Technologies

The invention provides novel compounds that enable visualization and tracking of phosphatidic acid (PA) production by phospholipase D (PLD) at thecellularandsubcellularlevels using a simple two-step strategy called IMPACT –ImagingPhospholipase DActivity withClickableAlcohols viaTransphophatidylation.

Phospholipase D (PLD) enzymes impact cell signaling by synthesizing the pleiotropic lipid phosphatidic acid (PA). PA alone has been associated with diverse physiological changes, but how PA causes these effects remains unknown due to the lack of suitable tools for visualizing its production within cells.

Cornell researchers have taken advantage of the enzymatic promiscuity of PLDs to develop a two-step strategy as shown in figure 1 involving novel, exogenous functionalized alcohols that enables the use of click chemistry to append imaging or other detection probes.

Figure 1: IMPACT

Proof of concept: Using various azidoalcohols as substrates, figure 2 shows live-cell imaging of PLD activity.

Figure 2: Evaluation of a panel of azidoalcohols for imaging PLD activity in HeLa cells. Cells were treated with PLDi (PLD inhibitor – bottom panel) or DMSO (top panel) followed by azidoalcohols AzET (A), AzProp (B), AzBut (C), AzPent (D) then incubated with BODIPY-cyclooctyne and imaged by confocal microscopy.