A Genetically Encoded Tool Kit for Manipulating and Monitoring Membrane Phosphatidylinositol 4,5-Bisphosphate in Intact Cells
Figure 2
Continuous recording of ECFP and EYFP fluorescence from a single HEK293 cell transfected with pShuttle- PIP2-tool.
The cell was voltage-clamped in whole cell mode. Fluorescence changes (A) were evoked by step depolarizations of 30 s in duration from −90 mV holding potential to the levels indicated. The resulting corrected fluorescence signals (FEYFP- yellow trace and FECFP- blue trace) were normalized to values before agonist application (F/Fo) or expressed as FRET ratio (FEYFP/FECFP, red trace). (B) Superimposed changes in FRET ratio from A on expanded timescale. (C) Changes in FRET ratio evoked by voltage-steps to −20 mV (blue) and +80 mV (black) were superimposed and scaled to match the steady-state levels. (D) Plot of t1/2 for rise time (depletion) and recovery of FRET signals against membrane potential. (E) Mean values±SEM of half times of FRET signal indicating PtdIns(4,5)P2-depletion and recovery by voltage steps to +80 mV (n = 6).