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First published online 27 February 2007
doi: 10.1242/jcs.03407

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Rtnl1 is enriched in a specialized germline ER that associates with ribonucleoprotein granule components

Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK

View larger version (119K): [in this window] [in a new window]   Fig. 1. The ER concentrates in the oocyte during oogenesis and is derived from fusome membranes representing a specialized ER. (A-D) The ER accumulates in the oocyte during oogenesis, as shown using an antibody against (A) the ER-retention signal HDEL and three different GFP-tagged ER proteins: (B) Rtnl1-GFP, (C) protein disulfide isomerase (PDI) and (D) translocon channel subunit Sec61 (Sec61-GFP). Arrows indicate HDEL labeling of the fusome in the germarium. Note that only Rtnl1-GFP is specifically enriched in the germline and mostly absent from the somatic follicular cell layer. (E) Concentration of Rtnl1-GFP in the fusome during different stages of cyst maturation in the germarium (for a scheme of stages within a germarium see Fig. 2A). Bars, 50 µm (A), 10 µm (B-E).

View larger version (66K): [in this window] [in a new window]   Fig. 2. The ER-membrane protein Rtnl1 is a marker of fusomal membranes during oogenesis. Examination of the fusome in the germaria during oogenesis reveals that the transmembrane protein Rtnl1 is highly enriched in fusomal membranes and very specific for the germline compared with PDI-GFP and Sec61-GFP. (A) Scheme of a germarium. (B) Scheme of Rtnl1, showing the two proposed topologies for reticulon proteins. (C-C'",D-D'", E-E'") Comparison of Rtnl1-GFP, PDI-GFP and Sec61-GFP, respectively, with antibody staining for HDEL (C",D",E") and -Spectrin (C'",D'",E'"). (F-F") Comparison of Rtnl1-GFP with labeling for acetylated -tubulin that marks the stable microtubule array, which is associated with the fusome and points into the oocyte (Röper and Brown, 2004). Note that Rtnl1 becomes very concentrated in the oocyte and at its posterior in region 3 of the germarium. Bars, 10 µm.

View larger version (135K): [in this window] [in a new window]   Fig. 3. Comparison of Rtnl1-GFP, PDI-GFP and Sec61-GFP in various embryonic tissues during development. (A-M') The labeling of Rtnl1, PDI and Sec61 by exon-trapping with GFP shows the endogenous expression patterns of all three proteins. All highlight similar ER structures in the early syncytial embryo before cellularization (A-C) and later within the embryonic epidermis (K-M, shown is the epidermis-amnioserosa interface), although levels differ in stage 14 embryos. Note the striking differences in expression within the CNS (D-F) where Rtnl1-GFP (D) is strongly concentrated in the axonal tracts but both PDI-GFP (E) and Sec61-GFP (F) are completely absent. Rtnl1-GFP is enriched in the SR (a specialization of smooth ER) of embryonic muscles (G), whereas PDI-GFP and Sec61-GFP strongly mark the perinuclear rough ER in muscles (H and I, respectively). G'-I' show higher magnifications of the areas indicated by red boxes in G-I. Arrows in G' and H' point to the SR, arrowheads in H' and I' point to the nuclear envelope. Note that the bright staining in G'-I' is the labeling within the embryonic epidermis and not the muscle. Bars, 10 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 4. The cytoskeleton recruits the membranes to the fusome. (A-F") Comparison of Rtnl1-GFP as a specific marker for the fusomal membranes with cytoskeletal components of the fusome: z-stack (A) or single confocal section (B) comparison of Rtnl1 (green in A and B and as a single channel in A' and B') with the adducin-like protein Hts (purple in A and B and as a single channel in A" and B"). Note that Hts is concentrated on spectrosomes and early fusomes and then rapidly declines in intensity during region 2b, respectively, whereas Rtnl1 becomes increasingly stronger with progress of the forming cyst through the germarium. (C,D) Two fusomes in early and later region 2b, demonstrating the increase in membrane fusome over cytoskeletal fusome. Note that the membranes (marked by Rtnl1-GFP in green in C and D and as a single channel in C' and D') are found throughout and also surrounding the structure labeled with the cytoskeletal fusome marker Hts (purple in C and D and as a single channel in C" and D"). (E) -Spectrin (purple in E and as a single channel in E") appears similar to Hts in its decline in intensity over the germarium compared with Rtnl1-GFP (green in E and as a single channel in E"), whereas Shot, F, remains very strong all throughout the germarium (purple in F and as a single channel in F"). Bars, 10 µm (A,B,E,F), 5 µm (C,D).

View larger version (102K): [in this window] [in a new window]   Fig. 5. Fusomal membranes are absent in hts1 and present in bam86 mutants. (A,A') hts1-mutant germaria that do not contain a cytoskeletal fusome (Lin et al., 1994) do not show any concentration of Rtnl1-GFP (green in A and as a single channel in A') in cysts in the germarium (cysts are labeled by Orb-staining in red in A). (B) Control germarium with the usual concentration of Rtnl1-GFP (green) on the fusome and Orb concentration into the oocyte (red). (C-C") In bam86-mutant germaria, which only contain spectrosomes and dump-bell shaped fusomes (McKearin and Ohlstein, 1995) marked by Hts (red in C and as a single channel in C"), Rtnl1-GFP associates with these fusomes (green in C and as a single channel in C'). Inset in C' shows Rtnl1-GFP in a wild-type germarium scanned at identical intensity to C'. The amount of Rnl1-GFP appears reduced. Bars, 10 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 6. The recruitment of fusomal membranes does not depend on Shot, Egl or microtubules, but the concentration of Rtnl1 into the oocyte does. (A-A") In germline clones of a null allele of shot (shot3) Rtnl1-GFP is still localized to the fusome, but does not concentrate into the oocyte. Germline clones are marked by the absence of GFP (green in A and as a single channel in A'; clones are indicated by the dotted lines). Rtnl1-GFP is also shown in green in A and also in the single channel in A'. As the germline clones lack GFP, Rtnl1-GFP distribution can be analyzed in these, but cannot be directly compared with adjacent wild-type germline cells. The cytoskeletal fusome is marked by Hts (red in A and as a single channel in A"). (B-D) In a heteroallelic combination of egl reported to be a null mutant, eglWU50/eglRC12, Shot (red in B) still localizes to the fusome and recruits the stable microtubule array marked by acetylated -tubulin (green in B), although Orb is completely mislocalized in all cysts (D). In this allelic combination, Rtnl1-GFP is recruited to the fusome at wild-type levels (C), but in the majority of cases the accumulation into the oocyte in region 3 of the germarium fails. (E-E") In the absence of microtubules in the germarium (by treatment of female flies with colchicine) Orb is completely mislocalized (red in E and as a single channel in E"), though some Rtnl1-GFP (green in E and as a single channel in E') still accumulates in the region of the fusome (white arrows), but it fails to concentrate in the oocyte. A-A",C and E-E" are stacks of confocal sections through the whole germarium, B and D are thick sections through the central portion of the germarium. Green arrows in E' point to the muscle sheet surrounding each ovariole that is also labeled by Rtnl1-GFP and partly visible in the projection. Bars, 10 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 7. Orb colocalizes with Rtnl1 on both the fusome-ER and the oocyte-ER. (A-B") The RNA-binding protein Orb (red in A and B and as a single channel in A" and B") colocalizes with Rtnl1-GFP (green in A and B and as a single channel in A' and B') on the central portion of the fusome. This is visible in both projections of confocal z-stacks (A-A") and also single confocal sections (B-B"). Arrows point to the central portion of the fusome in the germarium and the posterior crescent of Orb and Rtnl1-GFP in a budding egg chamber. (C-D") Orb and Rtnl1-GFP also colocalize in punctate structures in oocytes of stage 6 (C-C") and stage 8 (D-D") egg chambers. Rtnl1-GFP is shown in green in C and D and as a single channel in C' and D', Orb is in red in C and D and as a single channel in C" and D". Arrows in C',C",D' and D" point to colocalizing foci. Bars, 10 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 8. Rtnl1GFP colocalizes with components of RNP complexes on both the fusome-ER and the oocyte-ER. (A-A'") The DEAD-box protein Me31b, which is involved in translational silencing of mRNAs and a component of RNP complexes, colocalizes with Rtnl1-GFP and Orb on the central portion of the fusome in germaria. Shown is a z-stack projection of a germarium. Rtnl1GFP is green, Me31 is red, and Orb is blue in A, with the respective single channels shown in A'-A'". Arrowheads in A point to the colocalization on the central portion of the fusome. (B-B'") Sec61-GFP shows strongest labeling of the fusome in region 2a, where neither Orb nor Me31b are concentrated yet, and no further colocalization within the germarium is observed. Sec61-GFP is green, Me31 is red, and Orb is blue in B, with the respective single channels shown in B'-B'". (C-F) Rtnl1-GFP, Me31b and also Trailer Hitch (Tral), another component of RNP complexes, colocalize in punctate structures in the oocyte (C-C'" for Rtnl1GFP versus Me31b and Trailer Hitch, and E-E" for Rtnl1GFP versus Me31b alone) and nurse cells (D-D'" for Rtnl1GFP versus Me31b and Trailer Hitch, and F-F" for Rtnl1GFP versus Me31b alone). Shown are single confocal sections of a stage 6-7 egg chamber. In all composites Rtnl1GFP is in green, Me31b is in red and Tral is in blue. Arrowheads in C and D point to some of the colocalizing foci of Rtnl1GFP, Me31b and Tral. (G-K) Comparison of the localization of Me31b and Sec61-GFP and Me31b and PDI-GFP. Both Sec61-GFP and PDI-GFP are strongly concentrated in the entire oocyte and do not show foci of stronger labeling where Me31b is concentrated into foci (G for Sec61-GFP and I for PDI-GFP). Within the nurse cells, Me31b foci colocalize with labeling for Sec61-GFP and PDI-GFP (H for Sec61-GFP and K for PDI-GFP). In all composites Sec61-GFP and PDI-GFP are in green, Me31b is in red. Bars, 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 9. Flies carrying a hypomorphic or a null mutation in rtnl1 show only a mild phenotype in cyst maturation during oogenesis. (A) Scheme of potential Rtnl1 protein isoforms; red and orange boxes show protein-encoding exons. The green stars indicate the position where the GFP-exon is inserted and, thus, only the two largest protein isoforms are labeled in the Rtnl1-GFP line. The black bar indicates the deletion in the rtnl1null allele that eliminates all of the reticulon-homology domain encoding the membrane-association domain of the protein (Wakefield and Tear, 2006). (B) The rtnl11 allele, generated by F1-EMS mutagenesis screen (see Materials and Methods) appears to result in expression of a truncated protein. (Left) Western blot of Rtnl1-GFP exon-trap ovaries in the left panel; (right) immunoprecipitation with anti-GFP antibody followed by western blotting of Rtnl1-GFP exon-trap and rtnl11 ovaries. Red dots indicate the main Rtnl1-GFP protein band. (C) Compared with control germaria, germaria of both homozygous rtnl1null and rtnl11 mutant females show a less tight concentration of Orb on the fusome, and also sometimes even cysts completely lacking detectable concentration of Orb (arrows). Nonetheless, budding egg chambers in region 3 always have Orb concentrated within one single cell. Shown are z-stack projections through the whole germarium. Orb is in red and as a single channel shown in white in the middle panels, Shot, marking the fusome, is in blue and as a single channel shown in white in the right panels. Bar, 10 µm. (D) Germaria of homozygous rtnl1null mutant females still show concentration of Me31b into the oocyte in budding egg chambers, although similar to Orb, the concentration on the fusome is less tight (top panel, arrows point to budding egg chambers). In later egg chambers, Orb and Me31b still colocalize in foci within the oocyte (bottom panels, white arrows) and Me31b still accumulates in foci within the nurse cells (bottom panels, green arrowheads). Top panel is a z-stack projection through a whole germarium, bottom panel is a single confocal section. Me31b is in green and as a single channel shown in white in the middle panels, Orb is in red and as a single channel shown in white in the right panels.

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