外，标记有融合蛋白的条带位于传入神经末梢的近端。最后，融合蛋白标记斑马鱼不同发育时间点的毛细胞带。这些发现表明，融合蛋白是一种有效的标记活的和固定的毛细胞中的突触带的工具，这将有助于研究突触带的发育和表征具有突触形成缺陷的斑马鱼突变体。(C)2011爱思唯尔B.V.保留所有权利。
Synaptic ribbons are presynaptic cytomatrices that are required for efficient transfer of auditory information from hair cells to the central nervous system. In the hair cell, each electron-dense ribbon tethers numerous synaptic vesicles by fine filaments. The ribbon generally resides juxtaposed to the active zone plasma membrane. A dearth of appropriate tools to visualize the ribbon synapse has limited our knowledge of its development. Here we present the design and implementation of a method to visualize synaptic ribbons in hair cells. This scheme uses a tagged version of the protein Ribeye a, which is specific to ribbons. We generate the DNA construct Tg(pvalb3b:ribeye a-mCherry) to transgenically express the fusion protein Ribeye a-mCherry in zebrafish hair cells. The fusion protein localizes to the basolateral surface of the hair cell with a pattern similar to that of a hair cell labeled with an antiserum that recognizes ribeye proteins. Moreover, using this antiserum to label transgenics that express Ribeye a-mCherry, we demonstrate that the fusion protein and antibody-associated fluorescent signals overlap. In addition, ribbons labeled with the fusion protein are proximal to afferent nerve endings. Finally, the fusion protein labels hair-cell ribbons of zebrafish at different developmental time points. These findings indicate that the fusion protein is an effective tool to label ribbons in live and fixed hair cells, which will make it useful in the study of ribbon synapse development and to characterize zebrafish mutants with defects in synapse formation. (C) 2011 Elsevier B.V. All rights reserved.