Literature Review: Imaging Phosphoinositides

Phosphoinositides Are Important Because They Participate in almost all Cell-Signaling Events

Assays for phosphoinositides phosphates (PIP) have been constructed using PIP binding domains in cell lysates or expressed in cells. This paper describes a cell penetrating reporter peptide, which can act as cellular sensor of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) (Figure 1). The peptide is based the protein gelsolin which has 20aa sequence known to bind PIP with particular selectivity toward PI(4,5)P2. To engineer a fluorophore into this sequence, a cysteine residue was introduced at Val59, which is a region of the peptide that interacts with membranes. A 2-dimethylamino-6-acyl-naphthalene (DAN) fluorophore was then coupled to Cys59. The DAN fluorophore undergoes a 60–70 nm shift in emission spectra (a change from green to blue emission) upon binding to membranes. Therefore, a ratiometric assay could be constructed by measuring the fluorescence of the PIP bound versus the unbound peptide (F450/F520 values were measured). Furthermore, DAN can be excited using two-photon microscopes using 780 nm light, which enables imaging of biological samples without confounding absorbance or autofluorescence from the sample. The affinity of the PIP sensor peptide was found to be in the 4–6 μM range for PI(4,5)P2. In vitro studies using small unilamellar vesicles (SUVs) were initially performed to confirm that the PIP sensor peptide responded to different concentrations of PI(4,5)P2 content in membranes. A shorter DAN-13aa sensor was found to be capable of measuring PI(4,5)P2 localization in HEK293 cells. Variation of this peptide could improve the selectivity of this probe and potentially expand to other PIP species.

Ratiometric sensing scheme for phosphoinositide imaging by using cell-permeable 2-dimethylamino-6-acyl-naphthalene (DAN)-labeled peptide-based sensors.

* Abstract from ACS Chem Biol 2016, in press. DOI: 10.1021/acschembio.6b00067

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.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in CS Chemical Biology titled “Cell permeable ratiometric fluorescent sensors for imaging phosphoinositides.” Authors of the paper are Mondal S, Rakshit A, Pal S, Datta A.