Figure 2. Depletion and regeneration of... | Rockefeller University Press

Figure 2.

Depletion and regeneration of PtdIns(4,5)P2 during stimulation of M1R.(A) Schematic of homologous FRET measurements with PHPLCδ1. At the PM, CFP- and YFP-tagged PHPLCδ1 (blue and yellow, respectively) are bound to PtdIns(4,5)P2 and show a high FRET interaction; however, after the activation of PLCβ, they are released from PtdIns(4,5)P2 to the cytoplasm and do not generate a FRET signal. (B) Time course of PtdIns(4,5)P2 monitored as the homologous nFRETeff (normalized FRET efficiency) between PHPLCδ1-CFP and PHPLCδ1-YFP in tsA201 cells transfected with M1R. Activation of M1R by 10 µM Oxo-M for 10 min (gray bar) depletes PtdIns(4,5)P2 quickly, but then PtdIns(4,5)P2 regenerates slowly as fitted with a single-exponential function (red line). ΔnFRETeff is measured as the difference between the minimal level and the final level at 10 min during Oxo-M stimulation. Here and in the remaining figures, the presented time courses of FRET are representative traces from single cells. (C) Time course of PtdIns(4,5)P2 monitored as the normalized heterologous FRETeff between Lyn-CFP and TubbyR322H-YFP. (D) Mean of total PtdIns (left), PtdInsP (middle), and PtdInsP2 (right) in control and 1- or 10-min agonist stimulation measured by mass spectrometry in CHO cells stably expressing M1R (n = 4 independent lipid extractions). *, P < 0.05.

Depletion and regeneration of PtdIns(4,5)P₂ during stimulation of M₁R. (A) Schematic of homologous FRET measurements with PH_PLCδ1. At the PM, CFP- and YFP-tagged PH_PLCδ1 (blue and yellow, respectively) are bound to PtdIns(4,5)P₂ and show a high FRET interaction; however, after the activation of PLCβ, they are released from PtdIns(4,5)P₂ to the cytoplasm and do not generate a FRET signal. (B) Time course of PtdIns(4,5)P₂ monitored as the homologous nFRET_eff (normalized FRET efficiency) between PH_PLCδ1-CFP and PH_PLCδ1-YFP in tsA201 cells transfected with M₁R. Activation of M₁R by 10 µM Oxo-M for 10 min (gray bar) depletes PtdIns(4,5)P₂ quickly, but then PtdIns(4,5)P₂ regenerates slowly as fitted with a single-exponential function (red line). ΔnFRET_eff is measured as the difference between the minimal level and the final level at 10 min during Oxo-M stimulation. Here and in the remaining figures, the presented time courses of FRET are representative traces from single cells. (C) Time course of PtdIns(4,5)P₂ monitored as the normalized heterologous FRET_eff between Lyn-CFP and Tubby_R322H-YFP. (D) Mean of total PtdIns (left), PtdInsP (middle), and PtdInsP₂ (right) in control and 1- or 10-min agonist stimulation measured by mass spectrometry in CHO cells stably expressing M₁R (n = 4 independent lipid extractions). *, P < 0.05.

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