A Peptide-Based Fluorescent Sensor for Anionic Phospholipids

Abstract

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.

A change in metal cation switches selectivity of a phospholipid sensor from phosphatidic acid to phosphatidylserine

“…To account for any potential effects of physical or electronic interactions between the metal complex and fluorophore on the response, two different environment-sensitive fluorophores that have been previously used in phospholipid sensors-NBD and PRODAN-were employed. 36,41 Furthermore, to see whether any selectivity differences towards anionic phospholipids arise from the position of the fluorophores relative to the MDPA group, we varied the point of attachment of the NBD fluorophore to the peptide backbone. 42 The peptide backbone and DPA group of each sensor were prepared using solid phase peptide synthesis (SPPS) on Rink amide resin (details in ESI †).…”

Analytical Techniques for Single-Cell Biochemical Assays of Lipids

“…Genetic engineering of cells to express lipid binding domains (subunits of a larger protein) linked to a fluorescence protein (enhanced green fluorescent protein or others) also have found success. For example, a peptide-based sensor was developed to detect anionic phospholipids in live HeLa cells, an approach that was compatible with two-photon excitation (167).…”

Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining

“…This allows GUVs to be observed directly under the microscope, making them a convenient and accessible tool for lipid-binding studies. By preparing GUVs with defined lipid compositions, the specificity and sensitivity of lipid-binding probes can be evaluated and their accuracy and reproducibility in live cell experiments can be ensured ( Weingärtner et al, 2012; Chandra and Datta, 2022 ). GUVs with defined lipid compositions can be prepared by various methods, including swelling, PVA or agarose swelling, and electroformation using indium tin oxide glass slides and droplet transfer methods ( Angelova and Dimitrov, 1986 ; Weinberger et al, 2013; Bhatia et al, 2015 ; Shimane and Kuruma, 2022 ).…”