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

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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能够对磷脂酶 D 活性进行成像

化学成像技术在剖析信号转导途径的时空调节方面发挥了重要作用。磷脂酶 D (PLD) 酶通过水解磷脂酰胆碱产生多效性脂质第二信使磷脂酸，从而影响细胞信号传导。这种脂质信号如何导致如此多样化的生理和病理信号结果仍然是一个谜，这在很大程度上是由于缺乏合适的工具来可视化其在细胞内产生的空间和时间动态。在这里，我们报告了一种在活细胞中通过 PLD 酶对磷脂酸合成进行成像的化学方法。我们的方法利用了 PLD 的酶促混杂性，我们证明 PLD 可以接受叠氮基醇作为转磷脂酰化反应中的报告基因。然后使用菌株促进的叠氮-炔环加成对所得叠氮脂质进行荧光标记，从而实现带有活性 PLD 酶的细胞膜的可视化。我们的方法称为 IMPACT（通过转磷脂酰化作用使用可点击的醇成像磷脂酶 D活性），揭示了预期和意外位置的基础和刺激 PLD 活性池。此外，我们还揭示了细胞和亚细胞水平上 PLD 活性的显着异质性。总的来说，我们的研究强调了使用化学工具以高空间和时间分辨率直接可视化活性信号酶子集的重要性。

更新日期：2017-10-04

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