Chemical Tools for Understanding Phosphatidic Acid Signaling

Abstract

Phosphatidic acid (PA) is an important phospholipid biosynthetic intermediate and a lipid second messenger. Both the metabolism of PA and the interactions of PA with its effector proteins can occur on numerous organelle membranes, raising important questions about how cells direct specificity into the outcomes of PA signaling. We have endeavored to ‘uncouple the pleiotropy’ of PA signaling through the development of two complementary types of chemical biology toolsets for, respectively, visualizing and manipulating PA metabolism and signaling. The first of these, IMPACT, harnesses bioorthogonal chemistry to generate fluorescent lipids whose localization and abundance report on the activity of phospholipase D (PLD) enzymes that generate ‘signaling pools’ of PA. The second tool, optoPLD, involves the use of light‐controlled, or optogenetic, PLD enzymes capable of generating pools of PA at precise organelle locations upon blue light illumination. We will describe the development of these tools, including newer, second‐generation IMPACT probes and optimized ‘superPLD’ enzymes with much better catalytic properties than our original optoPLD systems. Further, we will describe select applications of IMPACT and optoPLD. These include the use of IMPACT as a readout in genome‐wide CRISPR screening to discover new regulators of PLD signaling and the use of these tools to examine the spatiotemporal dynamics of important pathways that intersect with PLD activation, such as GPCR signaling and the Hippo pathway. Collectively, this work highlights the power of applying diverse chemical biology approaches to shed light on cellular lipid signaling pathways.