Optically sensing phospholipid induced coil-helix transitions in the phosphoinositide-binding motif of gelsolin.

We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 µM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 µM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.

Cell-Permeable Fluorescent Sensors Enable Rapid Live Cell Visualization of Plasma Membrane and Nuclear PIP3 Pools.

Phosphoinositides, phospholipids that are key cell-signal mediators, are present at very low levels in cellular membranes and within nuclei. Phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), a phosphoinositide barely present in resting cell membranes, is produced when cells receive either growth, proliferation, or movement signals. Aberrant PIP3 levels are associated with the formation of cancers. PIP3 pools are also present in the nucleus, specifically in the nucleolus. However, questions related to the organization and function of this lipid in such membraneless intranuclear structures remain unanswered. Therefore, chemical sensors for tracking cellular PIP3 are invaluable not only for timing signal initiation in membranes but also for identifying the organization and function of membraneless nuclear PIP3 pools. Because PIP3 is present in the inner leaflet of cell membranes and in the nucleus, cell-permeable, rapid-response fluorescent sensors would be ideal. We have designed two peptide-based, water-soluble, cell-permeable, ratiometric PIP3 sensors named as MFR-K17H and DAN-NG-H12G. MFR-K17H rapidly entered into the cell cytoplasm, distinctly reporting rapid (<1 min) time scales of growth factor-stimulated PIP3 generation and depletion within cell membranes in living cells. Importantly, MFR-K17H lighted up inherently high levels of PIP3 in triple-negative breast cancer cell membranes, implying future applications in the detection of enhanced PIP3 levels in cancerous cells. On the other hand, DAN-NG-H12G targeted intranuclear PIP3 pools, revealing that within membraneless structures, PIP3 resided in a hydrophobic environment. Together, both probes form a unique orthogonally targeted combination of cell-permeable, ratiometric probes that, unlike previous cell-impermeable protein-based sensors, are easy to apply and provide an unprecedented handle into PIP3-mediated cellular processes.

A Peptide-Based Fluorescent Sensor for Anionic Phospholipids.

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.