Figure 1.
CHAT-1 and TAT-1 regulate plasma membrane PS asymmetry and express in the same tissues.
(A–C) DIC and confocal fluorescent images of wild-type (A), tat-1(qx30) (B) and chat-1(qx36) (C) embryos expressing ssGFP::Lact-C2 driven by heat-shock promoters. (D–F) DIC and fluorescent images of dissected gonads stained with FITC-Annexin V from wild type (D), tat-1(qx30) (E) and chat-1(qx36) (F). Arrows in (A–D) indicate apoptotic cells labeled by ssGFP::Lact-C2 or FITC-Annexin V. (G–K) DIC and fluorescent images of wild-type animals expressing both TAT-1::YFP and CHAT-1::CFP driven by endogenous promoters. TAT-1 and CHAT-1 expression can be observed in the same cell types (arrows). (L) Confocal fluorescent images of wild-type animals expressing TAT-1::YFP and CHAT-1::CFP driven by the intestine-specific promoter vha-6. TAT-1 and CHAT-1 colocalize to plasma membranes (arrows) and various intracellular compartments in the intestine. Insets show an amplified view with a magnification of X 1.8. Scale bars: 5 µm.
Figure 2.
Various endocytic compartments are defective in tat-1 and chat-1 mutants.
(A–O) Confocal fluorescent images of the intestine in wild type (A, D, G, J, M), tat-1(qx30) (B, E, H, K, N) and chat-1(qx36) (C, F, I, L, O) that express GFP::RAB-5 (A–C), GFP::RAB-7 (D–F), GFP::RAB-10 (G–I), GFP::RAB-11 (J–L) or mRFP::RME-1 (M–O). Arrowheads indicate labeling of abnormal vacuoles in mutant strains or membrane staining of mRFP::RME-1. Arrows show aggregated intracellular vesicles. (P–Q) Quantification of the average total intensity of GFP::RAB-5 (P) and GFP::RAB-7 (Q) per unit area. (R–S) Quantification of the average number of aggregated structures labeled by GFP::RAB-10 (R) and GFP::RAB-11 (S). (T) Quantification of basolateral mRFP::RME-1-positive structures in (M–O). In (P–T), data are shown as mean±SEM. *P<1.5×10−10. (U–X) Confocal fluorescent images of the intestine in wild type (U, W) and tat-1(qx30) (V, X) that coexpress mCHERRY::RAB-7 and GFP::RAB-5 (U, V) or mRFP::RME-1 and GFP::RAB-10 (W, X). Arrows indicate colocalization of RAB-5 and RAB-7 or RME-1 and RAB-10. Scale bars: 5 µm.
Figure 3.
Cargo recycling is disrupted in tat-1 and chat-1 mutants.
(A–I) Confocal fluorescent images of the intestine in wild type (A, D, G), tat-1(qx30) (B, E, H) and chat-1(qx36) (C, F, I) that express hTfR::GFP (A–C), hTAC::GFP (D–F) or GLUT1::GFP (G–I). GFP signal was mainly seen on plasma membranes in wild type (arrows) but accumulated intracellularly in tat-1 and chat-1 mutants (arrowheads). (J–L) Quantification of intracellular accumulation of hTfR::GFP (J), hTAC::GFP (K) and GLUT1::GFP as shown in (A–I). Data are shown as mean numbers of labeled structures ± SEM. *P<9.0×10−29. (M-Z2) hTfR::GFP (M–T) and GLUT1::GFP (U-Z2) are trapped in abnormal early endosomes in chat-1(RNAi) animals. Merged images of hTfR::GFP or GLUT1::GFP with mCHERRY RAB-5 (M, N, U, V), mCHERRY::RAB-10 (O, P, W, X), mCHERRY::RAB-7 (Q, R, Y, Z) or mRFP::RME-1 (S, T, Z1, Z2) in wild-type and chat-1(RNAi) intestines are shown. Overlap of hTfR or GLUT1 with different endocytic markers is indicated by arrows in wild type and arrowheads in chat-1(RNAi) animals. Scale bars: 5 µm.
Figure 4.
Yolk redistribution and degradation are defective in tat-1 and chat-1 mutants.
(A–D) tat-1 and chat-1 mutations do not affect yolk uptake in oocytes. DIC and confocal fluorescent images of wild type (A), tat-1(qx30) (B), tat-1(tm3110) (C) chat-1(qx36) (D) carrying VIT-2::GFP are shown. Arrows and arrowheads show yolk accumulation in mature oocytes and fertilized embryos, respectively. (F–U) Yolk degradation is affected in tat-1 and chat-1 mutants. Fluorescent images of wild-type (F–I), tat-1(qx30) (J–M), tat-1(tm3110) (N–Q) and chat-1(qx36) (R–U) embryos expressing VIT-2::GFP are shown. (W–Y) tat-1 and chat-1 mutants accumulate enlarged yolk granules in the intestine. DIC and fluorescent images of wild-type (W), tat-1(qx30) (X) and chat-1(qx36) (Y) intestine expressing VIT-2::GFP are shown. Enlarged yolk granules are indicated by arrows. (E, V, Z) Quantification of the average total intensity of VIT-2::GFP per unit area in fertilized early embryos (E), 4-fold stage embryos (V) and intestines (Z). The distribution of average total intensity in 96 (E, V) or 84 (Z) unit areas in each genotype is shown. Purple lines represent the average intensity of VIT-2::GFP in each strain. **P<1.8×10−7, *P<0.05. (Z1–Z3) tat-1 and chat-1 mutants accumulate LGG-1-positive structures in the intestine. DIC and fluorescent images of the intestine in wild type (Z1), tat-1(qx30) (Z2), and chat-1(qx36) (Z3) stained with anti-LGG-1 antibodies are shown. Arrows show LGG-1-positive structures. Average number of LGG-1 puncta in each strain was quantified in Z4. Data are shown as mean±SEM. *P<4.0×10−20. Scale bars: 5 µm.
Figure 5.
CHAT-1::GFP labels tubular structures in the intestine.
CHAT-1::GFP were coexpressed together with mCHERRY or mRFP fusions of various endolysosomal markers in the wild-type intestine. Arrows indicate colocalization of CHAT-1::GFP with different endocytic markers. Arrowheads show tubular structures labeled only by CHAT-1::GFP. Insets show an amplified view with a magnification of X 1.6. Scale bars: 5 µm.
Figure 6.
CHAT-1 tubules are disrupted in rab-10(lf) but are further extended in rme-1(lf) mutants.
(A–C) Confocal fluorescent images of CHAT-1::GFP in the intestine of wild type (A), rab-10(dx2) (B) and rme-1(b1045) (C). Insets have a magnification of X 1.5. (D and E) Confocal fluorescent images of CHAT-1::GFP and mCHERRY::RAB-5 in animals treated with control (D) or rab-10 RNAi (E). Arrows show RAB-5-positive early endosomes and arrowheads indicate CHAT-1-positive tubules. (F–H) Confocal fluorescent images of wild-type (F), rme-1(b1045) (G), rme-1(b1045);rab-5 RNAi (H) intestine expressing CHAT-1::GFP and mCHERRY::RAB-10. Tubular structures labeled by CHAT-1 and RAB-10 (arrows) became further extended in rme-1(b0145) mutants (G), but were abolished by rab-5 RNAi (H). Scale bars: 5 µm.
Figure 7.
Tubular extensions of sorting and recycling compartments are disrupted in tat-1 and chat-1 mutants.
(A) Confocal fluorescent images of wild-type intestine expressing both CHAT-1::GFP and mRFP::RME-1. Arrows indicate colocalization of CHAT-1 and RME-1. (B-D) Confocal fluorescent images of mRFP::RME-1 in wild-type (B), tat-1(qx30) (C) and chat-1(qx36) (D) intestines. RME-1-positive compartments are greatly reduced in number and become globular in tat-1 and chat-1 mutants. (E and F) Confocal fluorescent images of wild-type intestine expressing both CHAT-1::YFP and GLUT1::CFP (E) or GLUT1::GFP and mRFP::RME-1 (F). GLUT1 overlaps with CHAT-1 (E) or RME-1 (F) on tubular or tubulo-vesicular structures in the basolateral membrane area (arrows). (G–I) In wild-type intestine, GLUT1::GFP labels tubular structures (G), which are disrupted in tat-1(qx30) (H) or chat-1(qx36) (I) mutants. Insets have a magnification of X 1.6 in (A, E and F) and X 2 in (B–D and G–I). In all panels, images were taken at the top focus plane, which gives a better view of the basolateral membrane area. Scale bars: 5 µm.
Figure 8.
Endomembrane PS asymmetry is disrupted in tat-1 and chat-1 mutants.
(A–E) PS appears on the cytosolic surface of endocytic compartments. Confocal fluorescent images of wild type carrying GFP::Lact-C2 expressed specifically in the intestine (Pges-1GFP::Lact-C2) and mCHERRY fusions of RAB-5 (A), RAB-10 (B), RME-1 (C), RAB-7 (D) or stained with Lysotracker Red (E) are shown. Arrows show colocalization of GFP::Lact-C2 and endocytic markers. (F) CHAT-1 tubules are coated by PS. Confocal fluorescent images of wild-type intestine expressing both CHAT-1::GFP and mCHERRY::Lact-C2. CHAT-1 and Lact-C2 coincide on tubular membranes (arrows). Insets show an amplified view with a magnification of X 1.6. (G–I) DIC and fluorescent images of coelomocytes that take up and transport ssGFP::Lact-C2 and ssCHERRY endocytosed from the body cavity in wild type (G), tat-1(qx30) (H) and chat-1(qx36) (I). Arrows show endosomes; arrowheads show lysosomes. Internalized GFP::Lact-C2 is absent from endosomes in wild type but labels endomembanes in tat-1(qx30) and chat-1(qx36) mutants. Scale bars: 5 µm.
Figure 9.
Proposed functions of TAT-1/CHAT-1 in endocytic sorting and recycling.
TAT-1 and CHAT-1 localize to plasma membranes and to tubular early endosomes, ERCs and recycling endosomes where they regulate sorting and recycling events by restricting PS to the cytosolic membrane leaflet. TAT-1/CHAT-1 probably promotes tubule formation by enriching PS on the cytosolic leaflet, thereby affecting membrane curvature. RAB-10 and RAB-11 may also contribute to membrane tubulation in early endosomes and ERCs, whereas RME-1/AMPH-1 is involved in membrane fission and tubulation of recycling endosomes. PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomyelin.