Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides

Abstract

Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling.

Visualizing Temperature Mediated Activation of Gelsolin and Its Deactivation By Pip2: A Saxs Based Study

“…Interestingly, PIP 2 is primarily located at the cell membrane as well as perinuclear spaces, however the PIP kinase mediated synthesis of PIP 2 in plasma membrane is also reported 26, 27 . It is also proposed that certain proteins might serve as “buffers” which bind and sequester PIP 2 in the plasma, and subsequently concentrate PIP 2 in lateral membrane domains 26 .…”

Fluorescent Chemical Tools for Tracking Anionic Phospholipids

“…Because of the major roles played by phosphoinositides in mediating signalling and regulatory processes, fluorescent probes for monitoring phosphoinositide distribution and dynamics in living cells have emerged as valuable tools for cell biologists [4c,6e–g,15a,31c,d,36] . Tracking phosphoinositides in living cells is challenging due to their transient nature, presence in the inner leaflet of the plasma membrane and in intracellular organelle membranes, and low concentrations, necessitating the development of sensitive, reversible, fluorescent probes which should also preferably be cell‐permeable.…”

“…Reversible chemical probes will provide a handle into tracking phosphoinositide dynamics within living biological entities. The key limitation that has precluded the development of such small molecule based cell‐permeable probes for phosphoinositides barring a few recent examples [31d,64] has been the challenges associated with designing small molecule and peptide‐based scaffolds that can selectively bind to phosphoinositides. The critical requirement is that the scaffold should be selective not only toward a specific phosphoinositide over other members of the phosphoinositide family but also over other anionic lipids that are present in significantly higher concentrations in cell membranes [1b] .…”

“…Apart from intact protein domains like PLCδ1‐PH, ENTH, and the other domains listed in table 1, peptide sequences rich in positively charged amino acid residues from actin‐regulatory proteins like gelsolin, profilin, and cofilin bind to phosphoinositides like PI(4,5)P2 to regulate cytoskeletal rearrangements [8–9,64b,67] . Multiple binding studies with lipid vesicles have shown that these sequences can independently recognize PI(4,5)P2 and retain their selectivity when synthesized as separate entities [8–9,31d] . A study with a 10 amino acid sequence (QRLFQVKGRR) from the protein gelsolin conjugated to a Rhodamine B dye showed that the probe could afford a ‘turn‐off’ response in the presence of PI(4,5)P2 [64b] .…”

Recent Advancement in Fluorescent Probes for Sensing and Imaging Tyrosinase Activity in Living Cells

“…On the other hand, turn on/turn off type fluorescence probes often suffer in auto fluorescence, small stoke shifts and low penetration to living cells 29,30 along with some external interferences such as concentration, temperature, excitations wavelengths and resulting false reporting. But ratiometric fluorescent probes [31][32][33] overcomes such problems because such sensors give responses to the analytes at two different wavelengths, thus increasing the accuracy 34,35 of the result by eliminating external factors. The ratiometric signals produced by these probes are mostly based on the intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) process.…”