Triggered Golgi membrane enrichment promotes PtdIns(4,5)P2 generation for plasma membrane repair

Abstract

The maintenance of plasma membrane integrity and a capacity for efficiently repairing damaged membranes are essential for cell survival. Large-scale wounding depletes various membrane components at the wound sites, including phosphatidylinositols, yet little is known about how phosphatidylinositols are generated after depletion. Here, working with our in vivo C. elegans epidermal cell wounding model, we discovered phosphatidylinositol 4-phosphate (PtdIns4P) accumulation and local phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] generation at the wound site. We found that PtdIns(4,5)P2 generation depends on the delivery of PtdIns4P, PI4K, and PI4P 5-kinase PPK-1. In addition, we show that wounding triggers enrichment of the Golgi membrane to the wound site, and that is required for membrane repair. Moreover, genetic and pharmacological inhibitor experiments support that the Golgi membrane provides the PtdIns4P for PtdIns(4,5)P2 generation at the wounds. Our findings demonstrate how the Golgi apparatus facilitates membrane repair in response to wounding and offers a valuable perspective on cellular survival mechanisms upon mechanical stress in a physiological context.

Graphical abstract

Figure 1.

Wounding triggers PtdIns4P accumulation and PtdIns(4,5)P₂ generation at the wound site. (A) Illustration showing C. elegans epidermal cell hyp7 as a model for membrane repair. In this study, membrane components PtdIns(4,5)P2 were labeled by the PH domain from PLC-δ1, and PtdIns4P were labeled by the P4M domain from L. pneumophila SidM. (B) Time-lapse images showing the accumulation of PH::GFP at the wound site. “W” indicates the wound site. Pcol-19-PH::GFP (zjuSi175) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (C) Representative HiS-SiM single-plane images of PH::GFP before and after wounding. Pcol-19-PH::GFP(zjuSi175) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (D) Representative confocal time-lapse images of the recruitment of mKate2::P4M and PH::GFP between 5 to 90 min after wounding. Pcol-19-PH::GFP(zjuSi175);Pcol-19-mKate2::P4M (zjuSi333) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (E) Line graph showing the intensity change (ΔF_w/F_uw) of mKate2::P4M and PH::GFP at different time points after wounding. The plot indicates the mean ± SD (n = 6 for mKate2::P4M curve and 8 for PH::GFP curve) at each time point. (F) Fluorescent recovery after photobleaching (FRAP) analysis of the recovery capability of mKate2::P4M and PH::GFP before and 1 h after wounding. Pcol-19-PH::GFP(zjuSi175);Pcol-19::mKate2::P4M(zjuSi333) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (G) Mobile fraction (F_m) analysis of FRAP of mKate2::P4M and PH::GFP in F. Error bars represent the mean value ± SD (n = 27, 18, 27, 18 from the left to the right), Mann–Whitney test, ***P < 0.001. See also Fig. S1; and Videos 1, 2, 3, 4, 5, 6, and 7.

Figure S1.

Epidermal membrane wounding triggers PtdIns4P accumulation and PtdIns(4,5)P₂ generation at the wound site, related to Fig. 1. (A) Representative single-plane confocal images showing the colocalization of myr::mKate2 and PH::GFP before and after wounding. Pcol-19-myr::mKate2(zjuSi46); Pcol-19-PH::GFP(zjuSi175) transgenic animals were used for wounding and imaging. “W” indicates the wound site. White dotted boxes indicate the zoom-in region. Blue arrows pointed to the colocalized puncta, green arrows pointed to PH::GFP puncta, and magenta arrows pointed to myr::mKate2 puncta. Scale bar: 10 µm. (B) HiS-SiM time-lapse images of PH::GFP in wounded Pcol-19-PH::GFP(zjuSi175) animals. Scale bar: 10 µm. (C) Representative single-plane confocal images showing the colocalization of mKate2::P4M and PH::GFP before and after wounding. Pcol-19-PH::GFP(zjuSi175); Pcol-19-mKate2::P4M(zjuSi333) transgenic animals were used for wounding and imaging. White dotted boxes indicate the zoom-in region. Magenta arrows indicate the colocalized puncta. Scale bar: 10 µm. (D) Line graph showing the intensity change (ΔF_w/F_uw) of unwounded and wounded worms after FRAP. The mean ± SD at each time point is indicated (n = 27, 18, 27, 18 from the top to the bottom).

Figure 2.

PtdIns(4,5)P₂ 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.

Figure S2.

PtdIns(4,5)P₂ generation depends on PtdIns4P, PI4K, and PPK-1 activity, related to Fig. 2. (A) Representative single-plane confocal images showing GFP::FKBP::SAC-1, PH::mKate2, and tagBFP::FRB::P4M without rapamycin treatment before and after wounding. Pcol-19-PH::mKate2(zjuSi321) II; Pcol-19-P4M::FKBP12(mTOR)::tagBFP; Pcol-19-GFP::FKBP1A(FKBP 2-108AA)::SAC1(2-517AA)(zjuEx1859) transgenic animals were used for wounding and imaging. “W” indicates the wound site. Scale bar: 10 µm. (B) Representative single-plane confocal images showing GFP::FKBP::SAC-1, and PH::mKate2 with FRB, as well as tagBFP::FRB::P4M and PH::mKate2 without FKBP before and after wounding. Worms are treated with rapamycin 100 µM). Pcol-19-PH::mKate2(zjuSi321) II; Pcol-19-GFP::FKBP1A(FKBP 2-108AA)::SAC1(2-517AA)(zjuEx1865) and Pcol-19-PH::mKate2(zjuSi321); Pcol-19-GFP::FKBP1A(FKBP 2-108AA)::SAC1(2-517AA)(zjuEx1865) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (C) Representative confocal images of mKate2::P4M before and after wounding with ppk-1 RNAi treatment. Pcol-19-mKate2::P4M(zjuSi333) animals were used for wounding and imaging. Scale bar: 10 µm. (D) Representative confocal images of PH::GFP before and after wounding with ppk-1 RNAi treatment. Pcol-19-PH::GFP(zjuSi175) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (E) Quantitation analysis of the PH::GFP intensity ratio (ΔF_w/F_uw) at 3 h after wounding (D). The error bars represent the mean value ± SD (n = 41 and 33), one-way ANOVA multiple comparisons test, ***P < 0.001. (F) Comparison of the amino acid sequence of human PIP5K1A and C. elegans PPK-1. (G) Experimental design of ppk-1(syb6134) double point mutation based on the comparison of the nucleotide sequence of ppk-1(wt) and ppk-1(syb6134). Below is the secondary structure of ppk(syb6134) mutation. (H) Representative confocal images of Pppk-1::GFP showing the expression of ppk-1 in multiple tissues of young adult C. elegans. (I) Representative single-plane confocal images of myr::mKate2 and GFP::PPK-1 colocalization before and after wounding. Pcol-19-myr::mKate2(zjuSi46); Pcol-19-GFP::PPK-1(zjuSi367) transgenic animals were used for wounding and imaging. White dotted boxes indicate the zoom-in region. Magenta arrows indicate the colocalized puncta. Scale bar: 10 µm. (J) Representative time-lapse confocal images of the recruitment of GFP::PPK-1 and mKate2::P4M between 5 to 60 min after wounding. Pcol-19-GFP::PPK-1(zjuSi367); Pcol-19-mKate2::P4M(zjuSi333) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (K) Diagram of PPK-1 secondary structure and representative confocal images of the recruitment of truncated PPK-1 to the wound site. Pcol-19-PPK-1(1–453)::GFP(zjuEx2147), Pcol-19-GFP::PPK-1(c)(zjuEx2142), Pcol-19-PPK-1(1–85)::GFP(zjuEx2265,) Pcol-19-PPK-1(84–457)::GFP(zjuEx2267) transgenic animals were used for wounding and imaging. Scale bar: 10 µm.

Figure S3.

Wounding triggers the enrichment of the Golgi membrane at the wound site, related to Fig. 3. (A) Representative single-plane confocal images showing the localization of the Golgi apparatus (stained with Golgi-tracker-red) before and after wounding in N2 worms. “W” indicates the wound site. Scale bar: 10 µm. (B) Representative single-plane confocal images showing the colocalization of TGN-38::tagBFP, GFP::MANS-2, and mKate2::R12B2.2 before and after wounding. Pcol-19-TGN-38::tagBFP(zjuSi280); Pcol-19-GFP::MANS-2(zjuSi302); Pcol-19-mKate2::R12B2.2(zjuEx2075) transgenic animals were used for wounding and imaging. Magenta arrows indicate the colocalized puncta. Scale bar: 10 µm. (C) Representative HiS-SiM single-plane images showing the colocalization 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. “W” indicates the wound site. Scale bar: 10 µm. (D) Representative confocal images of mKate2::P4M with the treatment of rab-1 RNAi, rab-6.2 RNAi, and BFA (150 µg/ml), as well as in rab-6.2(ok2254) mutant animals before and after wounding. Pcol-19-mKate2::P4M(zjuSi333) transgenic animals were used for drug treatment, Pcol-19-mKate2::P4M(zjuSi333); Pcol-19-RDE-1(juIs346); rde-1(ne219) for RNAi treatments. Scale bar: 10 µm. (E) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) for the different treatments shown in (E). The error bars represent the mean value ± SD (n = 156, 19, 22, 138, 134 from the left to the right), Mann–Whitney test, ***P < 0.001. **P < 0.01. (F) Representative confocal images showing TGN-38::tagBFP and mKate2::P4M with the treatment of GSK-A1 (100 μM) before and after wounding. Pcol-19-TGN-38::tagBFP(zjuSi280); Pcol-19-mKate2::P4M(zjuSi333) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (G) Representative single-plane confocal images showing the colocalization of mKate2::ZC8.6 with TGN-38::tagBFP or GFP::MANS-2. Pcol-19-TGN-38::tagBFP(zjuSi280); Pcol-19-mKate2::ZC8.6(zjuEx2330), Pcol-19-GFP::MANS-2(zjuSi302); Pcol-19-mKate2::ZC8.6(zjuEx2320) transgenic animals were used for wounding and imaging. Scale bar: 10 µm.

Figure 3.

Wounding triggers the accumulation of the Golgi membrane at the wound site. (A) Diagram showing the location of Golgi apparatus compartment markers and the target of pharmacological inhibitors. (B) Representative confocal images showing the distribution of Golgi apparatus markers before and after wounding. The markers include GFP::RAB-1 (ERGIC), GFP::MANS-2 (medial), GFP::RAB-6.2 (trans), and TGN-38::tagBFP (TGN), respectively. Pcol-19-GFP::RAB-6.2(zjuSi313), Pcol-19-TGN-38::tagBFP(zjuSi280), Pcol-19-GFP::MANS-2(zjuSi302), and Pcol-19-GFP::RAB-1(zjuSi253) transgenic animals were used for wounding and imaging. “W” indicates the wound site. Scale bar: 10 µm. (C) HiS-SiM single-plane images showing the accumulation of TGN-38::tagBFP and GFP::MANS-2 at the wound site. Pcol-19-TGN-38::tagBFP(zjuSi280) and Pcol-19-GFP::MANS-2(zjuSi302) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (D) Representative single-plane confocal images showing the colocalization of TGN-38::tagBFP and myr::mKate2 before and after wounding. Pcol-19-TGN-38::tagBFP(zjuSi280); Pcol-19-myr::mKate2(zjuSi46) transgenic animals were used for wounding and imaging. “W” indicates the wound site. Scale bar: 10 µm. See also Fig. S3 and Video 9.

Figure 4.

The accumulation of the Golgi membrane at the wound site is required for membrane repair. (A) Representative confocal images showing TGN-38::tagBFP before and after wounding with the treatment of rab-1 and rab-6.2 RNAi, as well as rab-6.2(ok2254) deletion mutant. Pcol-19-TGN-38::tagBFP(zjuSi280) for RNAi, and Pcol-19-TGN-38::tagBFP(zjuSi280); Pcol-19-RDE-1(juIs346); rde-1(ne219) transgenic animals were used for wounding and imaging. Scale bar: 10 µm. (B) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of TGN-38::tagBFP after wounding (A). Error bars represent the mean value ± SD (n = 93, 15, 34, 58, 28 from the left to the right). One-way ANOVA multiple comparisons, ***P < 0.001. **P < 0.01. (C) Representative confocal images showing GFP::MANS-2 before and after wounding with the treatment of Blebbistatin (20 µM) and BFA (150 µg/ml). Pcol-19-GFP::MANS-2(zjuSi302) for drug treatment were used for wounding and imaging. Scale bar: 10 µm. (D) Quantitation analysis of the intensity ratio (ΔF_w/F_uw) of GFP::MANS-2 after wounding (C). Error bars represent the mean value ± SD (n = 33, 45, 15, 15 from the left to the right). One-way ANOVA multiple comparisons, ***P < 0.001. (E) Quantitation analysis of the percentage of trypan blue staining of worms at 6 h after wounding treated with BFA (150 µg/ml), rab-1 RNAi, and rab-6.2 RNAi, as well as rab-6.2(ok2254) deletion mutant. N2 strain was used for drug treatments. Pcol-19-RDE-1(juIs346); rde-1(ne219) strain was used for RNAi treatment. The error bars represent the mean value. ± SD (n = 3), Student’s t test, *P < 0.05. ***P < 0.001. (F) Quantitation analysis of the survival rates (%) of animals treated with Blebbistatin (20 µM), rab-1 RNAi, rab-6.2 RNAi, and GSK-A1(100 μM) after wounding. N2 for drug treatments and Pcol-19-RDE-1(juIs346); rde-1(ne219) transgenic animals for RNAi treatments, wounding and survival rate quantification. The error bars represent the mean value ± SD (n = 3), Student’s t test, ***P < 0.001. *P < 0.05. See also Fig. S3.

Figure 5.

The Golgi membrane enrichment provides PtdIns4P for PtdIns(4,5)P₂ 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.