Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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DOI：
10.3791/57690
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发表时间：
2018-06-01
影响因子：
1.2
通讯作者：
Busch, Karin B.
中科院分区：
文献类型：
--
作者：
Appelhans, Timo;Beinlich, Felix R. M.;Busch, Karin B.
关于蛋白质在细胞亚区室中的定位的知识对于理解它们的特定功能至关重要。在这里，我们提出了一种超分辨率技术，允许通过生成这些蛋白质的定位和跟踪图来确定蛋白质可访问的微区室。此外，通过多色定位显微镜，定位和跟踪不同的亚区室的蛋白质的配置文件，同时获得。该技术是活细胞特异性的，并且基于单个移动的膜蛋白的重复成像。感兴趣的蛋白质在基因上与特定的，所谓的自我标记标签融合。这些标签是以共价方式与底物反应的酶。与这些底物缀合的是荧光染料。酶标记的蛋白质与荧光标记的底物反应产生标记的蛋白质。这里，四甲基罗丹明（TMR）和硅罗丹明（SiR）用作附着于酶底物的荧光染料。通过使用pM至nM范围内的底物浓度，实现了亚化学计量标记，其产生不同的信号。这些信号以类似于15-27 nm的精度定位。该技术允许单个分子的多色成像，其中颜色的数量受到可用的膜渗透染料和标记酶的库的限制。我们通过确定质量控制酶（Pten）诱导的激酶1（PINK 1）在不同线粒体中的定位来展示该技术的可行性！在其处理过程中与其他膜蛋白的关系。然而，通过单分子FRET或共跟踪对不同标记的单个蛋白质之间的真实物理相互作用的测试受到限制，因为低标记度降低了同时标记两个相邻蛋白质的概率。虽然该技术对于膜隔室中的蛋白质成像是强有力的，但在大多数情况下，它不适合于确定高度移动的可溶性蛋白质的定位。
Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with similar to 15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of selflabeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondria! compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.