Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity

Abstract

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.

A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity

“…This low temporal resolution makes it nonideal for ascertaining the precise, subcellular localizations of active PLD enzymes because lipid diffusion and trafficking can occur on more rapid time scales (43)(44)(45). In particular, we noted the lack of PM-derived IMPACT fluorescence upon stimulation with phorbol 12-myristate 13-acetate (PMA), which is thought to directly stimulate PKC (23,33) and result in translocation of PLD1 to the PM (19).…”

“…2A). This method, termed IMPACT for Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation, enabled fluorescent labeling of cellular membranes within live cells, using SPAAC tagging, as a function of their PLD activities (33).…”

“…Our approach relies on the ability of endogenous PLD enzymes to accept unnatural substrate analogs that are rapidly converted, in sequential enzyme-mediated and bioorthogonal tagging steps, into fluorescent lipids whose subcellular location faithfully reports on the location(s) of active PLD enzymes. The major advances reported here address the key deficiency of earlier versions of this method, namely its poor temporal resolution that, because of trafficking of the fluorescent lipid reporters before imaging, prevented the visualization of the true localizations of PLD activation (32,33).…”

Spatiotemporal control of phosphatidic acid signaling with optogenetic, engineered phospholipase Ds

“…A subsequent click chemistry tagging step with an alkyne-quaternary ammonium tag (Alk-QA) enables highly sensitive identification and quantification of the phosphatidyl alcohol species (varying by acyl tail composition) by using liquid chromatography-mass spectrometry (LC-MS) analysis. Importantly, the identities of the phosphatidyl alcohols match those of the natural PA species produced by the PLDs (Bumpus and Baskin, 2017;Brown et al, 2007). Alternatively, click chemistry tagging using a bicyclononyne-fluorophore conjugate (e.g., bicyclononane BODIPY fluorophore [BCN-BODIPY]) generates fluorescent phosphatidyl alcohols, which can be visualized by fluorescence microscopy in live cells or quantified by HPLC from lipid extracts ( Fig.…”

Mapping Enzyme Activity on Tissue by Functional Mass Spectrometry Imaging

“…High‐resolution spatially resolved assays of enzyme activity would therefore present a powerful tool for elucidating or diagnosing the causes of a range of diseases . Targeted imaging of enzyme activity using chemically labelled probes or substrates has been demonstrated; however, no single imaging modality can routinely map the distribution of enzymes on tissue while simultaneously quantifying their catalytic efficacy and selectivity.…”