Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853.
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853.
Proc Natl Acad Sci U S A. 2019 Jul 30;116(31):15453-15462. doi: 10.1073/pnas.1903949116. Epub 2019 Jul 16.
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 -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 -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.
信号转导的保真度需要时空控制信号转导剂的产生。磷脂酸 (PA) 是一种多效脂质第二信使，其作用模式因上游刺激、生物合成来源和产生部位而异。细胞如何调节 PA 的局部产生以产生不同的信号转导结果仍然难以捉摸。与其他第二信使不同，PA 生物合成的部位不能以亚细胞精度准确可视化。在这里，我们描述了一种快速的化学酶促方法，用于成像磷脂酶 D (PLD) 酶的生理 PA 产生。我们的方法利用了一个惊人的发现，即大体积、亲水性的环辛烯含伯醇可以取代水作为 PLD 活性部位中 PLD 的脂质底物磷脂酰胆碱的转磷酸化反应中的亲核试剂。所得的环辛烯含脂质通过无冲洗的逆电子需求 Diels-Alder (IEDDA) 反应与荧光四嗪试剂标记，从而可以通过共焦显微镜实时立即可视化。引人注目的是，佛波酯刺激 PLD 产生的质膜 (PM) 上最初产生的荧光报告脂质通过明显的非胞吐途径而不是胞吞作用被迅速内化，这表明这种基于活性的成像工具包可用于研究细胞内磷脂转运的机制。通过将重点放在 IEDDA 反应的最初 10 秒上，我们准确地确定了生理激动剂诱导的内源性 PLD 活性的亚细胞位置，这些激动剂激活 G 蛋白偶联受体和受体酪氨酸激酶信号。这些工具有望揭示脂质运输途径以及局部 PLD 信号的生理和病理效应。
