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

Abstract

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.

Imaging and Editing the Phospholipidome

“…52−55 The alkyne-or azide-bearing PC analogues labeled via the Kennedy pathway are distributed in essentially all membranes without appreciable selectivity, 47,49 but organelle-targeting clickable dyes can be used to visualize subsets of azido PC analogues in specific organelle membranes, including the PM, ER, and mitochondria. 56,57 Beyond the Kennedy pathway, we showed that an alkynyl choline derivative can also be converted to the corresponding PC analogue through a single PLD transphosphatidylation step (Figure 6B). 37 Because PLD activity can be controlled by external stimuli to activate endogenous PLDs or via exogenous optoPLDs, we envisioned that diverse bioorthogonal choline analogues might permit differential organelle labeling if installed selectively by PLD enzymes.…”

Adding a Chemical Biology Twist to CRISPR Screening

“…Another recent example of using bioorthogonal labeling and CRISPR screening came from Tsuchiya et al, who applied an organelle-selective click chemistry labeling of the abundant phospholipid phosphatidylcholine (PC), followed by flow cytometry with CRISPRko screens, to identify regulators of PC biosynthesis and transport. [63] In this screen, CRISPRperturbed cells were metabolically labeled with an azidocholine analog to generate azido-functionalized PC lipids within several organelle membranes. Using cyclooctyne-fluorophore dyes which were targeted to specific organelle compartments, the azido-PC could be selectively tagged.…”

Organelle-Selective Membrane Labeling through Phospholipase D-Mediated Transphosphatidylation

“…Instead, a different strategy involves covalent tagging of abundant lipid constituents of membranes using metabolic labeling and bioorthogonal chemistry. , Notably, phosphatidylcholine (PC) is an attractive target because it is the most abundant molecule in eukaryotic membranes. , Indeed, metabolic labeling of PC was first achieved using propargylcholine, an alkynyl analogue of choline, which is biosynthetically converted to PC via the multistep Kennedy pathway. , Propargylcholine labeling produces alkynyl PC, which can be visualized in fixed cells by Cu-catalyzed azide–alkyne cycloaddition (CuAAC) tagging with azide-bearing fluorophores . For live-cell imaging, fluorescent PC derivatives can be generated by metabolic labeling with an azidocholine analogue ( 1 , Figure A) followed by strain-promoted azide–alkyne cycloaddition (SPAAC) tagging with cyclooctyne-fluorophores .…”

“… 12 , 13 Indeed, metabolic labeling of PC was first achieved using propargylcholine, an alkynyl analogue of choline, which is biosynthetically converted to PC via the multistep Kennedy pathway. 14 , 15 Propargylcholine labeling produces alkynyl PC, which can be visualized in fixed cells by Cu-catalyzed azide–alkyne cycloaddition (CuAAC) tagging with azide-bearing fluorophores. 16 For live-cell imaging, fluorescent PC derivatives can be generated by metabolic labeling with an azidocholine analogue ( 1 , Figure 1 A) followed by strain-promoted azide–alkyne cycloaddition (SPAAC) tagging with cyclooctyne-fluorophores.…”

“…A recent strategy harnessed 1 for selective staining of organelle membranes including the endoplasmic reticulum (ER), Golgi complex, mitochondria, and lysosomes, wherein 1 was incorporated globally via the Kennedy pathway but the targeting selectivity was enabled by bespoke cyclooctyne reagents endowed with organelle-targeting groups that restricted the SPAAC reaction to the target organelles defined by the biased localization of the cyclooctyne reagent. 18 Though highly useful, 15 this modular approach required a different cyclooctyne reagent for each organelle and thus involved non-trivial synthetic efforts to produce the panel of detection reagents. In addition, although this approach enabled visualization of inter-organelle phospholipid transfer, the lipids carried bulky fluorophore tags that could potentially interfere with translocation rates and directions of transport.…”