IMPACT: Imaging phospholipase d activity with clickable alcohols via transphosphatidylation

Bumpus, Timothy W.; Liang, Dongjun; Baskin, Jeremy M.

doi:10.1016/bs.mie.2020.04.037

Cited by 16 publications

(19 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our approach for visualizing lipid transport at ER-PM contact sites is, essentially, a repurposing of bioorthogonal chemistry tools originally developed for visualizing phospholipase D (PLD) signaling activity. IMPACT (Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation) involves metabolic labeling of cells with bioorthogonal primary alcohols that are used by PLDs in a transphosphatidylation reaction of phosphatidylcholine (PC) to form bioorthogonally labeled phosphatidyl alcohol lipids, followed by click chemistry tagging to generate fluorescent phosphatidyl alcohols [31][32][33][34][35] (Figure 1A). By controlling the PLD stimulus during the metabolic labeling step and using rapid and fluorogenic inverse electron-demand Diels-Alder (IEDDA) trans-cyclooctene-tetrazine click chemistry tagging, the initial sites of fluorescent lipids can be restricted to desired membranes.…”

Section: Resultsmentioning

confidence: 99%

Imaging interorganelle phospholipid transport by extended synaptotagmins using bioorthogonally tagged lipids

Liang

Luan

Baskin

2023

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Imaging interorganelle phospholipid transport by extended synaptotagmins using bioorthogonally tagged lipids

Liang

Luan

Baskin

2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Second, lentivirus containing this optoPLD library is generated and delivered into HEK 293T cells such that each cell expresses a different optoPLD mutant. Third, activity-based fluorescent labeling is performed using a bioorthogonal labeling method termed Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation (IMPACT) 31,32 (Fig. 2a).…”

Section: Resultsmentioning

confidence: 99%

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

Tei

Bagde

Fromme

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…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%

“…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

View full text Add to dashboard Cite

show abstract

IMPACT: Imaging phospholipase d activity with clickable alcohols via transphosphatidylation

Cited by 16 publications

References 27 publications

Imaging interorganelle phospholipid transport by extended synaptotagmins using bioorthogonally tagged lipids

Imaging interorganelle phospholipid transport by extended synaptotagmins using bioorthogonally tagged lipids

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

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology

Contact Info

Product

Resources

About