A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity
A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity
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DOI：
10.1073/pnas.1903949116
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发表时间：
2019-07-30
影响因子：
11.1
通讯作者：
Baskin, Jeremy M.
中科院分区：
文献类型：
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作者：
Liang, Dongjun;Wu, Kane;Baskin, Jeremy M.
信号转导的保真度要求对信号介质的产生进行时空控制。磷脂酸(PA)是一种多效性脂质第二信使，其作用方式因上游刺激、生物合成来源和生产地点而异。细胞如何调节局部PA的产生以影响不同的信号结果仍然难以捉摸。与其他第二信使不同，PA生物合成的位置不能以亚细胞的精度准确地可视化。在这里，我们描述了一种快速的，化学酶法成像磷脂酶D(PLD)酶生理性产生PA的方法。我们的方法利用了一个显著的发现，即在PLD的脂质底物磷脂酰胆碱的转磷脂酰化反应中，体积大、亲水性的含环辛烯的伯醇可以取代水作为PLD活性部位的亲核剂。通过无需漂洗的反向电子需求Diels-Alder(IEDDA)反应，用含氟四氮试剂标记生成的含反式环辛烯的脂类，使其能够通过共聚焦显微镜实时显示。值得注意的是，最初由佛波酯刺激PLD而在质膜(PM)产生的荧光报告脂通过明显的非囊泡途径而不是内吞作用迅速内化，这表明这一基于活动的成像工具集适用于探索细胞内磷脂运输的机制。相反，通过关注IEDDA反应的最初10个S，我们精确地定位了G蛋白偶联受体和受体酪氨酸激酶信号通路的生理激动剂引发的内源性PLD活性的亚细胞位置。这些工具有望阐明脂类转运途径以及局部PLD信号的生理和病理效应。
The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene-containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD's lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene-containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels-Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.