Figure 5. Rop acts with Syx1/Syx4 in... | Rockefeller University Press

Figure 5.

Rop acts with Syx1/Syx4 in trans-Golgi-to–plasma membrane recycling. (A) Confocal images of fat body from wild-type, BM-40-SPARC>Syx1i, >Syx4i and double >Syx1i+Syx4i L3 larvae (feeding stage) showing localization of Collagen IV (α2 chain Vkg-GFP, green). Plasma membrane labeled with BM-40-SPARC-GAL4–driven myr-RFP (magenta). Magnified inset shows intracellular Collagen IV accumulation in >Syx1i+Syx4i fat body, with the Collagen IV channel separated on the right. (B) Fat body from wild-type, BM-40-SPARC>Syx1i, >Syx4i, and >Syx1i+Syx4i larvae showing markers Lamp1-YFP (lysosomes, green) and Atg8a-mCherry (autophagosomes, magenta). Plasma membrane labeled with BM-40-SPARC-GAL4–driven myr-RFP (magenta). Magnified inset shows formation of autophagic vesicles in >Syx1i+Syx4i fat body, with the Atg8a channel separated on the right. (C) Fat body showing localization of Syx1-mRFP (magenta) and trans-Golgi marker GalT-GFP (green), both driven by Cg-GAL4. The graphs on the right quantify their proximity in 10 individual cells (upper graph) and average proximity (lower graphs, n = 10, error bars indicate SD). (D) Fat body showing localization of mRFP-Syx4 (magenta) and trans-Golgi marker GalT-GFP (green), both driven by Cg-GAL4. The graphs quantify their proximity as in C. (E) Fat body expressing Rop-GFP alone (control) or co-expressing Rop-GFP with Syx1-mRFP, Syx4-mRFP, Syx7-mRFP, and Syx17-mRFP, all driven by BM-40-SPARC-GAL4. GFP in green and mRFP in magenta. Rectangular insets are shown magnified below. (F) Quantification of the recruitment of Rop-GFP to the plasma membrane by Syx1, Syx4, Syx7, and Syx17. Each dot represents the ratio of Rop-GFP intensity between plasma membrane and cytoplasm in one cell, measured in images like those in E. n = 10. Horizontal lines mark the mean. P values from unpaired t tests for differences with the control are reported. n.s., P > 0.05; ***, 0.001 > P > 0.0001, and ****, P < 0.0001. (G) Localization of Syx1-mRFP (magenta) in wild-type, Cg >Ropi, and >Rab11i fat body. (H) Localization of Syx4-mRFP (magenta) in wild-type, Cg>Ropi, and >Rab11i fat body. (I) Localization of Rab11-GFP (green) in wild-type, BM-40-SPARC>Ropi, and >Syx1i+Syx4i fat body.

Rop acts with Syx1/Syx4 in trans-Golgi-to–plasma membrane recycling. (A) Confocal images of fat body from wild-type, BM-40-SPARC>Syx1ⁱ, >Syx4ⁱ and double >Syx1ⁱ+Syx4ⁱ L3 larvae (feeding stage) showing localization of Collagen IV (α2 chain Vkg-GFP, green). Plasma membrane labeled with BM-40-SPARC-GAL4–driven myr-RFP (magenta). Magnified inset shows intracellular Collagen IV accumulation in >Syx1ⁱ+Syx4ⁱ fat body, with the Collagen IV channel separated on the right. (B) Fat body from wild-type, BM-40-SPARC>Syx1ⁱ, >Syx4ⁱ, and >Syx1ⁱ+Syx4ⁱ larvae showing markers Lamp1-YFP (lysosomes, green) and Atg8a-mCherry (autophagosomes, magenta). Plasma membrane labeled with BM-40-SPARC-GAL4–driven myr-RFP (magenta). Magnified inset shows formation of autophagic vesicles in >Syx1ⁱ+Syx4ⁱ fat body, with the Atg8a channel separated on the right. (C) Fat body showing localization of Syx1-mRFP (magenta) and trans-Golgi marker GalT-GFP (green), both driven by Cg-GAL4. The graphs on the right quantify their proximity in 10 individual cells (upper graph) and average proximity (lower graphs, n = 10, error bars indicate SD). (D) Fat body showing localization of mRFP-Syx4 (magenta) and trans-Golgi marker GalT-GFP (green), both driven by Cg-GAL4. The graphs quantify their proximity as in C. (E) Fat body expressing Rop-GFP alone (control) or co-expressing Rop-GFP with Syx1-mRFP, Syx4-mRFP, Syx7-mRFP, and Syx17-mRFP, all driven by BM-40-SPARC-GAL4. GFP in green and mRFP in magenta. Rectangular insets are shown magnified below. (F) Quantification of the recruitment of Rop-GFP to the plasma membrane by Syx1, Syx4, Syx7, and Syx17. Each dot represents the ratio of Rop-GFP intensity between plasma membrane and cytoplasm in one cell, measured in images like those in E. n = 10. Horizontal lines mark the mean. P values from unpaired t tests for differences with the control are reported. n.s., P > 0.05; ***, 0.001 > P > 0.0001, and ****, P < 0.0001. (G) Localization of Syx1-mRFP (magenta) in wild-type, Cg >Ropⁱ, and >Rab11ⁱ fat body. (H) Localization of Syx4-mRFP (magenta) in wild-type, Cg>Ropⁱ, and >Rab11ⁱ fat body. (I) Localization of Rab11-GFP (green) in wild-type, BM-40-SPARC>Ropⁱ, and >Syx1ⁱ+Syx4ⁱ fat body.