Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity,ACS Central Science

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Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity
ACS Central Science ( IF 12.7 ) Pub Date : 2017-10-04 00:00:00 , DOI: 10.1021/acscentsci.7b00222
Timothy W. Bumpus ₁ , Jeremy M. Baskin ₁

Affiliation

Chemical imaging techniques have played instrumental roles in dissecting the spatiotemporal regulation of signal transduction pathways. Phospholipase D (PLD) enzymes affect cell signaling by producing the pleiotropic lipid second messenger phosphatidic acid via hydrolysis of phosphatidylcholine. It remains a mystery how this one lipid signal can cause such diverse physiological and pathological signaling outcomes, due in large part to a lack of suitable tools for visualizing the spatial and temporal dynamics of its production within cells. Here, we report a chemical method for imaging phosphatidic acid synthesis by PLD enzymes in live cells. Our approach capitalizes upon the enzymatic promiscuity of PLDs, which we show can accept azidoalcohols as reporters in a transphosphatidylation reaction. The resultant azidolipids are then fluorescently tagged using the strain-promoted azide–alkyne cycloaddition, enabling visualization of cellular membranes bearing active PLD enzymes. Our method, termed IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation), reveals pools of basal and stimulated PLD activities in expected and unexpected locations. As well, we reveal a striking heterogeneity in PLD activities at both the cellular and subcellular levels. Collectively, our studies highlight the importance of using chemical tools to directly visualize, with high spatial and temporal resolution, the subset of signaling enzymes that are active.

中文翻译：

可点击的底物模拟物使磷脂酶D活性成像

化学成像技术在剖析信号转导途径的时空调节中发挥了重要作用。磷脂酶D（PLD）酶通过磷脂酰胆碱的水解产生多效脂质第二信使磷脂酸，从而影响细胞信号传导。很大程度上由于缺乏合适的工具来可视化其在细胞内产生的时空动态，这一个脂质信号如何导致如此多样的生理和病理信号转导结果仍是一个谜。在这里，我们报告一种化学方法，用于通过活细胞中的PLD酶对磷脂酸合成进行成像。我们的方法利用了PLD的酶促混杂性，我们证明其可以接受叠氮醇作为转磷脂酰化反应的报告分子。然后使用应变促进的叠氮化物-炔烃环加成反应，对生成的叠氮脂质进行荧光标记，从而使带有活性PLD酶的细胞膜可视化。我们的方法称为IMPACT（林老化P hospholipase d甲ctivity与Ç lickable甲lcohols经由Ť ransphosphatidylation），揭示了基底的池和在预期和非预期的位置刺激PLD活性。同样，我们揭示了在细胞和亚细胞水平上PLD活性的显着异质性。总的来说，我们的研究突出了使用化学工具以高时空分辨率直接可视化活跃的信号传导酶子集的重要性。

更新日期：2017-10-25

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