Synthesis and Cellular Labeling of Caged Phosphatidylinositol Derivatives

Abstract

Phosphatidylinositol (PI) is the biosynthetic precursor for seven phosphoinositides, important signaling lipids in cells. A membrane‐permeant caged PI derivative featuring a photo‐removable coumarinyl group masking the negative charge of the phosphate, as well as two enzymatically removable butyrate esters for increased lipophilicity and for preventing phosphate migration, were synthesized. Rapid cell entry and cellular labeling in fixed cells was demonstrated by a photo‐cross‐linkable diazirine followed by attachment of a fluorophore through click chemistry. Using this technique, we found that the multifunctional caged PI derivative resided predominantly at internal membranes but rapidly changed to the plasma membrane after uncaging. Accordingly, a preliminary proteomic analysis of the lipid–protein conjugates revealed that the two major PI transport proteins PITPα and β were prime targets of the photo‐cross‐linked PI derivative.

Glycolysis regulates Hedgehog signalling via the plasma membrane potential

“…PAC‐NAE contains a C15 fatty acid featuring diazirine and alkyne modifications (Fig 6B). After incubation with tissue explants, PAC‐NAE intracellular localization can be visualized by attaching azide‐modified fluorescent dyes using click chemistry (Haberkant et al , 2013; Gaebler et al , 2016; Höglinger et al , 2017; Müller et al , 2020). To study N‐acylethanolamide trafficking, we loaded PAC‐NAE onto preparations of Drosophila Lpp at levels comparable to those observed in circulation and applied these lipoproteins to explanted wing discs.…”

“…Patterns of growth and differentiation during development are functionally associated with changes in cell metabolism (Kaelin & McKnight, 2013; Shyh‐Chang et al , 2013; Buck et al , 2015; Oginuma et al , 2017; Müller et al , 2020). Shifts between oxidative and glycolytic metabolism can alter the balance of differentiated T‐cell and macrophage populations (Buck et al , 2015), and regulate the balance of proliferation and differentiation in the mouse cerebellum (Gershon et al , 2013) and in Drosophila neuroblasts (Homem et al , 2014).…”

Fluorescent Chemical Tools for Tracking Anionic Phospholipids

“…The molecule becomes fluorescent upon undergoing a cyclo‐addition reaction with an alkyne. Addition of 3‐azidocoumarin following the cellular incorporation of alkyne‐tagged phosphoinositides has been applied to pin‐point the localization of these signalling lipids [22,43–44] . In situ tagging of phosphoinositides is an attractive concept provided the strategy is not limited by the modified properties of the lipid following the attachment of the fluorescent tag [42] .…”

Synthesis and Cellular Labeling of Multifunctional Phosphatidylinositol Bis‐ and Trisphosphate Derivatives

“…In the past, we and others equipped lipids such as cholesterol, sphingosine and diacylglycerol (DAG) with a photo‐crosslinking diazirines as well as a terminal alkyne group for click chemistry [10] . Very recently, we equipped phosphatidylinositol with all of the named functional groups [11] . In a typical experiment, cells were incubated with the lipid and illuminated with 350 nm light for crosslinking [12] .…”