Click chemistry–enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling

Bumpus, Timothy W.; Huang, Shiying; Tei, Reika; Baskin, Jeremy M.

doi:10.1073/pnas.2025265118

Cited by 14 publications

(14 citation statements)

References 61 publications

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“…O-ClickFC can directly measure the abundance of de novo -synthesized PC by FACS at organelle resolution, which allows high-throughput CRISPR screening focused on subcellular PC distribution. Compared with recent examples of FACS-based CRISPR screens using lipid metabolism reporters such as fluorescent lipid (cholesterol)-binding proteins (Lu et al, 2022), phospholipase D activity-based click labeling (Bumpus et al, 2021), or a fluorescent PC analog (Maruoka et al, 2021), O-ClickFC offers a unique and unprecedented platform to elucidate uncertain links between PC metabolism and the whole genome.…”

Section: Discussionmentioning

confidence: 99%

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Tsuchiya

Tachibana

Nagao

et al. 2022

Preprint

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Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain – especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

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Section: Discussionmentioning

confidence: 99%

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Tsuchiya

Tachibana

Nagao

et al. 2022

Preprint

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“…Separately, we have recently used IMPACT-enabled FACS enrichment as a phenotypic selection step in a genome-wide CRISPR interference screen to elucidate new regulators of PLD signaling (Figure 4A) (44). At a mechanistic level, these studies revealed glycogen synthase kinase 3 (GSK3) as a positive regulator of PKC-mediated PLD signaling via effects on de novo gene expression.…”

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%

Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

Tei

Baskin

2022

Journal of Biological Chemistry

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“…In practice, we chose to use CRISPR interference (CRISPRi) to perform knockdown due to the accessibility of genome-wide libraries and suitability for such screening, even for essential genes. Following FACS enrichment of desired cell populations, short guide RNAs (sgRNAs) enriched in each population were identified by next-generation sequencing, and this information led to the identification of genes whose knockdown modulated PLD activity (Figure C) …”

Section: Applications Of Impact For Biological Discoverymentioning

confidence: 99%

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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Click chemistry–enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling

Cited by 14 publications

References 61 publications

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

Imaging and Editing the Phospholipidome

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