Genome-tagging of Yl receptor and Yl-mediated Yolk endocytosis in vitellogenic oocyte.

(A) Schematics of Yl genome-tagging construct. The amino acid sequences of the encoded Yl protein that flank the eGFP-3xHA insertion is annotated on top of the illustration. Black lines: introns and 5’ and 3’ untranslated regions (UTR) of yl gene. Rectangular boxes: exons of yl gene with the coding regions shaded in gray. (B) Schematics of Yl-eGFP-3xHA fusion protein (left) and closeup view of the C-terminal cytoplasmic tail of Yl protein with the eGFP-3xHA insertion (right). TM, transmembrane domain. (C) Western blot analysis of homogenates from adult female flies probed independently with anti-HA or anti-GFP antibodies, as indicated. A single ~240kDa band positive for both GFP and HA was present in the flies transgenic for Yl-eGFP-3xHA genome-tagging construct (YL) but absent in w1118 non-transgenic (WT) control. (D) Stage 8 oocyte double-labeled by anti-GFP for Yl-eGFP-3xHA (green) and anti-Yolk (red) proteins, as annotated. Inserts are zoom-in view of the region highlighted in white box, showing enrichment of Yl along the oocyte cortex and the differential distribution of Yl and Yolk on granules of different sizes inside the oocyte. (E-G) Expression and subcellular localization of Yl-eGFP-3xHA in stage 10 oocytes imaged for Yl-GFP-3xHA by anti-GFP (green) and yolk granules (gray) by differential interference contrast (DIC). (E) Low-magnification view of whole egg chamber, showing (bottom) the localization of Yl along the oocyte plasma membrane, (middle) yolk granules inside the oocytes and (top) their overlaying view. (F, G) Surface (F) and cross-section (G) views of a plasma membrane area at high-magnifications. (H-J) TEM of stage 9 egg chambers. (H) Low-magnifications overview, showing an oocyte filled with many electron-dense yolk granules surrounded by follicle cells (FC) and nurse cells (NC), as annotated. (I) Closer-up view of the cortex region of an oocyte. Notice that along the plasma membrane, the presence of many small granules (red arrows) with characteristic electron-dense core in the middle of translucent space surrounded by a single layer of membrane, larger granules of growing sizes with irregular boundaries (marked with “i”) and large mature granules (marked with “m”). (J) High-magnification view of plasma membrane area, showing the presence of CCPs and CCVs (green arrows) and nearby early endosomes (red arrows). Genotypes for (D-G): w1118; p{mini-W+, yl-eGFP-3xHA}. (H-J) w1118. The sizes of the scale bars as annotated inside images.