Figure 6. Participation of Golgi PtdIns4... | Rockefeller University Press

Figure 6.

Participation of Golgi PtdIns4P in the regeneration of PM PtdIns(4,5)P2 during M1R activation.(A) Left: Time course of Golgi PtdIns4P level during M1R stimulation measured with photometry of PHFAPP1-YFP, a probe specific for Golgi PtdIns4P. The fluorescence signal normalized to the initial fluorescence (F/F0) was fitted by a single exponential function (red line). Confocal images of PHFAPP1-YFP on the right side show reduction of Golgi PtdIns4P after M1R activation for 10 min. Scale bar, 10 µm. Different experiment from the photometry time course on the left. (B) Time course of PtdIns(4,5)P2 concentration (density) during application of Oxo-M (gray bar) calculated from homologous FRETeff of CFP- and YFP-tagged PHPLCδ1 probes using Eq. 1. Regeneration of PtdIns(4,5)P2 was fitted with a single exponential curve (red line). (C) Mean time constants decrease for Golgi PtdIns4P (n = 3 cells) and increase for PM PtdIns(4,5)P2 (n = 8 cells). (D) Depression of PtdIns(4,5)P2 regeneration by Golgi recruitment of PJ-Sac1 phosphatase or PJ-dead with 1 µM rapamycin (blue shading, Rapa). nFRETeff between CFP- and YFP-tagged PHPLCδ1 domains measured during 10 µM Oxo-M (gray bar). (E) Comparison of PJ-Sac1 or PJ-dead recruitment to the Golgi by 1 µM rapamycin PM on PtdIns4P regeneration during receptor activation. nFRETeff between P4M-CFP and Lyn-YFP measured during application of rapamycin followed by 10 µM Oxo-M. (F and G) Simulation of PtdIns(4,5)P2 regeneration (F) and PtdIns4P regeneration (G) during Golgi action of PJ-Sac1 and PJ-dead using the expanded model also used for Fig. 7 and Fig. S9.

Participation of Golgi PtdIns4P in the regeneration of PM PtdIns(4,5)P₂ during M₁R activation. (A) Left: Time course of Golgi PtdIns4P level during M₁R stimulation measured with photometry of PH_FAPP1-YFP, a probe specific for Golgi PtdIns4P. The fluorescence signal normalized to the initial fluorescence (F/F₀) was fitted by a single exponential function (red line). Confocal images of PH_FAPP1-YFP on the right side show reduction of Golgi PtdIns4P after M₁R activation for 10 min. Scale bar, 10 µm. Different experiment from the photometry time course on the left. (B) Time course of PtdIns(4,5)P₂ concentration (density) during application of Oxo-M (gray bar) calculated from homologous FRET_eff of CFP- and YFP-tagged PH_PLCδ1 probes using Eq. 1. Regeneration of PtdIns(4,5)P₂ was fitted with a single exponential curve (red line). (C) Mean time constants decrease for Golgi PtdIns4P (n = 3 cells) and increase for PM PtdIns(4,5)P₂ (n = 8 cells). (D) Depression of PtdIns(4,5)P₂ regeneration by Golgi recruitment of PJ-Sac1 phosphatase or PJ-dead with 1 µM rapamycin (blue shading, Rapa). nFRET_eff between CFP- and YFP-tagged PH_PLCδ1 domains measured during 10 µM Oxo-M (gray bar). (E) Comparison of PJ-Sac1 or PJ-dead recruitment to the Golgi by 1 µM rapamycin PM on PtdIns4P regeneration during receptor activation. nFRET_eff between P4M-CFP and Lyn-YFP measured during application of rapamycin followed by 10 µM Oxo-M. (F and G) Simulation of PtdIns(4,5)P₂ regeneration (F) and PtdIns4P regeneration (G) during Golgi action of PJ-Sac1 and PJ-dead using the expanded model also used for Fig. 7 and Fig. S9.