Figure 6.
Endocytic recycling block leads to secretory cargo retention, autophagy, and trans-Golgi enlargement. (A) Confocal images of fat body from wild-type and BM-40-SPARC>Rab11i L3 larvae (feeding stage) showing intracellular accumulation of Collagen IV (α2 chain Vkg-GFP, green). Plasma membrane labeled with myr-RFP (magenta). Rectangular inset magnified on the right side. (B) Fat body from wild-type and BM-40-SPARC>Rab11i larvae showing autophagic vesicle formation (Atg8a-mCherry in magenta; Lamp1-YFP in green). Rectangular inset magnified on the right side. (C) Wild-type and Cg>Rab11i fat body expressing trans-Golgi marker GalT-TagRFP (magenta) and endocytic marker TfR-GFP (green). Upon Rab11 knockdown, TfR signal disappears from the plasma membrane and forms larger puncta connected by thread-like structures. Rectangular insets are shown magnified below. (D) Quantification of the proximity between TfR-GFP and GalT-TagRFP. The upper graph represents proximity in 10 cells measured in images like those in C. The lower graph represents average proximity values (n = 10, error bars indicate SD). 51.7% of trans-Golgi are proximal to TfR puncta in the wild type. Upon Rab11 knockdown, 97.2% of Golgi are proximal to TfR-positive structures (note that TfR signal could not be analyzed as individual puncta in this condition). (E) Wild-type, BM-40-SPARC>Rab11i, and >Ropi fat body expressing trans-Golgi marker GalT-TagRFP (magenta). Rectangular insets are shown magnified below. (F)BM-40-SPARC>Syx1i, >Syx4i and double >Syx1i+Syx4i fat body expressing trans-Golgi marker GalT-TagRFP (magenta). (G) Wild-type, BM-40-SPARC>Rab11i, >Ropi, >Syx1i, >Syx4i, and double >Syx1i+Syx4i fat body expressing mid-Golgi marker ManII-GFP (green). (H) Trans-Golgi and mid-Golgi size measured in images like those in E–G. The data are presented as violin plots where quartiles are marked, with dots representing individual measurements of punctum size. n = 100. P values from unpaired t tests are indicated for differences with the wild type. n.s., P > 0.05; *, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ****, P < 0.0001.