一种实时点击化学成像方法揭示了磷脂酶 D 活性的刺激特异性亚细胞位置。

IF 0.7 Q3 POLITICAL SCIENCE
RUSI Journal Pub Date : 2019-07-30 Epub Date: 2019-07-16 DOI:10.1073/pnas.1903949116
Dongjun Liang, Kane Wu, Reika Tei, Timothy W Bumpus, Johnny Ye, Jeremy M Baskin
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引用次数: 0

摘要

信号转导的保真度要求对信号物质的产生进行时空控制。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的不同而不同。细胞如何调节局部磷脂酸的产生以实现不同的信号转导结果仍是一个谜。与其他第二信使不同,PA 的生物合成位点无法以亚细胞精度精确观察。在这里,我们描述了一种快速的化学方法,用于对磷脂酶 D(PLD)产生的生理性 PA 进行成像。在 PLD 的脂质底物磷脂酰胆碱的反磷脂酰化反应中,体积大、亲水性强的反环辛烯伯醇可取代水成为 PLD 活性位点的亲核物,我们的方法正是利用了这一重大发现。通过免冲洗、逆电子需求 Diels-Alder (IEDDA) 反应,产生的含反式环辛烯的脂质被荧光四嗪试剂标记,从而可通过共聚焦显微镜实时观察。令人震惊的是,在光甘油酯刺激 PLD 的诱导下,最初在质膜(PM)上产生的荧光报告脂质通过明显的非囊泡途径而不是内吞途径迅速内化,这表明这种基于活性的成像工具集可用于探测细胞内磷脂的运输机制。通过聚焦 IEDDA 反应的最初 10 秒钟,我们精确定位了 G 蛋白偶联受体和受体酪氨酸激酶信号生理激动剂激发的内源性 PLD 活性的亚细胞位置。这些工具有望揭示脂质贩运途径以及局部 PLD 信号的生理和病理效应。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity.

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.

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来源期刊
RUSI Journal
RUSI Journal POLITICAL SCIENCE-
CiteScore
1.40
自引率
10.00%
发文量
39
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