Figure 5. The Golgi membrane enrichment... | Rockefeller University Press

Figure 5.

The Golgi membrane enrichment provides PtdIns4P for PtdIns(4,5)P2local generation in membrane repair. (A) Representative single-plane confocal images of mKate2::P4M and TGN-38::tagBFP before and after wounding. Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-TGN-38::tagBFP(zjuSi280) transgenic animals were used for wounding and imaging. “W” indicates the wound site. The white dotted box indicates the zoom-in region. Magenta arrows point to the colocalized puncta. Scale bar: 10 µm. (B) Mander's colocalization analysis of mKate2::P4M and TGN-38::tagBFP before and after wounding. The error bars represent the mean value ± SD (n = 24, 22, 60 from the left to the right), Mann—Whitney test, ***P < 0.001. (C) Representative single-plane confocal images showing the recruitment of PH::GFP and TGN-38::tagBFP before and after wounding. Pcol-19-PH::GFP(zjuSi175); Pcol-19-TGN-38::tagBFP(zjuSi280) transgenic animals were used for wounding and imaging. The white dotted box indicates the zoom-in region. Magenta arrows indicate the colocalized puncta. Scale bar: 10 µm. (D) Mander’s colocalization analysis of PH::GFP and TGN-38::tagBFP before and after wounding. The error bars represent the mean value ± SD (n = 28, 23, 24 from the left to the right), Mann—Whitney test, ***P < 0.001. (E) Representative confocal images showing PH::GFP before and after wounding with the treatments of BFA (150 µg/ml), rab-1 RNAi, and rab-6.2 RNAi, as well as rab-6.2(ok2254) mutant. Pcol-19-PH::GFP(zjuSi175) for drug treatments and Pcol-19-PH::GFP(zjuSi175);Pcol-19-RDE-1(juIs346); rde-1(ne219) for RNAi treatment. Scale bar: 10 µm. (F) Quantitation analysis of the intensity ratio (ΔFw/Fuw) of PH::GFP in BFA (150 µg/ml) and rab-1, rab-6.2 RNAi, and rab-6.2(ok2254) mutant animals after wounding (E). The error bars represent the mean value ± SD (n = 91, 19, 24, 44, 93 from the left to the right). One-way ANOVA multiple comparisons, ***P < 0.001. **P < 0.01. (G) Representative confocal images of TGN-38:: FRB::tagBFP and PH::mKate2 accumulation at 3 h after wounding after rapamycin (100 µM) treatment. Pcol-19-PH::mKate2(zjuSi321) II; Pcol-19-TGN-38::FRB::tagBFP; Pcol-19-GFP::FKBP::SAC1(zjuEx2307) animals were used for wounding and imaging. Scale bar: 10 µm. (H) Quantitation analysis of the intensity ratio (ΔFw/Fuw) of PH::mKate2 in rapamycin and DMSO-treated worms (G). The error bars represent the mean value ± SD (n = 13 and 16). Mann–Whitney test, ***P < 0.001. (I) Quantitation analysis of the intensity ratio (ΔFw/Fuw) of TGN-38::tagBFP and mKate2::P4M with GSK-A1 (100 μM) treatment. The error bar represents the mean value ± SD (n = 54, 47, 24, 17 from the left to the right). Mann—Whitney test, ***P < 0.001. (J) Representative confocal images of mKate2::P4M before and after wounding with the treatment of zc8.6 RNAi. Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-RDE-1(juIs346); rde-1(ne219) animal was used for wounding and imaging. Scale bar: 10 µm. (K) Quantitation analysis of the intensity ratio (ΔFw/Fuw) of mKate2::P4M. The error bar represents the mean value ± SD (n = 30 and 19), Mann–Whitney test, **P < 0.01. (L) Representative confocal images of PH::GFP before and after wounding with the treatment of zc8.6 RNAi. Pcol-19-PH::GFP(zjuSi175); Pcol-19-RDE-1(juIs346); rde-1(ne219) animal was used for wounding and imaging. Scale bar: 10 µm. (M) Quantitation analysis of the intensity ratio (ΔFw/Fuw) of PH::GFP. The error bar represents the mean value ± SD (n = 31 and 15), Mann–Whitney test, **P < 0.01. (N) Illustration of the proposed model on how the Golgi apparatus-originated translocation to the wound site to deliver PtdIns4P for PtdIns(4,5)P2 generation in C. elegans epidermal membrane repair. See also Fig. S3 and Video 10.

The Golgi membrane enrichment provides PtdIns4P for PtdIns(4,5)P ₂ local generation in membrane repair. (A) Representative single-plane confocal images of mKate2::P4M and TGN-38::tagBFP before and after wounding. Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-TGN-38::tagBFP(zjuSi280) transgenic animals were used for wounding and imaging. “W” indicates the wound site. The white dotted box indicates the zoom-in region. Magenta arrows point to the colocalized puncta. Scale bar: 10 µm. (B) Mander's colocalization analysis of mKate2::P4M and TGN-38::tagBFP before and after wounding. The error bars represent the mean value ± SD (n = 24, 22, 60 from the left to the right), Mann—Whitney test, ***P < 0.001. (C) Representative single-plane confocal images showing the recruitment of PH::GFP and TGN-38::tagBFP before and after wounding. Pcol-19-PH::GFP(zjuSi175); Pcol-19-TGN-38::tagBFP(zjuSi280) transgenic animals were used for wounding and imaging. The white dotted box indicates the zoom-in region. Magenta arrows indicate the colocalized puncta. Scale bar: 10 µm. (D) Mander’s colocalization analysis of PH::GFP and TGN-38::tagBFP before and after wounding. The error bars represent the mean value ± SD (n = 28, 23, 24 from the left to the right), Mann—Whitney test, ***P < 0.001. (E) Representative confocal images showing PH::GFP before and after wounding with the treatments of BFA (150 µg/ml), rab-1 RNAi, and rab-6.2 RNAi, as well as rab-6.2(ok2254) mutant. Pcol-19-PH::GFP(zjuSi175) for drug treatments and Pcol-19-PH::GFP(zjuSi175);Pcol-19-RDE-1(juIs346); rde-1(ne219) for RNAi treatment. Scale bar: 10 µm. (F) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of PH::GFP in BFA (150 µg/ml) and rab-1, rab-6.2 RNAi, and rab-6.2(ok2254) mutant animals after wounding (E). The error bars represent the mean value ± SD (n = 91, 19, 24, 44, 93 from the left to the right). One-way ANOVA multiple comparisons, ***P < 0.001. **P < 0.01. (G) Representative confocal images of TGN-38:: FRB::tagBFP and PH::mKate2 accumulation at 3 h after wounding after rapamycin (100 µM) treatment. Pcol-19-PH::mKate2(zjuSi321) II; Pcol-19-TGN-38::FRB::tagBFP; Pcol-19-GFP::FKBP::SAC1(zjuEx2307) animals were used for wounding and imaging. Scale bar: 10 µm. (H) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of PH::mKate2 in rapamycin and DMSO-treated worms (G). The error bars represent the mean value ± SD (n = 13 and 16). Mann–Whitney test, ***P < 0.001. (I) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of TGN-38::tagBFP and mKate2::P4M with GSK-A1 (100 μM) treatment. The error bar represents the mean value ± SD (n = 54, 47, 24, 17 from the left to the right). Mann—Whitney test, ***P < 0.001. (J) Representative confocal images of mKate2::P4M before and after wounding with the treatment of zc8.6 RNAi. Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-RDE-1(juIs346); rde-1(ne219) animal was used for wounding and imaging. Scale bar: 10 µm. (K) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of mKate2::P4M. The error bar represents the mean value ± SD (n = 30 and 19), Mann–Whitney test, **P < 0.01. (L) Representative confocal images of PH::GFP before and after wounding with the treatment of zc8.6 RNAi. Pcol-19-PH::GFP(zjuSi175); Pcol-19-RDE-1(juIs346); rde-1(ne219) animal was used for wounding and imaging. Scale bar: 10 µm. (M) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of PH::GFP. The error bar represents the mean value ± SD (n = 31 and 15), Mann–Whitney test, **P < 0.01. (N) Illustration of the proposed model on how the Golgi apparatus-originated translocation to the wound site to deliver PtdIns4P for PtdIns(4,5)P₂ generation in C. elegans epidermal membrane repair. See also Fig. S3 and Video 10.