A Peptide-Based Fluorescent Sensor for Anionic Phospholipids

[Image: see text] Anionic phospholipids are key cell signal mediators. The distribution of these lipids on the cell membrane and intracellular organelle membranes guides the recruitment of signaling proteins leading to the regulation of cellular processes. Hence, fluorescent sensors that can detect anionic phospholipids within living cells can provide a handle into revealing molecular mechanisms underlying lipid-mediated signal regulation. A major challenge in the detection of anionic phospholipids is related to the presence of these phospholipids mostly in the inner leaflet of the plasma membrane and in the membranes of intracellular organelles. Hence, cell-permeable sensors would provide an advantage by enabling the rapid detection and tracking of intracellular pools of anionic phospholipids. We have developed a peptide-based, cell-permeable, water-soluble, and ratiometric fluorescent sensor that entered cells within 15 min of incubation via the endosomal machinery and showed punctate labeling in the cytoplasm. The probe could also be introduced into living cells via lipofection, which allows bypassing of endosomal uptake, to image anionic phospholipids in the cell membrane. We validated the ability of the sensor toward detection of intracellular anionic phospholipids by colocalization studies with a fluorescently tagged lipid and a protein-based anionic phospholipid sensor. Further, the sensor could image the externalization of anionic phospholipids during programmed cell death, indicating the ability of the probe toward detection of both intra- and extracellular anionic phospholipids based on the biological context.