DEV02821 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Two classes of stage-7 rab6-null egg chambers. (A-C) Between stages 2 and 7, two classes of rab6^D23D egg chambers, defined on the basis of the integrity of the oocyte, are observed. In wild-type stage-7 egg-chambers (A), each nurse cell is compartmentalized by actin and communicates with neighboring cells through actin-rich ring canals (arrowhead). The oocyte (Oo) is found at the posterior of the egg chamber. Arrow in A′′ indicates the position of the germinal vesicle. (B) The first class of rab6^D23D egg chambers (32.5%) is characterized by the absence of all cortical actin delimiting the cystocytes, oocyte included, and loss of cyst organization. Typically, a single cluster of ring canals, surrounded by actin debris, is evident in the middle of the egg chamber (arrowhead). The oocyte most probably degenerates soon after, as most egg chambers contain only 15 polyploid nurse cell nuclei. However, in a few, the germinal vesicle can be detected in the vicinity of the cluster (B′′, arrow). Egg chambers of this class continue to grow until a ‘pseudo’ stage 7 (estimated by their size), and ultimately degenerate, because none of this type is observed beyond stage 7. The second class of rab6^D23D egg chamber (67.5%) is a more heterogeneous group. Although all egg chambers of this category contain an individualized oocyte, they display an actin organization ranging from wild type, to strongly disorganized cytoplasmic clusters of ring canals and actin debris (C, arrowhead). (C) A rab6^D23D egg chamber in which only the oocyte remains individualized is shown. As late-stage rab6^D23D egg chambers containing an oocyte and displaying actin defects are observed, we infer that some egg chambers of this second class develop beyond stage 7.

• Supplemental Figure 2 -

  Fig. S2. GFP-Rab6 is enriched in the oocyte during oogenesis. (A) Germarium expressing a GFP-Rab6 fusion protein (green) exclusively in the germ line, under control of the nanos promoter ;;nos:Gal4VP16/UAS:GFP-Rab6;, using the Gal4/UAS system (Rorth, 1998), stained to reveal BicD (red), F-actin (phalloidin, blue) and DNA (DAPI, white overlay). (A) Expression of GFP-Rab6 is apparent in region 2 of the germarium and the protein concentrates in the presumptive oocyte (A′) during its specification, as indicated by the presence of the oocyte fate marker, BicD protein (A′′). (B) rab6^D23D egg chambers (Ovo^D-selected germline clones) expressing a GFP-Rab6 fusion protein (green) exclusively in the germ line, under control of the nanos promoter hs::FLP/+;rab6^D23D,FRT40/Ovo^D,FRT40;nos:Gal4VP16/UAS:GFP-Rab6;, stained to reveal Staufen (red), F-actin (phalloidin, blue) and DNA (DAPI, white overlay). GFP-Rab6 expression is sufficient to rescue all the egg-chamber disorganization and mislocalization phenotypes observed, and to promote oogenesis and Staufen/oskar mRNA localization at the oocyte posterior at mid-oogenesis. This demonstrates that Rab6 activity is required in the germ line. GFP-Rab6 is detected in all cells of the germline cyst, concentrated in the oocyte (B′), first throughout the ooplasm (St. 4), then along the oocyte plasma membrane, most conspicuously in the anterior-dorsal corner near the oocyte nucleus (St. 6 and 9).

• Supplemental Figure 3 -

  Fig. S3. Rab6 is required in the germ line for egg-chamber organization. (A,B) Egg chambers containing rab6 follicular cell clones (FCC) or germline clones (GLC) stained to reveal F-actin (phalloidin, red) and BicD (blue). rab6^D23D clones are distinguished by the absence of nuclear GFP (green). In egg chambers with a completely mutant follicular epithelium (A), each cystocyte is individualized by the actin cytoskeleton (A′). The oocyte is localized at the posterior of the egg chamber and is enriched for BicD protein (A′′). Only egg chambers in which the germ line is mutant lose their organization (B). In these, all compartmentalization by actin is lost and a single central cluster of ring canals (B′) around which BicD protein accumulates (B′′) lies in the middle of the open syncytium.

• Supplemental Figure 4 -

  Fig. S4. Rab6-mediated membrane trafficking is required for nurse cell plasma membrane growth. (A) rab6^D23D egg chambers that reach mid-oogenesis and form open syncytia display a stereotypic organization. A stage-9 rab6^D23D egg chamber, labeled to reveal actin (phalloidin, orange) and DNA (DAPI, blue). The image consists of the projection of two focal planes, 3 μm apart. (A′′) The four ring canals of the oocyte are numbered. (A′) The nurse cell nuclei are numbered, the ring canals are represented by the orange ellipses, and the positions of residual membranes detected in the front and the back planes are indicated by continuous and dotted red lines, respectively. Nurse cells 1-8 and 9-12 form eight- and four-cell open syncytia, respectively. The exact grouping of nurse cells 13, 14 and 15 is not readily resolved. (B,C) Schematic representing the stereotypic organization of nurse cell open syncytia in rab6^D23D egg chambers (B) and a proposed explanation for their formation (C). (B) The oocyte is connected to the nurse cell open syncytia by its four ring canals (as shown in A); nurse cells sharing ring canals with the oocyte form syncytia with nurse cells more remote from the oocyte. (C) In wild-type egg chambers, new membrane material (NM) and associated Sec5 and Syx1A proteins are delivered (black arrows in C) to the nurse cell plasma membrane (PM) via Rab6-dependent and Rab6-independent pathways. There, the exocyst complex is organized and promotes addition of new membranes during plasma membrane growth (red arrow in C). (C′) In rab6^D23D egg chambers, Syx1A localizes to nurse cell plasma membranes via a Rab6-independent pathway (black arrow in C′), whereas lack of Rab6 impairs Rab6-dependent localization of Sec5 and reduces the general flow and trafficking of vesicles and new membranes required for membrane growth (crossed dashed black lines). Furthermore, addition of new membranes is impaired by the absence or dysfunction of the exocyst complex (cross and dashed red line on C′). Syx1A and associated membranous material, delivered and added to the nurse cell membranes via Rab6-independent pathways, do not support nurse cell plasma membrane growth, such that the weakened membranes collapse, leading to the formation of nurse cell open syncytia. In the cytoplasm, collapsed plasma membranes, actin cytoskeleton, ring canals (RC), and Syx1A-positive membranes as well as post-Golgi vesicles form clusters in the open syncytial cytoplasm. Syx1A (but not Sec5) is detected on the remaining plasma membranes delimiting the open syncytia.

• Supplemental Figure 5 -

  Fig. S5. Post-Golgi vesicles are detected around the cytoplasmic cluster of rab6-null open syncytia. Wild-type (A) and rab6^D23D (B) egg chambers stained to reveal the distribution of Lava lamp, a golgin-family protein that serves as a reporter of the Golgi (Papoulas et al., 2005; Sisson et al., 2000) (Lava lamp, green), F-actin (phalloidin, red) and DNA (DAPI, blue). In affected rab6^D23D egg chambers at early and later stages, Lava lamp is detected around the cluster of ring canals and actin debris (B, arrowheads) revealing that some portion of the FM4-64-staining vesicles detected around the ring canal clusters (Fig. 2) are Golgi-derived. This observed enlargement of the Golgi compartment resembles the situation in S. cerevisiae mutant for Ypt6 (Rab6 homolog), for other Golgi-localized Rab proteins involved in exocytosis, or for Exocyst components in which exocytosis is impaired at the post-Golgi level (Antony et al., 1992; Martinez et al., 1994; Matanis et al., 2002; Novick et al., 1981; Novick et al., 1980; Short et al., 2002; White et al., 1999; Yeaman et al., 2001; Young et al., 2005). The accumulation of Golgi-derived vesicles in the cytoplasm of rab6^D23D egg chambers suggests an impairment of exocytosis at the post-Golgi level.

• Supplemental Figure 6 -

  Fig. S6. Enlargement of the Golgi compartment in rab6-null egg chambers. (A,B) Sections of wild-type and rab6 mutant egg chambers subjected to high-pressure freezing observed by electron microscopy (procedure available upon request). Four nurse cell nuclei, individualized by plasma membranes in the wild-type control (A) or regrouped into two syncytia in the rab6^D23D mutant (B) are shown. In wild-type egg chambers, at higher magnification (A′), the plasma membranes of the nurse cells appear regularly linked by electron-dense junctions (A′, arrows). In rab6^D23D egg chambers, the plasma membranes of the nurse cell syncytia appear loose and lacking regularly spaced adhesion foci (B′, arrows). In the cytoplasm of rab6^D23D cells, large globular elements (B′, arrowheads) are observed. These are positive for the Golgi markers Lva, FWS and GM130 (data not shown) (Farkas et al., 2003; Papoulas et al., 2005; Sisson et al., 2000; Trucco et al., 2004), indicating that they correspond to abnormally large Golgi units and Golgi-derived membranes (B′′); compare to wild-type Golgi units (A′′). ER compartments, distinguished by their specific morphology and using PDI marker (Bobinnec et al., 2003; Kondylis et al., 2001), appear normal in rab6^D23D egg chambers. The enlargement of the Golgi compartment in rab6^D23D is strongly reminiscent of what is observed in yeast or mammalian cell mutants for regulators of exocytosis (Antony et al., 1992; Martinez et al., 1994; Matanis et al., 2002; Novick et al., 1981; Novick et al., 1980; Short et al., 2002; White et al., 1999; Yeaman et al., 2001; Young et al., 2005) and supports the data obtained by light microscopy (Fig. S5). Taken together, these data indicate that, in rab6 mutant egg chambers, exocytosis is impaired at the Golgi level. RC, ring canal; ER, endoplasmic reticulum.

• Supplemental Figure 7 -

  Fig. S7. Detailed percentages and description of the phenotypic classes observed in rab6-null egg chambers. Graph showing the quantification of the various phenotypes observed in rab6^D23D egg chambers. Bars indicate the percentage of each class of phenotype. (1, 2) Pre-stage-7 egg-chambers. (1) Egg-chambers containing an oocyte. (2) Strongly affected egg-chambers (containing a central cluster of ring canals and actin debris), without an individualized oocyte. (B-H) Stage 9 and 10 egg chambers (data in Fig. 6 and text). (B-D) Egg chambers classified according to localization of Kin:βGal and Staufen in the oocyte. (B) Wild-type-like; (C) Kin:βGal/Staufen mislocalized in a blob and residually at the posterior pole; (D) Kin:βGal/Staufen entirely mislocalized (no posterior crescent). (F-H) Egg chambers classified according to localization of oskar mRNA and Oskar protein in the oocyte. (F) Wild-type-like; (G) oskar mRNA in a cloud, with a residual posterior crescent of oskar mRNA and Oskar protein; (H) all oskar mRNA mislocalized (no Oskar protein detected).