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

Tei, Reika; Baskin, Jeremy M.

doi:10.1016/j.jbc.2022.101810

Cited by 15 publications

(12 citation statements)

References 67 publications

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“…5A) within cells, and that it could be used for gel, fluorescence microscopy and LC-MS/MS-based chemoproteomics applications in the mapping of protein-lipid interactions in mammalian cells. More recently, Baskin and co-workers pioneered the development of IMaging Phospholipase D (PLD) Activity with Clickable alcohols via Transphosphatidylation (IMPACT) assays [80][81][82][83] for monitoring the activity of PLD (Fig. 5B), as well as assessing the production of different phospholipids (e.g., phosphatidic acid, phosphatidyl alcohols) in mammalian cells via PLD enzyme activity and studying the interactions of these phospholipids with diverse proteins in particular physiological contexts.…”

Section: In Vivo Synthesized Lipid Probesmentioning

confidence: 99%

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Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

2023

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Section: In Vivo Synthesized Lipid Probesmentioning

confidence: 99%

“…More recently, Baskin and co-workers pioneered the development of IMaging Phospholipase D (PLD) Activity with Clickable alcohols via Transphosphatidylation (IMPACT) assays 80–83 for monitoring the activity of PLD (Fig. 5B), as well as assessing the production of different phospholipids ( e.g.…”

Section: Photoreactive Bioorthogonal Lipid Probesmentioning

confidence: 99%

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

2023

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“…32 We have developed a method termed IMPACT to visualize the activity of phospholipase Ds (PLDs), which generate PA, by leveraging the ability of these enzymes to attach bioorthogonally labeled primary alcohols onto phospholipids in a transphosphatidylation, or head group exchange, reaction (Figure 4A). [33][34][35][36] To establish LExM as a more general strategy for ExM-based imaging of alkyne-labeled lipids, we subjected cells to IMPACT labeling with the PLD substrate hexynol, CuAAC tagging with 1, and the LExM protocol. Consistent with the ProCho-based LExM studies, IMPACT labeling generated samples with a 5.6x expansion factor (at the 4 h hydrolysis timepoint) and fluorescence signal that correlated well with the pre-LExM image (Figure 4B).…”

Section: Super-resolution Imaging Techniques Including Stimulated Emi...mentioning

confidence: 99%

Lipid Expansion Microscopy

White

Conwell

et al. 2022

Preprint

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Strategies to visualize cellular membranes with light microscopy are restricted by the diffraction limit of light, which far exceeds the dimensions of lipid bilayers. Here, we describe a method for super-resolution imaging of metabolically labeled phospholipids within cellular membranes. Guided by the principles of expansion microscopy, we develop an approach featuring cell-permeable reagents that enables direct chemical anchoring of bioorthogonally labeled phospholipids into a hydrogel network and is capable of tunable, isotropic expansion, thus facilitating super-resolution imaging of cellular membranes. We apply this method, termed lipid expansion microscopy, to visualize organelle membranes with precision, including a unique class of membrane-bound structures known as nuclear invaginations. As it is compatible with standard confocal microscopes, lipid expansion microscopy will be widely applicable for super-resolution imaging of phospholipids and cellular membranes in numerous physiological contexts.

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“…Phospholipase D (PLD) is an ideal starting point for such a general phospholipid membrane editor. PLD catalyzes hydrolysis of PC to form a signaling lipid, phosphatidic acid (PA) 14,15 , and it can also catalyze transphosphatidylation with exogenous alcohols to swap out head groups to form a variety of natural and unnatural phospholipids [16][17][18][19] . Though mammalian cells have endogenous PLD enzymes, they are dispensable for viability and exhibit low levels of basal activity 16 .…”

Section: Introductionmentioning

confidence: 99%

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

Tei

Bagde

Fromme

et al. 2022

Preprint

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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 upon 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 developed and structurally characterized a family of "superPLDs" with up to 100-fold higher activity than wildtype PLD. We demonstrated 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.

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

Cited by 15 publications

References 67 publications

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

Lipid Expansion Microscopy

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

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