Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity

Area of Science:

• Cell Biology
• Biochemistry
• Chemical Biology

Background:

• Signal transduction pathways rely on spatiotemporal regulation, often involving lipid second messengers.
• Phospholipase D (PLD) enzymes produce phosphatidic acid, a key signaling lipid, but its dynamic production is poorly understood.
• Existing tools lack the resolution to visualize PLD activity in real-time within cells.

Purpose of the Study:

• To develop a novel chemical method for imaging phosphatidic acid synthesis by PLD enzymes in live cells.
• To overcome limitations in visualizing the spatial and temporal dynamics of PLD activity.
• To investigate the localization and heterogeneity of PLD activity.

Main Methods:

• Utilized the enzymatic promiscuity of PLDs to accept azidoalcohols as reporters.
• Employed strain-promoted azide-alkyne cycloaddition for fluorescent tagging of azidolipids.
• Developed the IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation) method for live-cell imaging.

Main Results:

• Successfully visualized phosphatidic acid synthesis by active PLD enzymes in live cells.
• Identified both expected and unexpected cellular locations of basal and stimulated PLD activity.
• Revealed significant heterogeneity in PLD activity at cellular and subcellular levels.

Conclusions:

• The IMPACT method provides high spatial and temporal resolution for visualizing active PLD enzymes.
• Direct visualization of PLD activity is crucial for understanding its diverse physiological and pathological roles.
• This chemical imaging approach advances the study of signaling enzyme dynamics.