Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans
Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans
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DOI：
10.1016/j.jneumeth.2005.11.016
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发表时间：
2006-06-30
影响因子：
3
通讯作者：
Schafer, WR
中科院分区：
文献类型：
--
作者：
Gottschalk, A;Schafer, WR
为了研究突触中特异性受体和其他细胞表面蛋白的丰度，仅在插入质膜时对这些蛋白进行特异性标记将是有利的。我们描述了一种方法，允许荧光标记细胞表面蛋白在活的和行为的动物，即在线虫秀丽隐杆线虫。左旋唑敏感烟碱乙酰胆碱受体（nAChR）亚基等蛋白在其细胞外c末端被标记为表位，并将针对这些标记的荧光抗体注射到体液中。这些抗体特异性标记肌肉和神经元细胞表面的突触区域，同时使用不同的标签促进了共定位研究。通过证明ric3和unc-38的突变会导致对胆碱能激动剂的行为抗性，从而强烈降低甚至消除nAChR细胞表面的表达，可以对荧光进行量化。我们还使用这种方法可视化了细胞外外膜蛋白ODR-2，该蛋白与调节脊椎动物神经元nachr的神经毒素样蛋白有关。同样地，当注射荧光a-bungaro毒素时，与咽和神经系统中的某些nachr结合。这表明，从理论上讲，任何具有足够亲和力的分子相互作用都可以用于特异性标记活线虫的细胞表面结构。(c) 2005 Elsevier B.V.版权所有
To study the abundance of specific receptors and other cell surface proteins at synapses, it would be advantageous to specifically label these proteins only when inserted in the plasma membrane. We describe a method that allows to fluorescently label cell surface proteins in live and behaving animals, namely in the nematode Caenorhabditis elegans. Proteins such as subunits of the levamisole sensitive nicotinic acetylcholine receptor (nAChR) were epitope-tagged at their extracellular C-termini, and fluorescent antibodies against those tags were injected into the body fluid. These antibodies specifically labelled synaptic regions on the cell surface of muscles and neurons, and simultaneous use of different tags facilitated co-localization studies. Quantification of the fluorescence is possible, as verified by demonstrating that mutations in ric-3 and unc-38, which cause behavioural resistance to cholinergic agonists, strongly reduce or even abolish nAChR cell surface expression. We also used this method to visualize the extracellular peripheral membrane protein ODR-2, which is related to a neurotoxin-like protein regulating vertebrate neuronal nAChRs. Likewise, fluorescent a-bungarotoxin, when injected, bound to certain nAChRs in the pharynx and the nervous system. This showed that, theoretically, any molecular interaction of sufficient affinity may be used to specifically label cell surface structures in live nematodes. (c) 2005 Elsevier B.V. All rights reserved.