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First published online 17 December 2003
doi: 10.1242/dev.00931

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The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary

¹ Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
² Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA

View larger version (137K): [in a new window]   Fig. 1. sec5E10 mutant germlines display defects in membrane addition and egg chamber polarity. (A-C) Wild-type development of the ovariole. Membranes are marked with phalloidin (red), which binds to F-actin, and nuclei with Hoechst 33342 (blue). The oocyte (*) and egg chamber stage are indicated. At stage 7 (B), the oocyte nucleus (*) has moved to an anterior corner of the oocyte. At stage 10 (C), the oocyte has grown disproportionately to the nurse cells while its nucleus (*) remains at the dorsal anterior corner. (D-F) Homozygous sec5E10 germline clones. (D) Mutant egg chambers often fail to exit the germarium (*). Those that do possess multi-nucleate cells, lacking phalloidin-labeling between nuclei (arrowhead) and possessing aggregated ring canals (arrow). (E) By stage 4 or 5, all ring canals are clumped together in the multi-nucleate chamber (arrow). (F) Clones arrest by stage 6. Ring canals are aggregated (arrow) and follicle cells begin to die. Wild-type (G) and sec5E10 (H) egg chambers labeled with fluorescein-conjugated tomato lectin (green) and phalloidin (red). Wild-type (I) and sec5E10 (J) egg chambers stained for Syntaxin (green) and phalloidin (red). (K,K') The oocyte (*), labeled with antibodies to Dynein Heavy Chain (green), is incorrectly positioned in sec5E10 germline clones. Wild-type egg chambers (L) and two z sections through one sec5E10 egg chamber (M). E-cadherin (green) concentrates at the adherens junctions between follicle cells in both mutant and wild type. Polar cells (arrows), labeled by antibodies to FasIII (red), are located at the posterior of the egg chamber in wild type, but are mispositioned in sec5E10 germline clones. All images are single z sections of confocal image stacks. Scale bars: 20 µm.

View larger version (84K): [in a new window]   Fig. 2. Dorsal patterning defects in sec5E13. Stage 10 egg chambers with wild-type (A) or sec5E13 (B-D) germline clones. Some mutants lack phalloidin (red) between nuclei (blue) and have aggregated ring canals (C, arrow). However, many sec5E13 germlines (D) more closely resemble wild type. Wild-type eggs (E) have two stereotypically spaced appendages on the dorsal side. All eggs laid by sec5E13 germline mothers display fused or closely spaced dorsal appendages (F). Scale bars: 20µm.

View larger version (104K): [in a new window]   Fig. 3. Sec5 localization and the establishment of the anteroposterior axis. (A-D,I-K,M) Egg chambers, stage and genotype indicated, were stained with phalloidin (red), Hoechst (blue) and anti-Sec5 monoclonal 22A2 (green). (A,B) Sec5 concentrates posteriorly, at the boundary between the follicle cells and the oocyte. Particularly at stage 5, sec5 resides at the apical end of the two posterior polar cells (arrow in merged image). (C,D) Sec5 persists in the posterior follicle cells (arrows) when the germline is homozygous for sec5E13. (E-H) Follicle cell clones of sec5E13 (marked by the absence of GFP). (E,F) The oocyte is labeled with anti-Orb (also green), and membranes are marked with phalloidin (red). When the posterior follicle cells are homozygous for sec5E13 (F; arrow), the oocyte (*) no longer occupies the posterior-most position. (G) Two z sections through one egg chamber. Again, the sec5E13 follicle cell clone at the posterior (arrows) causes the oocyte (*), labeled with anti-Gurken (green), to reside too anteriorly. Adherens junctions between follicle cells are labeled with anti-E-cadherin (red). (H) In larger sec5E13 follicle cell clones (arrowhead), the epithelium is disordered compared with heterozygous cells (arrow). (I-K) Wild type. At stages 6-8, Sec5 appears increasingly at the anterior of the oocyte (arrows). (L) A colcemid-treated stage 10 egg chamber wherein the oocyte nucleus (*) did not migrate correctly. Sec5 (green) still concentrates at the anterior membranes of the oocyte. (M,N) At stage 10, Sec5 concentrates at the anterior corners (arrows), and is nearly absent from the posterior membrane. (O) Syntaxin (red) is uniformly localized on the oocyte membrane. The nucleus is marked with an asterisk. Scale bars: 20 µm.

View larger version (125K): [in a new window]   Fig. 4. Gurken and yolkless trafficking are disrupted in sec5E13 oocytes. (A-G,J-M) Egg chambers, genotype indicated, were stained with phalloidin (red), Hoechst (blue), and anti-Gurken (green, A-G) or anti-Yolkless (green, J-M). (A,D) At early stages, Gurken accumulates in the oocyte in both wild-type and sec5E13germlines. (B,E) At stage 8, Gurken is mislocalized within the cytoplasm of sec5E13 oocytes. (C,F) Gurken localizes at the anterodorsal membrane in stage 10 wild-type oocytes (arrow and enlargement), but in sec5E13 mutant oocytes accumulates in puncta within the ooplasm. Little Gurken is observed in the membrane (arrow and enlargement). (G) A compressed z stack of a sec5E13 stage 10 egg chamber, displaying mislocalized Gurken protein throughout the ooplasm. (H,I) Oocyte yolk granules visualized by Nomarski optics. (J,K) Yolkless (green) accumulates in the cytosol of stage 7 oocytes. (L,M) By stage 10, Yolkless shifts to the plasma membrane of wild-type but not sec5E13 oocytes. Scale bars: 20 µm.

View larger version (110K): [in a new window]   Fig. 5. Posterior markers are normal in sec5E13 oocytes. (A) Wild-type and (B) sec5E13 oocytes labeled with anti-Oskar (green). (C) Par-1 (green) localizes properly in sec5E13 clones (arrow). Phalloidin (red) and the oocyte nucleus(*) are shown. (D) A Kinesin-ßgal fusion (green) localizes to the posterior end of the sec5E13 oocyte. In wild-type (E,F) and sec5E13 (G,H) egg chambers, Dynein Heavy Chain (green) accumulates in the oocyte between stages 1-6 (E,G) and subsequently (F,H) at its posterior end (arrow). Scale bars: 20 µm.

View larger version (137K): [in a new window]   Fig. 6. The microtubule cytoskeleton is oriented correctly in sec5E13 oocytes, although the oocyte nucleus is displaced from the cortex. (A) Wild-type and (B) sec5E13 stage 7 egg chambers expressing Nod-GFP (green) under the control of nanos-Gal4 and stained with phalloidin (red). (C-F) Stage 10 egg chambers, stained as above. In sec5E13 oocytes, Nod-GFP properly concentrates anteriorly (D). The oocyte nucleus (*) is also often at the dorsal anterior corner. However, the overexpression of Nod-GFP in the germline enhances the sec5E13 phenotype (E,F). (G-H) Single z sections of stage 10 egg chambers labeled with phalloidin (red) and nuclei with Hoechst (blue), and the position of the oocyte nucleus shown with an arrow. (I,J) The full z stack of G and H were rotated in three dimensions and the nurse cells optically removed to reveal the close apposition of the nucleus to the surface in wild type (I), but not in the sec5E13 chamber (J). (K-O) Egg chambers stained for Bicaudal-D (green), phalloidin (red) and with Hoechst (nuclei, blue), with genotypes indicated. (M-O) Bicaudal-D (arrows) remains associated with the nucleus (*) whether in wild type or sec5E13 oocytes. Scale bars: 20 µm.

View larger version (35K): [in a new window]   Fig. 7. Sec5 localization and function during oocyte development. (A) At stage 2, Sec5 is present on all egg chamber membranes at low levels, but concentrates between the posterior follicle cells and the oocyte, particularly at the apical membrane of the polar cells. Throughout the egg chamber, Sec5 is required to preserve the integrity of the growing cells. Sec5 in the posterior follicle cells and oocyte is essential to position the oocyte at the posterior of the egg chamber. (B) By stage 5, Sec5 concentrates also along the anterior oocyte membrane, at a time when this membrane grows disproportionately to the nurse cells. The enrichment of Sec5 in the polar cells persists, and is probably required there to maintain the posterior position of the oocyte. (C) At stage 8, Yolkless rapidly inserts in the oocyte membrane in a Sec5-dependent manner. At this time, Sec5 is present along the oocyte membrane. Sec5 is also moderately enriched at the anterior rim of the oocyte, and is required there for the initial trafficking of Gurken. (D) By stage 10, Sec5 is concentrated only at the anterior rim of the oocyte membrane. At this time, the majority of Gurken is trafficked to the dorsoanterior membrane to signal to the EGFR in the adjacent follicle cells. The enrichment of Sec5 along all the anterior rim of the oocyte indicates that Sec5 alone cannot specify to which region of that domain Gurken is targeted.