Imaging and Editing the Phospholipidome

Abstract

Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.

Figure 1.

Overview of mammalian phospholipid metabolism. (A) Cartoon structure of a typical lipid bilayer, highlighting the chemical structure of glycerophospholipids. R₁ and R₂ denote fatty acyl tails at the sn-1 and sn-2 positions that can be saturated, monounsaturated, or polyunsaturated, and a phosphate-linked headgroup is located at the sn-3 position. (B) Simplified metabolic pathway showing biosynthesis and interconversion of the major glycerophospholipids and selected roles in signaling.

Figure 2.

Small-molecule and engineered enzyme-based tools for optical control of PA signaling. (A) Isomerization of the photoswitchable PA analogues AzoPA and dAzoPA (dAzoPA is shown) from the trans configuration to the cis configuration by 365 nm light leads to stimulation of mTOR signaling and suppression of Hippo signaling. The trans configuration can be regenerated by exposure to 450 nm light. (B) Optogenetic phospholipase Ds (optoPLDs) enable light-dependent generation of PA by recruitment of a PLD enzyme from Streptomyces sp. PMF to a desired organelle membrane mediated by CRY2–CIBN heterodimerization, followed by PLD-catalyzed hydrolysis of PC to generate PA.

Figure 3.

Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). (A) Activity-based directed evolution of superPLDs with higher catalytic activities in mammalian cells using error-prone PCR and an IMPACT-based enrichment strategy. (B) The concept underlying IMPACT. PLDs can catalyze two reactions of PC: hydrolysis to form PA and transphosphatidylation with primary alcohols to form phosphatidyl alcohols. By using transphosphatidylation with bioorthogonal alcohols such as 3-azido-1-propanol followed by tagging with a bicyclononyne (BCN)-BODIPY fluorophore via the strain-promoted azide–alkyne cycloaddition (SPAAC) bioorthogonal reaction, IMPACT enables generation of fluorescent phosphatidyl alcohol lipids as reporters of cellular PLD activity. (C) Platform for discovery of new activators and inhibitors of mammalian PLD signaling by coupling pooled, genome-wide CRISPR interference (CRISPRi) screening with IMPACT labeling, fluorescence-activated cell sorting (FACS) enrichment, and short guide RNA (sgRNA) sequencing.

Figure 4.

XL-IMPACT, a photoaffinity labeling variant for discovery of phospholipid–protein interactions. PLD-mediated transphosphatidylation of PC with a minimalist diazirine alkyne alcohol yields a photo-cross-linkable mimic of phosphatidylethanol (PEth), a phospholipid formed following alcohol consumption. Following CuAAC tagging with azido-biotin, streptavidin (SA)-based enrichment, and LC–MS/MS-based chemoproteomics, XL-IMPACT enabled the identification of the protein interactome of PEth, a biomarker of alcohol consumption.

Figure 5.

Real-time IMPACT (RT-IMPACT) using the tetrazine ligation for rapid visualization of PLD and GPCR–G_q signaling. (A) RT-IMPACT involves a rapid inverse-electron-demand Diels–Alder (IEDDA) reaction between a strained alkene, (S)-oxoTCO, and a fluorogenic tetrazine-BODIPY (Tz-BODIPY). (B) RT-IMPACT is a useful tool for reporting on the spatiotemporal dynamics of GPCR–G_q signaling, and we applied it to elucidate the relationship between G_s and G_q signaling triggered by the parathyroid hormone (PTH) receptor PTHR1. βAr, β-arrestin.

Figure 6.

Imaging phosphatidylcholine analogues at super-resolution with organelle-level precision. (A) Lipid expansion microscopy (LExM) uses clickable, polymerizable fluorophores that can tag alkyne-PC analogues metabolically labeled via the Kennedy pathway. Following hydrogel generation and expansion, super-resolution imaging of lipids can be achieved using standard confocal microscopes. Shown are (top) conventional confocal microscopy and (bottom) LExM images of adjacent regions of a HeLa cell, where lipids metabolically labeled with propargylcholine (ProCho) and then tagged with an azido-BODIPY-methacrylamide reagent via CuAAC are shown in green and an ER marker is shown in magenta. Scale bar: 400 nm. Adapted from ref 51. Copyright 2022 American Chemical Society. (B) Clickable choline analogues can metabolically label PC via either the Kennedy pathway or PLD-mediated transphosphatidylation. (C) Organelle-selective labeling with PC analogues generated via PLD transphosphatidylation. IMPACT labeling using N,N-dimethylazidocholine and BCN-BODIPY tagging results in ER- and Golgi-selective labeling, whereas the extension to N,N-dipropylazidocholine leads to selective mitochondrial and lysosomal labeling despite using the same BCN-BODIPY tagging reagent.