Reika Tei

All Publications

• Membrane editing with proximity labeling reveals regulators of lipid homeostasis. Nature chemical biology Tei, R., Li, X. L., Luan, L., Baskin, J. M. 2026

  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.

  View details for DOI 10.1038/s41589-025-02104-x

  View details for PubMedID 41501183

  View details for PubMedCentralID 3233269

• Author Correction: Synthetic GPCRs for programmable sensing and control of cell behaviour. Nature Kalogriopoulos, N. A., Tei, R., Yan, Y., Klein, P. M., Ravalin, M., Cai, B., Soltesz, I., Li, Y., Ting, A. Y. 2025

  View details for DOI 10.1038/s41586-025-08607-w

  View details for PubMedID 39870926

• Synthetic GPCRs for programmable sensing and control of cell behaviour. Nature Kalogriopoulos, N. A., Tei, R., Yan, Y., Klein, P. M., Ravalin, M., Cai, B., Soltesz, I., Li, Y., Ting, A. 2024

  Abstract

  Synthetic receptors that mediate antigen-dependent cell responses are transforming therapeutics, drug discovery and basic research1,2. However, established technologies such as chimeric antigen receptors3 can only detect immobilized antigens, have limited output scope and lack built-in drug control3-7. Here we engineer synthetic G-protein-coupled receptors (GPCRs) that are capable of driving a wide range of native or non-native cellular processes in response to a user-defined antigen. We achieve modular antigen gating by engineering and fusing a conditional auto-inhibitory domain onto GPCR scaffolds. Antigen binding to a fused nanobody relieves auto-inhibition and enables receptor activation by drug, thus generating programmable antigen-gated G-protein-coupled engineered receptors (PAGERs). We create PAGERs that are responsive to more than a dozen biologically and therapeutically important soluble and cell-surface antigens in a single step from corresponding nanobody binders. Different PAGER scaffolds allow antigen binding to drive transgene expression, real-time fluorescence or endogenous G-protein activation, enabling control of diverse cellular functions. We demonstrate multiple applications of PAGER, including induction of T cell migration along a soluble antigen gradient, control of macrophage differentiation, secretion of therapeutic antibodies and inhibition of neuronal activity in mouse brain slices. Owing to its modular design and generalizability, we expect PAGERs to have broad utility in discovery and translational science.

  View details for DOI 10.1038/s41586-024-08282-3

  View details for PubMedID 39633047

  View details for PubMedCentralID 10225594

• The dynamic regulatory network of phosphatidic acid metabolism: a spotlight on substrate cycling between phosphatidic acid and diacylglycerol. Biochemical Society transactions Tei, R. 2024

  Abstract

  Mammalian cells utilize over 1000 different lipid species to maintain cell and organelle membrane properties, control cell signaling and processes, and store energy. Lipid synthesis and metabolism are mediated by highly interconnected and spatiotemporally regulated networks of lipid-metabolizing enzymes and supported by vesicle trafficking and lipid-transfer at membrane contact sites. However, the regulatory mechanisms that achieve lipid homeostasis are largely unknown. Phosphatidic acid (PA) serves as the central hub for phospholipid biosynthesis, acting as a key intermediate in both the Kennedy pathway and the CDP-DAG pathway. Additionally, PA is a potent signaling molecule involved in various cellular processes. This dual role of PA, both as a critical intermediate in lipid biosynthesis and as a significant signaling molecule, suggests that it is tightly regulated within cells. This minireview will summarize the functional diversity of PA molecules based on their acyl tail structures and subcellular localization, highlighting recent tools and findings that shed light on how the physical, chemical, and spatial properties of PA species contribute to their differential metabolic fates and functions. Dysfunctional effects of altered PA metabolism as well as the strategies cells employ to maintain PA regulation and homeostasis will also be discussed. Furthermore, this review will explore the differential regulation of PA metabolism across distinct subcellular membranes. Our recent proximity labeling studies highlight the possibility that substrate cycling between PA and DAG may be location-dependent and have functional significance in cell signaling and lipid homeostasis.

  View details for DOI 10.1042/BST20231511

  View details for PubMedID 39417337

• Imaging Interorganelle Phospholipid Transport by Extended Synaptotagmins Using Bioorthogonally Tagged Lipids ACS CHEMICAL BIOLOGY Luan, L., Liang, D., Chiu, D., Tei, R., Baskin, J. M. 2024

  Abstract

  The proper distribution of lipids within organelle membranes requires rapid interorganelle lipid transport, much of which occurs at membrane contact sites and is mediated by lipid transfer proteins (LTPs). Our current understanding of LTP mechanism and function is based largely on structural studies and in vitro reconstitution. Existing cellular assays for LTP function use indirect readouts, and it remains an open question as to whether substrate specificity and transport kinetics established in vitro are similar in cellular settings. Here, we harness bioorthogonal chemistry to develop tools for direct visualization of interorganelle transport of phospholipids between the plasma membrane (PM) and the endoplasmic reticulum (ER). Unnatural fluorescent phospholipid analogs generated by the transphosphatidylation activity of phospholipase D (PLD) at the PM are rapidly transported to the ER dependent in part upon extended synaptotagmins (E-Syts), a family of LTPs at ER-PM contact sites. Ectopic expression of an artificial E-Syt-based tether at ER-mitochondria contact sites results in fluorescent phospholipid accumulation in mitochondria. Finally, in vitro reconstitution assays demonstrate that the fluorescent lipids are bona fide E-Syt substrates. Thus, fluorescent lipids generated in situ via PLD activity and bioorthogonal chemical tagging can enable direct visualization of the activity of LTPs that mediate bulk phospholipid transport at ER-PM contact sites.

  View details for DOI 10.1021/acschembio.4c00345

  View details for Web of Science ID 001272780800001

  View details for PubMedID 39023576

• Ultralow Background Membrane Editors for Spatiotemporal Control of Phosphatidic Acid Metabolism and Signaling ACS CENTRAL SCIENCE Li, X., Tei, R., Uematsu, M., Baskin, J. M. 2024; 10 (3): 543-554

  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.

  View details for DOI 10.1021/acscentsci.3c01105

  View details for Web of Science ID 001161283700001

  View details for PubMedID 38559292

  View details for PubMedCentralID PMC10979500

• Ultralow background membrane editors for spatiotemporal control of lipid metabolism and signaling. bioRxiv : the preprint server for biology Li, X., Tei, R., Uematsu, M., Baskin, J. M. 2023

  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 LOV domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and non-perturbative 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.

  View details for DOI 10.1101/2023.08.31.555787

  View details for PubMedID 37693485

• NME3 binds to phosphatidic acid and mediates PLD6-induced mitochondrial tethering JOURNAL OF CELL BIOLOGY Su, Y., Chiu, H., Chang, Y., Sung, C., Chen, C., Tei, R., Huang, X., Hsu, S., Lin, S., Wang, H., Lin, Y., Hsu, J., Bauer, H., Feng, Y., Baskin, J. M., Chang, Z., Liu, Y. 2023; 222 (10)

  Abstract

  Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

  View details for DOI 10.1083/jcb.202301091

  View details for Web of Science ID 001077350500001

  View details for PubMedID 37584589

  View details for PubMedCentralID PMC10432850

• Activity-based directed evolution of a membrane editor in mammalian cells NATURE CHEMISTRY Tei, R., Bagde, S. R., Fromme, J., Baskin, J. M. 2023; 15 (7): 1030-+

  Abstract

  Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

  View details for DOI 10.1038/s41557-023-01214-0

  View details for Web of Science ID 000993681200003

  View details for PubMedID 37217787

  View details for PubMedCentralID PMC10525039

• Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling JOURNAL OF BIOLOGICAL CHEMISTRY Tei, R., Baskin, J. M. 2022; 298 (4): 101810

  Abstract

  The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking. Because PA metabolism and trafficking are rapid, approaches to accurately visualize and manipulate its levels require high spatiotemporal precision. Here, we describe recent efforts to create a suite of chemical tools that enable imaging and perturbation of PA signaling. First, we describe techniques to visualize PA production by phospholipase D (PLD) enzymes, which are major producers of PA, called Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). IMPACT harnesses the ability of endogenous PLD enzymes to accept bioorthogonally tagged alcohols in transphosphatidylation reactions to generate functionalized reporter lipids that are subsequently fluorescently tagged via click chemistry. Second, we describe two light-controlled approaches for precisely manipulating PA signaling. Optogenetic PLDs use light-mediated heterodimerization to recruit a bacterial PLD to desired organelle membranes, and photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. We highlight select applications of these tools for studying GPCR-Gq signaling, discovering regulators of PLD signaling, tracking intracellular lipid transport pathways, and elucidating new oncogenic signaling roles for PA. We envision that these chemical tools hold promise for revealing many new insights into lipid signaling pathways.

  View details for DOI 10.1016/j.jbc.2022.101810

  View details for Web of Science ID 000823106100014

  View details for PubMedID 35276134

  View details for PubMedCentralID PMC9006657

• Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bumpus, T. W., Huang, S., Tei, R., Baskin, J. M. 2021; 118 (48)

  Abstract

  Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

  View details for DOI 10.1073/pnas.2025265118

  View details for Web of Science ID 000723881800002

  View details for PubMedID 34810254

  View details for PubMedCentralID PMC8640934

• Induced proximity tools for precise manipulation of lipid signaling CURRENT OPINION IN CHEMICAL BIOLOGY Tei, R., Baskin, J. M. 2021; 65: 93-100

  Abstract

  Lipids are highly dynamic molecules that, due to their hydrophobicity, are spatially confined to membrane environments. From these locations, certain privileged lipids serve as signaling molecules. For understanding the biological functions of subcellular pools of signaling lipids, induced proximity tools have been invaluable. These methods involve controlled heterodimerization, by either small-molecule or light triggers, of functional proteins. In the arena of lipid signaling, induced proximity tools can recruit lipid-metabolizing enzymes to manipulate lipid signaling and create artificial tethers between organelle membranes to control lipid trafficking pathways at membrane contact sites. Here, we review recent advances in methodology development and biological application of chemical-induced and light-induced proximity tools for manipulating lipid metabolism, trafficking, and signaling.

  View details for DOI 10.1016/j.cbpa.2021.06.005

  View details for Web of Science ID 000712375400012

  View details for PubMedID 34304140

• Optical Control of Phosphatidic Acid Signaling ACS CENTRAL SCIENCE Tei, R., Morstein, J., Shemet, A., Trauner, D., Baskin, J. M. 2021; 7 (7): 1205-1215

  Abstract

  Phosphatidic acids (PAs) are glycerophospholipids that regulate key cell signaling pathways governing cell growth and proliferation, including the mTOR and Hippo pathways. Their acyl chains vary in tail length and degree of saturation, leading to marked differences in the signaling functions of different PA species. For example, in mTOR signaling, saturated forms of PA are inhibitory, whereas unsaturated forms are activating. To enable rapid control over PA signaling, we describe here the development of photoswitchable analogues of PA, termed AzoPA and dAzoPA, that contain azobenzene groups in one or both lipid tails, respectively. These photolipids enable optical control of their tail structure and can be reversibly switched between a straight trans form and a relatively bent cis form. We found that cis-dAzoPA selectively activates mTOR signaling, mimicking the bioactivity of unsaturated forms of PA. Further, in the context of Hippo signaling, whose growth-suppressing activity is blocked by PA, we found that the cis forms of both AzoPA and dAzoPA selectively inhibit this pathway. Collectively, these photoswitchable PA analogues enable optical control of mTOR and Hippo signaling, and we envision future applications of these probes to dissect the pleiotropic effects of physiological and pathological PA signaling.

  View details for DOI 10.1021/acscentsci.1c00444

  View details for Web of Science ID 000679934200016

  View details for PubMedID 34345670

  View details for PubMedCentralID PMC8323247

• ESCRT-III and ER-PM contacts maintain lipid homeostasis MOLECULAR BIOLOGY OF THE CELL Jorgensen, J. R., Tei, R., Baskin, J. M., Michel, A. H., Kornmann, B., Emr, S. D. 2020; 31 (12): 1302-1313

  Abstract

  Eukaryotic cells are compartmentalized into organelles by intracellular membranes. While the organelles are distinct, many of them make intimate contact with one another. These contacts were first observed in the 1950s, but only recently have the functions of these contact sites begun to be understood. In yeast, the endoplasmic reticulum (ER) makes extensive intermembrane contacts with the plasma membrane (PM), covering ∼40% of the PM. Many functions of ER-PM contacts have been proposed, including nonvesicular lipid trafficking, ion transfer, and as signaling hubs. Surprisingly, cells that lack ER-PM contacts grow well, indicating that alternative pathways may be compensating for the loss of ER-PM contact. To better understand the function of ER-PM contact sites we used saturating transposon mutagenesis to identify synthetic lethal mutants in a yeast strain lacking ER-PM contact sites. The strongest hits were components of the ESCRT complexes. The synthetic lethal mutants have low levels of some lipid species but accumulate free fatty acids and lipid droplets. We found that only ESCRT-III components are synthetic lethal, indicating that Vps4 and other ESCRT complexes do not function in this pathway. These data suggest that ESCRT-III proteins and ER-PM contact sites act in independent pathways to maintain lipid homeostasis.

  View details for DOI 10.1091/mbc.E20-01-0061

  View details for Web of Science ID 000537320400009

  View details for PubMedID 32267208

  View details for PubMedCentralID PMC7353149

• Spatiotemporal control of phosphatidic acid signaling with optogenetic, engineered phospholipase Ds JOURNAL OF CELL BIOLOGY Tei, R., Baskin, J. M. 2020; 219 (3)

  Abstract

  Phosphatidic acid (PA) is both a central phospholipid biosynthetic intermediate and a multifunctional lipid second messenger produced at several discrete subcellular locations. Organelle-specific PA pools are believed to play distinct physiological roles, but tools with high spatiotemporal control are lacking for unraveling these pleiotropic functions. Here, we present an approach to precisely generate PA on demand on specific organelle membranes. We exploited a microbial phospholipase D (PLD), which produces PA by phosphatidylcholine hydrolysis, and the CRY2-CIBN light-mediated heterodimerization system to create an optogenetic PLD (optoPLD). Directed evolution of PLD using yeast membrane display and IMPACT, a chemoenzymatic method for visualizing cellular PLD activity, yielded a panel of optoPLDs whose range of catalytic activities enables mimicry of endogenous, physiological PLD signaling. Finally, we applied optoPLD to elucidate that plasma membrane, but not intracellular, pools of PA can attenuate the oncogenic Hippo signaling pathway. OptoPLD represents a powerful and precise approach for revealing spatiotemporally defined physiological functions of PA.

  View details for DOI 10.1083/jcb.201907013

  View details for Web of Science ID 000525735300015

  View details for PubMedID 31999306

  View details for PubMedCentralID PMC7054994

• A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liang, D., Wu, K., Tei, R., Bumpus, T. W., Ye, J., Baskin, J. M. 2019; 116 (31): 15453-15462

  Abstract

  The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene-containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD's lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene-containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels-Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

  View details for DOI 10.1073/pnas.1903949116

  View details for Web of Science ID 000477812400029

  View details for PubMedID 31311871

  View details for PubMedCentralID PMC6681737