spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 28 February 2007
doi: 10.1242/dev.02821

Development 134, 1419-1430 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Coutelis, J.-B.
Right arrow Articles by Ephrussi, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Coutelis, J.-B.
Right arrow Articles by Ephrussi, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis

Jean-Baptiste Coutelis and Anne Ephrussi*

Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.


Figure 1
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 1. Loss of actin organization in rab6D23D egg chambers. rab6D23D egg chambers (OvoD-selected germline clones) with or without a hs::rab6 transgene were stained to reveal DNA (DAPI, blue) and F-actin (phalloidin, yellow). In rab6D23D egg chambers (A), the actin cytoskeleton disappears progressively between stage 2 and stage 7. Expression of hs::rab6 (B) fully rescues the loss-of-actin phenotype of the rab6D23D mutant (compare A and B).

 

Figure 2
View larger version (64K):
[in this window]
[in a new window]

  		Fig. 2. rab6D23D egg chambers form open syncytia. (A-C) Egg chambers stained to reveal F-actin (phalloidin, red) and all membranes (FM4-64, green). rab6D23D clones are distinguished by the absence of nuclear GFP (blue). In wild-type stage-7 egg chambers (A), actin cytoskeleton and plasma membranes overlap (arrow). A membranous continuum decorating a nucleus (*) appears to span the ring canals, linking adjacent cells (arrowhead). (B,C) In mildly affected rab6D23D egg chambers (B), remaining plasma membranes and actin delimit two nurse cell `open' syncytia (arrow). Within these syncytia, clusters of actin debris and ring canals are embedded in dense membranous material (arrowhead in bottom syncytium; not visible in the upper syncytium in this focal plane). The membranous continuum passes through the ring canals in the remaining plasma membranes separating the large open syncytia and the oocyte. In strongly affected rab6D23D egg chambers (C), all compartmentalization by actin and plasma membrane is lost, and nurse cell nuclei lie in a common cytoplasm containing a single central cluster of ring canals, membranes and actin debris (arrowhead). The nurse cell nuclei (*) found in the syncytia away from the membranes appear disconnected from the membranous continuum (B,B') and are occasionally stripped of vesicular material (C,C').

 

Figure 3
View larger version (75K):
[in this window]
[in a new window]

  		Fig. 3. Trafficking of Yolkless and Gurken is impaired in rab6D23D oocytes. Egg chambers stained for Yolkless (A,B, green) or Gurken (C-E, green), F-actin (phalloidin, red) and DNA (DAPI, blue). In wild type (A), Yolkless concentrates in the oocyte cytoplasm before stage 8 (arrowhead). At stage 10, Yolkless is restricted to the oocyte membrane (A'). In rab6D23D egg chambers (B), Yolkless concentrates in the oocyte normally (arrowhead). However, at stage 10, an important proportion of Yolkless remains cytoplasmic, indicating that trafficking is impaired (B'). In wild type (C), Gurken is detected as a small arc between the oocyte nucleus and the antero-dorsal plasma membrane. Secreted Gurken is detected in the cytoplasm of the overlying follicle cells (C'', arrow). In the majority of affected rab6D23D stage-10 egg chambers (55.5%), large amounts of ectopic Gurken protein are detected in the ooplasm (D,E). In some (29.8%) affected rab6D23D egg chambers, secreted Gurken is still detected in the cytoplasm of the overlying follicle cells (D'', arrow). In others (25.7%), Gurken is detected exclusively in the ooplasm and is not detected in the follicle cell cytoplasm (E'').

 

Figure 4
View larger version (62K):
[in this window]
[in a new window]

  		Fig. 4. Sec5 and Syx1A localization on membranes is differentially impaired in rab6 egg chambers. Egg chambers stained to reveal Syx1A (A,B, green) or Sec5 (C,D, green), F-actin (phalloidin, red) and DNA (DAPI, blue). In wild type (A), Syx1A is detected on nurse cell and oocyte membranes (arrows). After stage 9, Syx1A is predominantly detected on nurse cell membranes. In rab6D23D egg chambers (B), Syx1A is detected on the remaining plasma membranes (arrow) and around the ring canal cluster (arrowheads), in both strongly and in mildly affected egg chambers. In stage-8 wild-type egg chambers (C), Sec5 and actin colocalize at nurse cell (arrow) and oocyte (Oo) plasma membranes. In rab6D23D egg chambers at these stages, Sec5 is absent from residual membranes that delimit nurse cell `open' syncytia (D, arrow) but is readily detected on the oocyte plasma membrane (Oo). Sec5 is not detected in the aggregates of ring canals and actin debris (D, arrowhead).

 

Figure 5
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 5. oskar mRNA localization is affected at mid-oogenesis in rab6-null egg chambers. (A-D) Stage-9 egg chambers stained to reveal F-actin (phalloidin, red), Kin:ß-gal (green), Staufen (blue) and DNA (DAPI, white). (E-H) Co-detection of oskar mRNA (red) and Oskar protein (green), and DNA (DAPI; blue). In wild type, Kin:ß-gal and Staufen (A), and oskar mRNA and Oskar protein (E) colocalize in a sharp crescent at the oocyte posterior. All rab6D23D egg chambers evaluated in this assay displayed a similarly affected actin cytoskeleton and the stereotypical pattern of eight- and four-nurse cell open syncytia (B-D). In nearly one third of these, localization of Kin:ß-gal and Staufen (B), and of oskar mRNA and Oskar protein (F), appears normal. In the remaining affected rab6D23D egg chambers, Kin:ß-gal and Staufen/oskar are detected in blobs either partially (C,G) or completely (D,H) delocalized from the oocyte cortex. Oskar protein is detected at the posterior cortex only (G''), and not with unlocalized oskar mRNA (G'',H'').

 

Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 6. Staufen localization is normal in sec5E13 egg chambers. Egg chambers stained for Staufen (green), F-actin (phalloidin, red) and DNA (DAPI, blue). (A) In wild-type egg chambers, Staufen concentrates in the oocyte during early oogenesis (egg-chamber 1). After stage 7 and repolarization of the oocyte MT cytoskeleton, Staufen is transiently detected in the middle of the oocyte (egg-chamber 2). From stage 9 onwards, Staufen forms a tight crescent at the posterior pole (egg-chamber 3). (B) In sec5E13 egg chambers, all steps of Staufen localization appear normal.

 

Figure 7
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 7. Rab6 and BicD are in a complex and act together in Staufen localization at mid-oogenesis. (A) BicD co-immunoprecipitates specifically with Rab6 in ovarian extracts. Ovarian extracts of transgenic flies expressing Myc-tagged Rab6 or Rab7 were immunoprecipitated using a monoclonal anti-Myc antibody. Western blots were probed using anti-BicD or anti-Myc antibodies. (B) BicD and rab6 interact genetically in Staufen localization. The graph shows the percentage of stage-9 and -10 egg chambers of different genotypes displaying mislocalized Staufen. An average of 215 egg chambers per genotype were counted. Compared with rab6 single mutants, the proportion of egg chambers displaying defects in Staufen localization increases additively in rab6, par-1 double mutants, and synergistically in rab6, BicD double mutants. (C,D) Egg chambers stained for BicD (green), F-actin (phalloidin, red) and DNA (DAPI, blue). In wild-type egg chambers (C), after anterior migration of the oocyte nucleus (asterisk), BicD is detected between the oocyte cortex and the nucleus (C', arrow). In rab6D23D egg chambers (D), BicD appears to be associated with the oocyte nucleus (D, asterisk), as in wild-type (D', arrow), and ectopically aggregates with ring canal clusters in nurse cell syncytia (D, arrowhead). I, input; U, unbound; B, bound.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007