Curated Optogenetic Publication Database

Abstract: Deficits in levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] mediate Alzheimer's disease (AD) pathogenesis and etiology. The depletion of PI(4,5)P2 has been reported in AD in both human brain and animal models. Repletion of the synaptic pool of PI(4,5)P2, through haploinsufficiency of the degrading enzyme, Synaptojanin 1, ameliorated behavioral deficits in a mouse model of AD, in spite of accumulating amyloid. In order to refine the contribution of PI(4,5)P2 to AD, we used optogenetic translocation of the PI(4,5)P2-synthesizing enzyme, phosphoinositide phosphate 4 kinase2A (PIP4K2A) to the plasma membrane using light inducible dimerizable cryptochrome 2 (CRY-2) and the transcription factor CRY2-binding domain (CIBN) fused to the plasma membrane-targeting motif (CAAX). Spatiotemporally controlled production of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] at the plasma membrane induced amelioration of behavioral deficits in a mouse model of AD. Imaging mass spectrometry confirmed alteration of specific PI(4,5)P2 acyl species, di-oleate, indicating that precise PI(4,5)P2 species may ultimately be leveraged for therapeutic intervention.

Abstract: Cellular lipid metabolism is subject to strong homeostatic regulation, but the players involved in and mechanisms underlying these pathways remain largely uncharacterized. Here we develop a 'feeding-fishing' approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics through proximity labeling (fishing) to elucidate molecular players and pathways involved in the homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. This approach identified several PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Mechanistic analysis revealed that PA homeostasis in the cytosolic leaflets of the plasma membrane and lysosomes is mediated by both local PA metabolism and the action of lipid transfer proteins that carry out interorganelle lipid transport before subsequent metabolism. More broadly, the interfacing of membrane editing to controllably modify membrane lipid composition with organelle membrane proteomics using proximity labeling represents a strategy for revealing mechanisms governing lipid homeostasis.

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.