A Miniature Peptide-based Ratiometric, Cell Permeable Sensor Images PI(3,5)P2 Lipid Pools in Organelle Membranes of Living Cells

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phosphoinositide, constituting ~0.05–0.1 mol % of total cellular phosphoinositides, yet plays crucial roles in innate immunity, endolysosomal trafficking, autophagy, and ion channel regulation. Owing to its transient occurrence, fast metabolic turnover, and presence in membranes of acidic intracellular compartments, selective and rapid visualization of PI(3,5)P2 pools in living cells has remained a challenge. Here, we report DAN–TRPML1-PEP , a cell-permeable, peptide-based, ratiometric fluorescent sensor for PI(3,5)P2. The sensor comprises of a 20-amino acid polybasic segment from the N-terminal domain of TRPML1 channel, a native PI(3,5)P2-binding protein. The peptide has been conjugated to a polarity-sensitive dye, 6-dimethylamino-2-acetylnaphthalene (DAN), to afford the sensor. DAN–TRPML1-PEP exhibits a selective ratiometric fluorescence response upon PI(3,5)P2 binding. Importantly, the sensor not only selectively detects PI(3,5)P2 over other physiologically relevant phospholipids but can also distinguish PI(3,5)P2 from closely related phosphoinositides, including PI(3)P and PI(5)P. DAN–TRPML1-PEP rapidly and directly enters living cells and selectively images PI(3,5)P2 within late endosomes, lysosomes, autophagosomes, and endocytic vesicles. The sensor could also be applied for wash-free live-cell imaging of PI(3,5)P2. Hence, DAN–TRPML1-PEP successfully reveals PI(3,5)P2 localization in living cells bringing in a much-required chemical tool for interrogating this key signaling lipid.