Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains
Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains
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DOI：
10.1177/2472555219830086
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发表时间：
2019-07-01
期刊：
影响因子：
3.1
通讯作者：
Zweifach, Adam
中科院分区：
文献类型：
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作者：
Yang, Xiuyi Alexander;Zweifach, Adam
在细胞中表达的分子内CFP-YFP荧光共振能量转移（FRET）传感器是强大的研究工具，但在筛选中使用相对较少。我们发现，当细胞在室温下孵育时，CFP-YFP FRET二酰基甘油传感器（DAGR）的表达随时间增加，以评估使用Molecular Devices Spectramax i3x荧光板读取器进行稳健测量的要求。YFP荧光>的表达水平比未转染的细胞高10倍，荧光素酯刺激的FRET比率变化60%或更多，才能持续产生稳健的Z ' >0.5。作为确认这些条件适合筛选的一种手段，我们开发了一种新的多读方案来测定NCI的C1结构域激活的激动剂和拮抗剂的机制集III。16种化合物阻止C1结构域易位。然而，没有阻断磷酯刺激的蛋白激酶C （PKC）活性，使用磷酸化特异性抗体评估- 6实际上刺激PKC活性。对于给定的FRET比率变化，流式细胞术产生更高的Z′，可能是发现拮抗剂的更好方法，因为它允许使用较低的磷酸酯浓度。我们得出结论，在Spectramax i3x平板阅读器上测量的CFP-YFP FRET可以在我们定义的条件下用于筛选。我们改变表达水平和FRET比率的策略可以用于确定在其仪器上进行稳健的基于细胞的分子内CFP-YFP FRET测量所需的条件。
Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z ' > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI's Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody-six actually stimulated PKC activity. Cytometry, which produces higher Z ' for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.