Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling

TL;DR

Researchers developed a new optogenetic tool to precisely control phosphatidic acid metabolism in specific cell locations, revealing how its production affects signaling pathways.

Contribution

A novel optogenetic system with ultralow background activity was created to control phospholipase D activity with spatiotemporal precision.

Findings

PA metabolism rates vary depending on subcellular location.

PA pools on different membranes influence AMPK signaling activation.

LOV domain-based photoswitches enable nonperturbative organelle targeting.

Abstract

Phosphatidic acid
(PA) is a multifunctional lipid with
important
metabolic and signaling functions, and efforts to dissect its pleiotropy
demand strategies for perturbing its levels with spatiotemporal precision.
Previous membrane editing approaches for generating local PA pools
used light-mediated induced proximity to recruit a PA-synthesizing
enzyme, phospholipase D (PLD), from the cytosol to the target organelle
membrane. Whereas these optogenetic PLDs exhibited high activity,
their residual activity in the dark led to undesired chronic lipid
production. Here, we report ultralow background membrane editors for
PA wherein light directly controls PLD catalytic activity, as opposed
to localization and access to substrates, exploiting a light–oxygen–voltage
(LOV) domain-based conformational photoswitch inserted into the PLD
sequence and enabling their stable and nonperturbative targeting to
multiple organelle membranes. By coupling organelle-targeted LOVPLD
activation to lipidomics analysis, we discovered different rates of
metabolism for PA and its downstream products depending on the subcellular
location of PA production. We also elucidated signaling roles for
PA pools on different membranes in conferring local activation of
AMP-activated protein kinase signaling. This work illustrates how
membrane editors featuring acute, optogenetic conformational switches
can provide new insights into organelle-selective lipid metabolic
and signaling pathways.

Ultralow background,
photoswitchable membrane editors enable
acute activation of phosphatidic acid metabolism and signaling.

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