Emission Ratiometric Indicators of Phosphoinositides
cellular processes via activation of the serine/threonine kinase, Akt. JHU inventors have developed an emission ratiometric reporter, InPAkt, as research tool for monitoring the production and degradation of PIP(3,4)2 and PIP3 in living cells after transfection with plasmid constructs encoding the reporter protein, using a Fluorescent Resonance Energy Transfer (FRET) assay. The reporter molecule comprises a pair of fluorescent proteins linked by a region containing a phosphoinositide binding domain, the pleckstrin homology domain of Akt, and a ?pseudoligand? of acidic amino acids. In the absence of PIP(3,4)2 and PIP3, the pseudoligand binds to the pleckstrin homology domain. Binding of PIP(3,4)2 and PIP3 results in a conformational change in the reporter and a change in the FRET emission ratio, between yellow and cyan, in a manner dependent on local phosphoinositide concentration. The reporter can also be targeted to different subcellular sites to monitor specific pools of PIs. Description (Set) Proposed Use (Set) This reporter can be used to monitor the production and degradation of PIP(3,4)2 and PIP3 with high temporal and spatial resolution. This reporter offers several advantages over existing technologies, including the ability to target the reporter to subcellular organelles to monitor specific pools of phosphatidylinositols, and the ability of the sensor to undergo a conformational change without membrane anchoring. In addition, the reporter does not require large amounts of cells or tissue, eliminates artifacts caused by cell shape changes, and provides a dual readout of FRET change and translocation in its untargeted form.
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