Figure 2. PtdIns(4,5) P 2 local... | Rockefeller University Press

Figure 2.

PtdIns(4,5)P2local generation depends on PtdIns4P, PI4K, and PI4P5K. (A) A diagram illustrating the synthesis of PtdIns(4,5)P2 from PI and PtdIns4P by PI4K and PI4P5K. Phenyl arsine oxide (PAO), KDU691, and GSK-A1 are inhibitors of PI4K. PPK-1 is the C. elegans homolog of mammalian PI4P-5 kinase. (B) Representative confocal images showing the effects of PAO (100 μM), KDU691 (500 μM), and GSK-A1 (100 μM) on mKate2::P4M expression before and after wounding. Pcol-19-mKate2::P4M(zjuSi333) transgenic animals were used for the experiment. “W” indicates the wound site. Scale bar: 10 µm. (C) Quantitation analysis of the mKate2::P4M intensity ratio (ΔFw/Fuw) at 3 h after wounding in animals treated with PAO, KDU691, and GSK-A1 (B). The error bars represent the mean value. ± SD (n = 32, 38, 54, 18 from the left to the right), Mann–Whitney test, ***P < 0.001. (D) Representative confocal images showing the effects of PAO (100 μM), KDU691 (500 μM), and GSK-A1 (100 μM) on PH::GFP expression before and after wounding. Pcol-19-PH::GFP(zjuSi175) transgenic animals were used for the experiments. Scale bar: 10 µm. (E) Quantitation analysis of the PH::GFP intensity ratio (ΔFw/Fuw) at 3 h after wounding in animals treated with PAO, KDU691, and GSK-A1 (D). The error bars represent the mean value. ± SD (n = 33, 36, 37, 21 from the left to the right), Mann–Whitney test, ***P < 0.001. (F) Quantitation analysis of trypan blue staining % in N2 worms 6 h after wounding and treatment with PAO (100 μM) and GSK-A1 (100 μM). The error bars represent the mean value. ± SD (n = 3), Student’s t test, ***P < 0.001. (G) An illustration shows the mechanism of the rapamycin-induced FRB-FKBP dimerization system to deplete PtdIns4P. (H) Representative confocal images of GFP::FKBP::SAC1, tagBFP::FRB::P4M, and PH::mKate2 after rapamycin (100 μM) treatment on the accumulation of tagBFP::FRB::P4M and PH::mKate2 at 3 h after wounding. Pcol-19-PH::mKate2(zjuSi321) II; Pcol-19-P4M::FRB::tagBFP; Pcol-19-GFP::FKBP::SAC1(zjuEx2307) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (I) Quantitation analysis of the mKate2::P4M intensity ratio (ΔFw/Fuw) at 3 h after wounding on rapamycin-treated worms (H). The error bars represent the mean value ± SD (n = 17 and 16). Mann–Whitney test, *P < 0.05. (J) Representative confocal images showing the PH::GFP on ppk-1(L218,226I) mutant worms before and after wounding. The leucines at positions 218 and 226 in the PPK-1 protein have been mutated to isoleucine in ppk-1(syb6134) mutant animals. Scale bar: 10 µm. (K) Quantitation analysis of the intensity change (ΔFw/Fuw) of PH::GFP after wounding (J). Error bars represent the mean value ± SD (n = 44 and 115). Mann–Whitney test, ***P < 0.001. (L) Representative confocal single-plane images showing the colocalization GFP::PPK-1 and mKate2::P4M before and after wounding. Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-GFP::PPK-1(zjuSi367) transgenic animals were used for wounding and imaging. The white dotted box indicates the zoom-in region. Light blue arrows indicate the colocalized puncta. Scale bar: 10 µm. (M) Representative confocal single-plane images showing the colocalization GFP::PPK-1 and mKate2::P4M, as well as GFP::PPK-1 and PH::mKate2, before and after wounding. Pcol-19-PH::mKate2(zjuSi321); Pcol-19-GFP::PPK-1(zjuSi367), Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-GFP::PPK-1(zjuSi367) transgenic animals were used for wounding and imaging. The white dotted box indicates the zoom-in region. Light blue arrows indicate the colocalized puncta. Scale bar: 10 µm. See also Fig. S2 and Video 8.

PtdIns(4,5)P ₂ local generation depends on PtdIns4P, PI4K, and PI4P5K. (A) A diagram illustrating the synthesis of PtdIns(4,5)P₂ from PI and PtdIns4P by PI4K and PI4P5K. Phenyl arsine oxide (PAO), KDU691, and GSK-A1 are inhibitors of PI4K. PPK-1 is the C. elegans homolog of mammalian PI4P-5 kinase. (B) Representative confocal images showing the effects of PAO (100 μM), KDU691 (500 μM), and GSK-A1 (100 μM) on mKate2::P4M expression before and after wounding. Pcol-19-mKate2::P4M(zjuSi333) transgenic animals were used for the experiment. “W” indicates the wound site. Scale bar: 10 µm. (C) Quantitation analysis of the mKate2::P4M intensity ratio (ΔF_w/F_uw) at 3 h after wounding in animals treated with PAO, KDU691, and GSK-A1 (B). The error bars represent the mean value. ± SD (n = 32, 38, 54, 18 from the left to the right), Mann–Whitney test, ***P < 0.001. (D) Representative confocal images showing the effects of PAO (100 μM), KDU691 (500 μM), and GSK-A1 (100 μM) on PH::GFP expression before and after wounding. Pcol-19-PH::GFP(zjuSi175) transgenic animals were used for the experiments. Scale bar: 10 µm. (E) Quantitation analysis of the PH::GFP intensity ratio (ΔF_w/F_uw) at 3 h after wounding in animals treated with PAO, KDU691, and GSK-A1 (D). The error bars represent the mean value. ± SD (n = 33, 36, 37, 21 from the left to the right), Mann–Whitney test, ***P < 0.001. (F) Quantitation analysis of trypan blue staining % in N2 worms 6 h after wounding and treatment with PAO (100 μM) and GSK-A1 (100 μM). The error bars represent the mean value. ± SD (n = 3), Student’s t test, ***P < 0.001. (G) An illustration shows the mechanism of the rapamycin-induced FRB-FKBP dimerization system to deplete PtdIns4P. (H) Representative confocal images of GFP::FKBP::SAC1, tagBFP::FRB::P4M, and PH::mKate2 after rapamycin (100 μM) treatment on the accumulation of tagBFP::FRB::P4M and PH::mKate2 at 3 h after wounding. Pcol-19-PH::mKate2(zjuSi321) II; Pcol-19-P4M::FRB::tagBFP; Pcol-19-GFP::FKBP::SAC1(zjuEx2307) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (I) Quantitation analysis of the mKate2::P4M intensity ratio (ΔF_w/F_uw) at 3 h after wounding on rapamycin-treated worms (H). The error bars represent the mean value ± SD (n = 17 and 16). Mann–Whitney test, *P < 0.05. (J) Representative confocal images showing the PH::GFP on ppk-1(L218,226I) mutant worms before and after wounding. The leucines at positions 218 and 226 in the PPK-1 protein have been mutated to isoleucine in ppk-1(syb6134) mutant animals. Scale bar: 10 µm. (K) Quantitation analysis of the intensity change (ΔF_w/F_uw) of PH::GFP after wounding (J). Error bars represent the mean value ± SD (n = 44 and 115). Mann–Whitney test, ***P < 0.001. (L) Representative confocal single-plane images showing the colocalization GFP::PPK-1 and mKate2::P4M before and after wounding. Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-GFP::PPK-1(zjuSi367) transgenic animals were used for wounding and imaging. The white dotted box indicates the zoom-in region. Light blue arrows indicate the colocalized puncta. Scale bar: 10 µm. (M) Representative confocal single-plane images showing the colocalization GFP::PPK-1 and mKate2::P4M, as well as GFP::PPK-1 and PH::mKate2, before and after wounding. Pcol-19-PH::mKate2(zjuSi321); Pcol-19-GFP::PPK-1(zjuSi367), Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-GFP::PPK-1(zjuSi367) transgenic animals were used for wounding and imaging. The white dotted box indicates the zoom-in region. Light blue arrows indicate the colocalized puncta. Scale bar: 10 µm. See also Fig. S2 and Video 8.