mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes

Abstract

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-alpha synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. Here, we show, by (immuno)electron microscopy that the exocytic pathway in stage 9-10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, we show that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way, by which the oocyte implements polarized deposition of transmembrane/secreted proteins. We propose that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons.

Figure 1.

Examination of the exocytic pathway in Drosophila oocytes. (A) Schematic representation of a stage 9–10 egg chamber with the oocyte abutting the 15 nurse cells and surrounded by a layer of somatic follicle cells. The dorsal/anterior corner is depicted in black (see Materials and Methods) and is where both Gurken protein and gurken RNA are known to localize. WT (B–D) and dCOG5-GFP (E and F) Drosophila egg chambers were processed for gurken RNA in situ hybridization (B), immunoelectron microscopy (C and E), conventional electron microscopy (D), and immunofluorescence (F). (C) Representation of a tER-Golgi unit (in brackets), according to the criteria described in the text, exhibiting a portion of an ER cisterna, one ER exit site, and a Golgi complex comprising a Golgi stack (G). (D) All tER-Golgi units visualized on the ultrathin resin stage 9 oocyte section are marked by an asterisk (*). (E) Ultrathin cryosections of dCOG5-GFP–expressing egg chambers were labeled for GFP (10-nm gold). The tER-Golgi unit (in brackets) is positive for dCOG5-GFP as were all the other in the oocyte cryosection. (F) Confocal section of a stage 9 dCOG5-GFP expressing egg chambers were labeled for GFP. The dots represent the tER-Golgi units in the oocyte, in the surrounding follicle cells and nurse cells. Note that the tER-Golgi units are randomly distributed within the ooplasm without concentration around the oocyte nucleus (N). The center of the oocyte shows a dimmer labeling intensity, due to penetration problems of the GFP antibody. Posterior, anterior, dorsal, and ventral are indicated by P, A, D, and V, respectively. N, nucleus. Bars, 200 nm (C and E); 5 μm (D and F).

Figure 7.

The ER is continuous in the Drosophila oocyte. (A–D) FRAP experiment on PDI-GFP egg chambers shows that the ER pervades the oocyte and comprises a single lumen. Photobleaching was performed for 30 s at 100% laser power in a selected area (square box, B), but diffusion from the surrounding ER enables recovery of the GFP signal (C), which is fully restored after 5 min (D). Cornichon mutants (strong allelic combination cni^AR55/cni^AA12) were labeled for Gurken protein by immunofluorescence (E and F) or IEM (G—J; 10-nm gold, arrows) and for mRNA (K and L). In this genetic background, the nucleus fails to move to the D/A corner in 70% of the oocytes and is localized at the posterior pole (F and L). The IEM micrographs (G–J) were generated from such an oocyte, and Gurken is present throughout the ER in all areas, reaching the anterior side after being synthesized at the posterior pole (L). The tER-Golgi units were not labeled. The egg chambers are oriented as in Figure 1A. N, oocyte nucleus. Bars, 10 μm (E and F); 100 nm (G–J).

Figure 2.

Gurken in the tER-Golgi units at the D/A corner. WT egg chamber cryosections were double labeled for Gurken (10-nm gold) and dSec23p (15-nm gold). Different areas of the oocyte sections were examined. (A) Area 1 corresponds to the D/A corner (see Materials and Methods, inset; the nucleus serves as a landmark). Gurken protein (arrows) is present in all the tER-Golgi units in this area that are also labeled for dSec23p. (B) Area 2 corresponds to the anterior side of the oocyte (inset) toward the middle. (C) Area 3 corresponds to the V/A corner (inset). In areas 2 and 3, the tER-Golgi units are negative for Gurken protein but positive for dSec23p. Bar, 200 nm (inset, 5 μm).

Figure 3.

Gurken colocalizes with dCOG5-GFP at the D/A corner. (A) Stage 9–10 WT egg chambers were processed for immunofluorescence and labeled for Gurken (red). Gurken is present in dots around the oocyte nucleus (N), in the ER at the D/A corner (white arrows), and in the intercellular space between the oocyte and the follicle cells at the D/A corner (white arrowheads). (B–D) dCOG5-GFP–expressing egg chambers were double labeled for Gurken (B, red) and GFP (C, green). Note that the Gurken-positive dots show complete overlap with dCOG5-GFP (D, merge) and therefore represent individual tER-Golgi units. Note that the Gurken in the ER (white arrows) does not overlap with dCOG5-GFP. The number of Gurken-positive tER-Golgi units is higher than 10 (see Results), because the thickness of the confocal section presented here is 3 times higher than this of cryosections. The egg chambers are oriented as in Figure 1A. Bar, 5 μm (A and D).

Figure 4.

Yolkless is transported in all tER-Golgi units. Cryosections of stage 9–10 WT egg chambers were labeled for the Gurken (15 nm) and Yolkless (10 nm). Yolkless, on its way to the plasma membrane, passes through all the tER-Golgi units of the ooplasm, at the D/A corner (A, together with Gurken), at the V/A corner (B) and at the posterior pole (P) (C). Arrows mark the specific Yolkless labeling. The 10-nm gold in A that is not marked by arrows is an artifact due, in this instance, to the double labeling technique. Bar, 200 nm.

Figure 5.

Gurken protein in the posterior tER-Golgi units of merlin mutants. Homozygous stage 9–10 merlin egg chambers were processed for gurken mRNA in situ hybridization (A), immunofluorescence of Gurken protein (B), and cryosectioned and labeled for Gurken (15-nm gold) (C and D). Note that only the tER-Golgi units next to the oocyte nucleus (N) at the posterior pole are Gurken positive (C), whereas these of the anterior side are negative (D). The white arrows in B point to Gurken-positive structures in posterior follicle cells, representing presumably endosomes, and the white arrowheads point to tER-Golgi units at the oocyte posterior pole. The dashed white line represents the contours of the oocyte. The egg chambers are oriented as in Figure 1A. P, posterior pole. Bar, 200 nm.

Figure 6.

Gurken protein in the tER-Golgi units of K10 and squid¹ mutants. Homozygous K10 (A–D) and squid¹ (E–H) egg chambers were processed for gurken mRNA in situ hybridization (A and E), or cryosectioned and labeled for Gurken (15-nm gold) (B–D, F–H). Different areas of the oocyte sections were examined. Area 1 (B and F) corresponds to the D/A corner (the oocyte nucleus [N] serves as a landmark), area 2 (C and G) corresponds to the anterior side of the oocyte, and area 3 (D and H) corresponds to the V/A corner (see legend for Figure 2). The tER-Golgi units (in brackets) were all positive for Gurken protein. Bar, 200 nm.

Figure 8.

Gurken diffusion in the ER can be induced. Gurken protein null background (grk^2B6/grk^2E12) egg chambers expressing the truncated form of Gurken lacking its transmembrane and cytoplasmic domain (gΔTC) (A and B), WT egg chambers (C and D), and cni^CF5/cni^AR55 (E and F), mock- (C and E) and brefeldin A-treated (D and F) were labeled for Gurken for immunofluorescence. Note that Gurken has diffused in the ER when it is truncated and when the egg chambers are treated with BFA (arrows in D and F). The egg chambers are oriented as in Figure 1A. N, oocyte nucleus. Bar, 10 μm.