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image/svg+xml Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao Closed Access logo, derived from PLoS Open Access logo. This version with transparent background. http://commons.wikimedia.org/wiki/File:Closed_Access_logo_transparent.svg Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao https://doi.org/10.1...arrow_drop_down
image/svg+xml Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao Closed Access logo, derived from PLoS Open Access logo. This version with transparent background. http://commons.wikimedia.org/wiki/File:Closed_Access_logo_transparent.svg Jakob Voss, based on art designer at PLoS, modified by Wikipedia users Nina and Beao
https://doi.org/10.1016/bs.mie...
Part of book or chapter of book . 2020 . Peer-reviewed
License: Elsevier TDM
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IMPACT: Imaging phospholipase d activity with clickable alcohols via transphosphatidylation

Authors: Timothy W. Bumpus; Dongjun Liang; Jeremy M. Baskin;

IMPACT: Imaging phospholipase d activity with clickable alcohols via transphosphatidylation

Abstract

Phospholipase Ds (PLDs) are multifunctional and disease-relevant enzymes operating at the center of phospholipid metabolism and signaling. Physiologically, they hydrolyze abundant phospholipids into phosphatidic acid (PA), a potent lipid second messenger and central biosynthetic intermediate. Given the pleiotropic nature of PA, the multiple locations of PLD activity within single cells, and differences in PLD activities across cell types in vivo, tools with spatiotemporal precision are urgently needed to dissect the signaling functions of PLDs. Here, we describe a toolset for visualizing and quantifying cellular PLD activity with high spatial and temporal resolution. Our approach capitalizes on the ability of PLDs to catalyze transphosphatidylation reactions with exogenous alcohols to generate phosphatidyl alcohols, lipids whose location and abundance report on the extent of PLD-mediated PA synthesis. Our key innovation is to employ functionalized, "clickable," alcohols as PLD substrates, which enables subsequent tagging of the resultant phosphatidyl alcohols with fluorophores or other functional probes for detection via highly selective click chemistry reactions. In this chapter, we describe this method, termed IMPACT (Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation), which can be coupled to downstream analysis by fluorescence microscopy, flow cytometry, HPLC, or mass spectrometry. We describe two variants of IMPACT, one with greater sensitivity, for detecting PLD activity at single-cell and population levels, and one with greater spatiotemporal resolution ("real-time," or RT-IMPACT), for accurately visualizing PLD activity at the subcellular, individual-organelle level. Together, IMPACT represents a major advance in our ability to dissect PLD-mediated PA signaling in native biological settings.

Related Organizations
Keywords

Alcohols, Phospholipase D, Phosphatidic Acids, Second Messenger Systems, Signal Transduction

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    13
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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
13
Top 10%
Average
Top 10%