Abstract
Phosphoinositides are rare membrane lipids that mediate cell signaling and membrane dynamics. PI(4)P and PI(3)P are two major phosphoinositides crucial for endolysosomal functions and dynamics, making them the lipids of interest in many studies. The acute modulation of phosphoinositides at a given organelle membrane can reveal important insights into their cellular function. Indeed, the localized depletion of PI(4)P and PI(3)P is a viable tool to assess the importance of these phosphoinositides in various experimental conditions. Here, we describe a live imaging method to acutely deplete PI(4)P and PI(3)P on endolysosomes. The depletion assay utilizes the GAI-GID1 or the FRB-FKBP inducible dimerization system to target the catalytic domain of the PI(4)P phosphatase, Sac1, or the PI(3)P phosphatase domain of MTM1 to the endolysosome for localized depletion of these phosphoinositides. By using the fluorescently tagged biosensors, 2xP4M and PX, we can validate and monitor the depletion of PI(4)P and PI(3)P, respectively, on endolysosomes in real-time. We discuss a method for normalizing the fluorescence measurements to appropriate the relative amount of these phosphoinositides in the organellar membranes (endolysosomes), which is required for monitoring PI(4)P or PI(3)P levels during the acute depletion assay. Since the localization of the dimerization partners is specified by the membrane targeting signal, our protocol will be useful for studying the signaling and functions of phosphoinositides at any membrane. Key features • Acute depletion and real-time monitoring of PI(3)P and PI(4)P on the endolysosomal membrane using chemically inducible dimerization systems. • Modifiable and adaptable to modulate other phosphoinositides on different organellar membranes.