Curated Optogenetic Publication Database

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.

Abstract: Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.

Abstract: OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.