Imaging Interorganelle Phospholipid Transport by Extended Synaptotagmins Using Bioorthogonally Tagged Lipids.

The proper distribution of lipids within organelle membranes requires rapid interorganelle lipid transport, much of which occurs at membrane contact sites and is mediated by lipid transfer proteins (LTPs). Our current understanding of LTP mechanism and function is based largely on structural studies and in vitro reconstitution. Existing cellular assays for LTP function use indirect readouts, and it remains an open question as to whether substrate specificity and transport kinetics established in vitro are similar in cellular settings. Here, we harness bioorthogonal chemistry to develop tools for direct visualization of interorganelle transport of phospholipids between the plasma membrane (PM) and the endoplasmic reticulum (ER). Unnatural fluorescent phospholipid analogs generated by the transphosphatidylation activity of phospholipase D (PLD) at the PM are rapidly transported to the ER dependent in part upon extended synaptotagmins (E-Syts), a family of LTPs at ER-PM contact sites. Ectopic expression of an artificial E-Syt-based tether at ER-mitochondria contact sites results in fluorescent phospholipid accumulation in mitochondria. Finally, in vitro reconstitution assays demonstrate that the fluorescent lipids are bona fide E-Syt substrates. Thus, fluorescent lipids generated in situ via PLD activity and bioorthogonal chemical tagging can enable direct visualization of the activity of LTPs that mediate bulk phospholipid transport at ER-PM contact sites.

Activity-based, bioorthogonal imaging of phospholipase D reveals spatiotemporal dynamics of GPCR-Gq signaling.

Canonically, G-protein-coupled receptor (GPCR) signaling is transient and confined to the plasma membrane (PM). Deviating from this paradigm, the parathyroid hormone receptor (PTHR1) stimulates sustained Gs signaling at endosomes. In addition to Gs, PTHR1 activates Gq signaling; yet, in contrast to the PTHR1-Gs pathway, the spatiotemporal dynamics of the Gq branch of PTHR1 signaling and its relationship to Gs signaling remain largely ill defined. Recognizing that a downstream consequence of Gq signaling is the activation of phospholipase D (PLD) enzymes, we leverage activity-based, bioorthogonal imaging tools for PLD signaling to visualize and quantify the Gq branch of PTHR1 signaling. We establish that PTHR1-Gq signaling is short lived, exclusively at the PM, and antagonized by PTHR1 endocytosis. Our data support a model wherein Gq and Gs compete for ligand-bound receptors at the PM and more broadly highlight the utility of bioorthogonal tools for imaging PLDs as probes to visualize GPCR-Gq signaling.

IMPACT: Imaging phospholipase d activity with clickable alcohols via transphosphatidylation.

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.

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.