Activity-based directed evolution of a membrane editor in mammalian cells

Tei, Reika; Bagde, Saket R.; Fromme, J. Christopher; Baskin, Jeremy M.

doi:10.1101/2022.09.26.509516

Cited by 5 publications

(15 citation statements)

References 67 publications

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“…When both constructs are expressed in mammalian cells, CRY2–CIBN heterodimerization upon blue-light stimulation recruits PLD to the desired membrane to produce PA (Figure B). OptoPLD enabled PA generation on several organelle membranes, including the PM, endosomes, the endoplasmic reticulum (ER), and the trans-Golgi network in our initial study and in subsequent work additional organelles such as mitochondria and lysosomes . To demonstrate the biological relevance of the optoPLD-derived PA, we established that PM-targeted optoPLD caused translocation of YAP into the nucleus under starvation, providing evidence of a suppressive effect of PM pools of PA on Hippo signaling.…”

Section: An Optogenetic Phospholipase D Enables Spatiotemporally Cont...mentioning

confidence: 85%

“…OptoPLD enabled PA generation on several organelle membranes, including the PM, endosomes, the endoplasmic reticulum (ER), and the trans-Golgi network in our initial study 3 and in subsequent work additional organelles such as mitochondria 32 and lysosomes. 33 To demonstrate the biological relevance of the optoPLD-derived PA, we established that PM-targeted optoPLD caused translocation of YAP into the nucleus under starvation, providing evidence of a suppressive effect of PM pools of PA on Hippo signaling. Interestingly, this finding not only corroborated a previous study indicating that PA can antagonize the Hippo pathway 24 but also concluded that PA generated at the PM and not on other organelle membranes is the main driver of this phenotype, driving home the importance of spatial regulation of PA-dependent signaling.…”

Section: Spatiotemporally Controlled Pa Productionmentioning

confidence: 99%

“…To address this shortcoming, we developed an activity-based directed enzyme evolution strategy to increase the activity of optoPLD in mammalian cells (Figure 3A). 33 Key to this strategy was the use of a method for fluorescent labeling of PLD activity within intact cells that we developed, termed IMPACT (for Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation; Figure 3B), which we will describe further in the following section. The directed evolution strategy began with generating a plasmid library of mutant PLDs using error-prone PCR and expression of this library in HEK 293 cells following lentiviral delivery.…”

Section: ■ Directed Evolution Of Superplds As Highly Efficient Membra...mentioning

confidence: 99%

“…Despite the power, precision, and mild nature of optogenetic recruitment, the first-generation optoPLD had modest activity due to its use of an unoptimized secreted bacterial enzyme in the mammalian cytosolic compartment. To address this shortcoming, we developed an activity-based directed enzyme evolution strategy to increase the activity of optoPLD in mammalian cells (Figure A) . Key to this strategy was the use of a method for fluorescent labeling of PLD activity within intact cells that we developed, termed IMPACT (for Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation; Figure B), which we will describe further in the following section.…”

Section: Directed Evolution Of Superplds As Highly Efficient Membrane...mentioning

confidence: 99%

“…A key unique feature of IMPACT relative to traditional PLD assays is its ability to enable single-cell measurements of PLD activity. One application that capitalizes upon this property was our use of IMPACT labeling and FACS enrichment of IMPACT high cells for the directed evolution of superactive forms of Streptomyces PLDs (Figure A). In another line of study, we combined IMPACT with pooled, genome-wide CRISPR screens to identify genes that regulate endogenous PLD signaling (Figure C).…”

Section: Applications Of Impact For Biological Discoverymentioning

confidence: 99%

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Imaging and Editing the Phospholipidome

Chiu

Baskin

2022

Acc. Chem. Res.

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Conspectus Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells. In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT. IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid–protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling. PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling. Collectively, the strategies described here summarize our progress to date in tac...

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Section: An Optogenetic Phospholipase D Enables Spatiotemporally Cont...mentioning

confidence: 85%

Section: Spatiotemporally Controlled Pa Productionmentioning

confidence: 99%

Section: ■ Directed Evolution Of Superplds As Highly Efficient Membra...mentioning

confidence: 99%

Section: Directed Evolution Of Superplds As Highly Efficient Membrane...mentioning

confidence: 99%

Section: Applications Of Impact For Biological Discoverymentioning

confidence: 99%

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Imaging and Editing the Phospholipidome

Chiu

Baskin

2022

Acc. Chem. Res.

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Imaging interorganelle phospholipid transport by extended synaptotagmins using bioorthogonally tagged lipids

Liang

Luan

Baskin

2023

Preprint

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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 large 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.

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Optogenetic strategies for optimizing the performance of biosensors of membrane phospholipids in live cells

Yao,

Lou,

Jin

et al. 2023

Preprint

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High-performance biosensors are crucial for elucidating the spatiotemporal regulatory roles and dynamics of membrane lipids, but there is a lack of improvement strategies for biosensors with low sensitivity and low-content substrates detection. Here we developed universal optogenetic strategies to improve a set of membrane biosensors by trapping them into specific region and further reducing the background signal, or by optically-controlled phase separation for membrane lipids detection and tracking. These improved biosensors were superior to typical tools and light simulation would enhance their detection performance and resolution, which might contribute to the design and optimization of other biosensors.

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Activity-based directed evolution of a membrane editor in mammalian cells

Cited by 5 publications

References 67 publications

Imaging and Editing the Phospholipidome

Imaging and Editing the Phospholipidome

Imaging interorganelle phospholipid transport by extended synaptotagmins using bioorthogonally tagged lipids

Optogenetic strategies for optimizing the performance of biosensors of membrane phospholipids in live cells

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