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First published online September 7, 2007
doi: 10.1242/10.1242/dev.008078

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Rab6 and the secretory pathway affect oocyte polarity in Drosophila

¹ Institut Jacques Monod, Unité Mixte de Recherche 7592, CNRS, Universités Paris 6 et Paris 7, 2 place Jussieu, F-75251, Paris Cedex 05, France.
² Cell Division Group, IRB, Parc Cientific de Barcelona, c/Josep Samitier 1-5, 08028 Barcelona, Spain.
³ Hybrigenics SA, 3-5 impasse Reille, 75014 Paris, France.
⁴ Unité Mixte de Recherche 144 CNRS/Institut Curie, Institut Curie, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

View larger version (86K): [in this window] [in a new window]   Fig. 1. Drosophila Rab6 shows a dynamic localization and is enriched on Golgi membranes. (A) Drab6 mutant oocytes rescued by GFP-Drab6 expression showed a stage-dependent distribution. Drab6 was central during stages 7 and 8 (arrow, in 40% of cases Drab6 expression was central, n=112), uniform during stage 9 (86%, n=81) and always juxtaposed to the oocyte cortex from the end of stage 9 onward (n=64). (B) RFP-Drab6 and PDI-GFP co-expressing egg chamber. (C) Colocalization of Drab6 and effects of its loss on different Golgi markers in control (I,II,IV,V,VII,VIII) and rab6D23D (III,VI,IX) oocytes. Lva (I) colocalized with Drab6 in a rescued egg chamber mainly at the cortex (II, arrows), but global Lva localization did not depend on Drab6 (III). GalT (IV) colocalized with RFP-Drab6 in the center during stage 8 (V, arrow; inset in IV is a stage 10 egg chamber), but did not accumulate in the center in rab6D23D (VI). WGA was central during stage 8 (VII arrow), colocalized with GalT (VIII, arrow), but formed abnormal ring-like aggregates in rab6D23D (IX, arrow in inset, which is a magnified view of the boxed area). (D) Immunoblots of fractions from a membrane density gradient of GalT-expressing ovaries tested with markers specific to the Golgi (Dynactin), the ER (KDEL and Syntaxin 5) and the plasma membrane (Syntaxin 5). GalT was predominantly enriched in fractions containing Golgi membranes, but was additionally found in fractions reflecting the plasma membrane. Vertical bars to the left indicate the sedimentation profile: ER, endoplasmatic reticulum; PM, plasma membrane. Scale bars: 20 µm.

View larger version (118K): [in this window] [in a new window]   Fig. 2. Grk is mislocalized to post-Golgi compartments in Drosophila Rab6 mutants. (A,B) Low magnification electron micrographs of control (A) and rab6D23D mutant (B) egg chamber. (A',A'') Magnified view of boxed area from A. (B',B'') Magnified view of boxed area from B. nc, nurse cells; oo, oocyte; fc, follicle cells. ER morphology in control (arrows in A') and in rab6D23D (arrows in B'). Normal tER-Golgi unit morphology in the control (A'') and onion-like shaped morphology in mutant (B''). Cisternae (arrowheads) were not altered in number, although severely swollen, in the mutant. (C) Drab6 and Grk colocalization in GFP-Drab6-rescued oocyte. (D,E) Projection of optical sections (20 µm) of control (D) and rab6D23D (E) stage 9 oocytes, stained for Grk and F-actin. Upper insets, confocal sections at the nucleus. Lower insets, control egg and ventralized egg in the mutant. In contrast to the control, Grk formed larger particles close to and ring-like structures remote from the nucleus (arrowhead) in the mutant. (F,G) Grk and Lva colocalized marginally to small particles close to the nucleus in the control (F) and in rab6D23D (G) oocytes. Colocalization was not observed for the large Grk-positive ring-like structures (G, arrowheads in enlarged view shown in inset) in the mutant. (H,I) Control and Drab6 mutant egg chamber, respectively, labeled with Grk and LysoTracker (Lyso) to reveal endosomal compartments and lysosomes. (H',I') Magnified view of upper boxed area from H and I, respectively. (H'',I'') Magnified view of lower boxed area from H and I, respectively. Grk colocalized in a ring-like manner with Lyso-positive structures close to and distal to the nucleus only in rab6D23D. Asterisk, oocyte nucleus. Scale bars: 20 µm; 500 nm in B''.

View larger version (48K): [in this window] [in a new window]   Fig. 3. Drab6 interacts with BicD in Drosophila. (A) Schematic of BicD. Exemplary truncated BicD clones containing the C-terminal H4 coiled-coil domain that interacted with Drab6 in the two-hybrid screen. (B) Interaction of Drab6 and BicD. (I) Western blot. GST-Drab6 specifically retained BicD. Preloading GST-Drab6 with the non-hydrolyzable GTP analog, GTP--S, yielded an improved interaction. In addition to BicD (89 kDa), a polypeptide of lower molecular weight (probably a degradation product, 60 kDa) was specifically retained on GST-Drab6 beads and could be revealed with the 1B11 but not the 4C2 antibody (II). (III-V) Frames taken from time-lapse recording of BicD-GFP (III) and RFP-Drab6 (IV) co-expressing egg chamber, which colocalized to several aggregates in nurse cells and the oocyte (V). (C) Drab6 accumulates in the center (arrow) in GFP-Drab6-rescued egg chambers (I). In BicDmom egg chambers of equal age (II), GFP-Drab6 accumulation in the center was abolished (arrow). In BicDmom oocytes, Grk protein accumulated in ring-like structures remote from the nucleus (III, arrow in inset, which is a magnification of the upper boxed area). Asterisk, position of oocyte nucleus. Scale bars: 20 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Drab6 is actively transported along microtubules. Frames taken from time-lapse recordings of GFP-Drab6-expressing Drosophila egg chambers. Untreated (A) GFP-Drab6-rescued egg chamber and (B) colchicine-treated egg chamber. (C) GFP-Drab6-expressing egg chamber overexpressing Dynamitin and (D) Khc7.288 germ line clone expressing GFP-Drab6. Below each panel, parameters of vesicle movement derived from the time-lapse recordings are indicated (A'-D'). Particle parameters were determined using ImageJ. Particles in oocytes and nurse cells were traced in a single optical plane in three different egg chambers for each: control, Khc clones and overexpression of Dynamitin. (A,B) Colchicine treatment abolished accumulation in the center as seen in controls (arrow) and particles seemed to form bigger clusters. (C) Overexpressing Dynamitin reduced accumulation of Drab6 at the cortex (arrow). (D) Stage 8 Khc788 oocyte expressing GFP-Drab6. Drab6 did not accumulate in the center and formed clusters close to the oocyte nucleus (asterisks). Scale bars: 20 µm.