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First published online 28 February 2007
doi: 10.1242/dev.02821
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Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
* Author for correspondence (e-mail: ephrussi{at}embl.de)
Accepted 25 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila oogenesis, Rab6, oskar mRNA, Exocyst, Microtubules, Polarity
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This asymmetry is the result of a succession of processes
that take place during oogenesis. The ovary consists of ovarioles, which are
composed of egg chambers of increasing developmental stages
(Fig. 1B). In the germarium,
germline stem cells divide asymmetrically, generating a new stem cell and a
cystoblast (Huynh and St Johnston,
2004). The cystoblast undergoes four rounds of mitosis with
incomplete cytokinesis, producing a germline cyst of 16 cells interconnected
via 15 actin-rich cytoplasmic bridges, the ring canals. Each cystocyte
displays a specific number of ring canals reflecting the mitosis at which it
was generated: the two first-born cystocytes possess four ring canals, and the
eight last-born possess one. Oocyte specification occurs via the progressive
and selective concentration of cell fate determinants to the two four-branched
cells and, finally, to just one of these cells, the oocyte. This process
requires an organized and polarized microtubule (MT) network spanning the
germline cyst and connecting the cystocytes
(Grieder et al., 2000;
Theurkauf et al., 1993;
Vaccari and Ephrussi, 2002).
The remaining 15 cells adopt the nurse cell fate and provide molecules
required for oocyte development. The germarium also contains somatic stem
cells. Some of these differentiate into follicular epithelial cells that
encapsulate the germline cyst, thus forming the stage-1 egg chamber, which
exits the germarium. Oogenesis has been subdivided into 14 stages (reviewed in
King, 1970;
Spradling, 1993). During the
first six stages, the 16 cystocytes grow at roughly identical rates and
contribute almost equally to the increase in size of the egg chamber. At stage
8, vitellogenesis begins and the increase in egg chamber size mainly reflects
the major growth of the oocyte.
Antero-posterior axis specification occurs at stage 7, with the secretion
by the oocyte of the Gurken protein, a Drosophila TGF
homolog
and EGF-receptor ligand (reviewed in
Nilson and Schupbach, 1999).
This signal induces a group of overlying follicle cells to adopt the posterior
fate (Gonzalez-Reyes et al.,
1995; Roth et al.,
1995). In return, these cells emit an unknown signal, causing the
disassembly of the unique MT-organizing center (MTOC) of the germline cyst.
This MTOC is located at the posterior of the oocyte until stage 7. New MT then
nucleate from the anterior and lateral cortex of the oocyte, whose nucleus
moves in a MT-dependent manner from its posterior location to an anterior
corner of the cell. Here, a second Gurken signal causes the overlying follicle
cells to adopt a dorsal fate, thus specifying the dorsoventral axis.
Concomitantly, MT-dependent localization of bicoid and oskar
mRNAs to the anterior and posterior of the oocyte, respectively, establishes
the antero-posterior axis of the future embryo (reviewed in
Riechmann and Ephrussi,
2001).
Genetic analysis has revealed the involvement of proteins such as
Bicaudal-D (BicD) and Dynein, a minus-end-directed MT motor, together with the
MT network, in germline cyst development, oocyte specification, asymmetric
mRNA localization within the oocyte and egg-chamber formation
(Navarro et al., 2004;
Oh and Steward, 2001;
Suter and Steward, 1991;
Swan et al., 1999;
Vaccari and Ephrussi, 2002).
Thus, in BicD mutant germaria, the MT cytoskeleton fails to maintain
its polarization and none of the cystocytes enter meiosis, resulting in a cyst
of 16 nurse cells (Huynh and St Johnston,
2000; Oh and Steward,
2001).
It has been shown that the mammalian homologs of BicD (BICD1 and BICD2)
physically interact with Dynamitin, a component of the Dynactin complex (the
regulatory and cargo-loading subunit of Dynein), and with Dynein intermediate
chain (Hoogenraad et al.,
2001) to control Dynein-based motility
(Hoogenraad et al., 2003). In
addition, BICD1 and BICD2 bind RAB6a and RAB6a'
(Matanis et al., 2002;
Short et al., 2002), members
of the Rab family of small monomeric GTPases, which are among the major
regulators of vesicular trafficking. Rab proteins segregate over the various
organelle membranes in the cell, and their specific interactions with
effectors promote asymmetric and polarized vesicle transport
(van Ijzendoorn et al., 2003;
Zerial and McBride, 2001).
RAB6a' also directly binds the Dynactin complex via its
p150Glued subunit and promotes the recruitment of this complex to
the trans-Golgi Network (TGN) (Short et
al., 2002). Finally, RAB6a, RAB6a', BICD2 and Dynactin
appear to cooperate to mediate the recycling and retrograde transport of
proteins from the TGN (Young et al.,
2005), indicating that, in mammals, these three proteins are
physically and functionally linked.
Polarized intracellular transport and vesicular trafficking can be used to
create and/or maintain asymmetry during Drosophila development
(Dudu et al., 2004;
Gonzalez-Gaitan, 2003).
However, little is known about the involvement of vesicular trafficking and of
Rab proteins in cell polarization during oogenesis. One Rab protein, Rab11,
has been shown to act in oskar mRNA localization at mid-oogenesis
(Dollar et al., 2002;
Jankovics et al., 2001). Given
the interactions in mammalian cells of BICD1 and BICD2, and of the
Dynactin-Dynein complex, whose homologs have essential functions in
Drosophila, with RAB6a and RAB6a', we investigated the
involvement of this protein in Drosophila oogenesis. Our study
reveals that Rab6 is required at several steps during oogenesis. Depletion of
Rab6 in the germ line causes membrane-trafficking defects that affect
egg-chamber plasma membranes and the proper organization of the germline cyst.
At mid-oogenesis, lack of Rab6 results in an abnormally polarized oocyte MT
network and in the mis-localization of oskar mRNA.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For the
generation of germline clones by FRT/FLP-mediated recombination,
rab6D23D (Purcell and
Artavanis-Tsakonas, 1999) was recombined with
[w;FRT40AneoR]. hs::rab6 is described by Purcell
and Artavanis-Tsakonas (Purcell and
Artavanis-Tsakonas, 1999). To generate
rab6D23D and sec5E13 somatic or
germline clones, [w;rab6D23D,FRT40A/Cyo;] flies (some
carrying the KinlacZ transgene)
(Clark et al., 1994), or
[w;sec5E13,FRT40A/Cyo;] flies
(Murthy and Schwarz, 2004)
were crossed with [yw,hs::FLP;OvoD,FRT40A/Cyo;] or with
[w,f,hs::FLP;GFP,FRT40A/Cyo] flies. Progeny were heat-shocked daily
for 1 hour at 37°C during the third larval instar. For rescue experiments, [yw,hs::FLP/w;OvoD,FRT40A/rab6D23D, FRT40A;hs::rab6/+;] females and [yw,hs::FLP/w;OvoD,FRT40A/rab6D23D,FRT40A;] control females were heat-shocked at 37°C for 1 hour each day during the third larval instar, and then three times for 30 minutes every 2 days until emergence.
Evaluation of hatching-rate, fertility and the embryonic requirement for rab6
Virgin rab6
[yw,hs::FLP/w;OvoD,FRT40A/rab6D23D,FRT40A]
females and [yw,hs::FLP/w;OvoD,FRT40A/FRT40A] and OreR
control females were collected and crossed to wild-type OreR males or to males
heterozygous for either rab6D23D
[w;rab6D23D/Cyo,UbiGFP;] or for a genomic deletion
covering the rab6 locus
[;Df(2L)prd1.7,b1Adhn2pr1cn1sca1/Cyo,UbiGFP;]
(Bloomington #3344). rab6D23D eggs were obtained after
OvoD selection. After aging the females for 3 days at 25°C,
eggs were collected on apple juice plates and hatch rates were scored. From
rab6D23D eggs that hatched, both GFP-positive and
GFP-negative second-instar larvae were observed; however, only GFP-positive
third-instar larvae were observed, and all hatched flies displayed the CyO
dominant marker, revealing the larval lethality of the rab6 mutation.
To test the fertility of flies hatched from rab6D23D eggs
that lacked the maternal contribution of Rab6, female offspring were mated
with wild-type OreR males.
Whole-mount ovary staining
Antibody stainings were performed as previously described
(Tomancak et al., 2000).
Antibodies and dilutions were as follows: rabbit anti-Staufen (1:2000), rabbit
anti-Oskar (1:3000), mouse anti-Sec5 (22A2; 1:200), mouse anti-Syx1A (8C3;
1:20), mouse anti-Gurken (1D12; 1:200), rat anti-Yolkless (1:100), mouse
anti-BicD (1B11; 1:20) and rabbit anti-Lava lamp (1:500). Double-labeling of
ovaries using RNA probes and antibodies was performed as previously described
(Vanzo and Ephrussi,
2002).
To observe membranes and membranous organelles in their native organization using lipophilic styryl compounds (FM-dyes, Molecular Probes), ovaries were dissected in PBS, fixed in 4% formaldehyde for 20 minutes and washed twice in PBS (10 minutes each). Next, they were incubated with rhodamine-conjugated phalloidin (1:500; Molecular Probes) in PBS for 6 hours at room temperature or overnight at 4°C. Subsequently, they were incubated for 10 minutes in PBS containing FM4-64 (1:2000), then for 5 minutes in PBS with DAPI (1:2500). They were then washed twice for 5 minutes in PBS and equilibrated in N-propylgallate medium before mounting. Confocal microscopy was performed on a Leica TCS-SP and images edited using Adobe Photoshop.
Cloning
N-terminal fusions of Myc-tag or GFP to Rab6 or Rab7 were constructed
according to standard procedures (details available upon request).
Co-immunoprecipitation and western blotting
Ovarian extracts were prepared from 3-day-old well-fed flies expressing
Myc-tagged Rab6 or Rab7 under maternal-tubulin promoter control
[;;UASp::Myc-Rab/Mattub::Gal4Vp16;] using the Gal4/UAS system
(Rorth, 1998). Ovaries were
washed twice in 1:1 PBS:Extraction Buffer [20 mM HEPES pH 7.5, 400 mM NaCl, 1
mM PMSF, 1xProtein Inhibitor without EGTA/EDTA (Roche catalogue number
1873580) 10% Glycerol] and lysed in 5 µl extraction buffer per 20 ovaries.
After centrifugation (10 minutes at 16,000 g at 4°C), the
supernatant was collected and 9 µl of 2xIP buffer (20 mM HEPES pH
7.5, 300 mM NaCl, 5 mM MgCl2, 2xProtein Inhibitor without
EGTA/EDTA and 500 mM Sucrose) plus 2 µl PBS and 2 µl of mouse monoclonal
anti-c-Myc (Sigma, Clone 9E10) was added per 5 µl supernatant, mixed and
rocked for 2 hours at 4°C. In total, 40 µl of 1:1 Protein G
beads:Washing Buffer (20 mM HEPES pH 7.5, 400 mM NaCl, 1xProtein
Inhibitor without EGTA/EDTA) were added and samples rocked for 1 hour at
4°C. The bead pellet and supernatant were collected individually. Beads
were washed four times in washing buffer and resuspended in SDS-buffer.
Proteins were detected by 10% SDS-PAGE followed by western blotting, using
either mouse monoclonal anti-c-Myc (9E10; 1:1000) or mouse anti-BicD (4C2;
1:100), following standard procedures.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, to analyze the role of rab6 in
oogenesis, we generated homozygous-mutant rab6 germline clones in
otherwise heterozygous females. Upon mitotic recombination,
rab6D23D germline clones were identified by either the
absence of nuclear GFP or were selected using the dominant female-sterile
OvoD technique (Chou and
Perrimon, 1996).
Loss of actin organization in rab6-null egg chambers
In wild-type egg chambers, each nurse cell is delimited by a plasma
membrane and an underlying actin cytoskeleton, but communicates with its
neighboring cells through the ring canals (see Fig. S1A in the supplementary
material). In rab6D23D mutant germaria, cyst formation and
maturation appeared normal (data not shown). However, from stages 2 to 7,
80.6% (n=191) of rab6D23D egg chambers displayed
a progressive disappearance of the nurse cell cortical actin cytoskeleton,
aberrantly spaced nurse cell nuclei (Fig.
1A), and a concomitant aggregation of ring canals and actin debris
in the egg chamber (see Fig. S1B,C in the supplementary material). At stage 7,
rab6D23D egg chambers of two classes, based on the
integrity of the oocyte, are observed (see Fig. S1 in the supplementary
material). The first class (32.5%) consisted of strongly affected egg chambers
and is characterized by the absence of all cortical actin, which usually
delimits the cystocytes, oocyte included. Such egg chambers continue to grow
but ultimately degenerate, as none of this type is observed beyond stage 7.
The second class (67.5%; see Fig. S1 in the supplementary material) is more
heterogeneous. All egg chambers of this second class possess a delimited
oocyte.
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rab6-null egg chambers form open syncytia
The disappearance of the cortical actin cytoskeleton and abnormal spacing
of the nurse cell nuclei in rab6D23D egg chambers
suggested the possible loss of plasma membranes within the mutant cysts. We
therefore evaluated the integrity of the membranes, staining them with
lipophilic styryl compounds (FM4-64, Molecular Probes) that fluoresce upon
insertion into membranes. To resolve plasma membranes, membranous organelles
and vesicles in their native organization, we used the rapid-diffusion
property of the dye to mark all membranes of fixed egg chambers without
permeabilization.
In wild-type cysts, the actin cytoskeleton and the plasma membrane appear to colocalize (Fig. 2A, arrow) and delimit each nurse cell. The majority of rab6D23D egg chambers, rather than forming a compartmentalized cyst, form `open' syncytia (Fig. 2B,C) in which some or all nurse cells lack delimiting plasma membranes and the nuclei are encompassed within a single actin and plasma membrane boundary (Fig. 2B, arrow). In strongly affected rab6D23D egg chambers, all nuclei are found within a single open syncytium (Fig. 2C). In these chambers, the nuclei are found at the periphery of a common cytoplasm containing, in its center, a single mass of membranes, ring canals and actin debris (Fig. 2C, arrowhead). Noticeably, the oocyte is consistently the last cell of such rab6-null germline cysts to display plasma membrane loss. rab6D23D egg chambers in which membrane loss was observed, but that nevertheless developed past stage 7, displayed a stereotypic organization at mid-oogenesis. In these chambers, 12 out of the 15 nurse cell nuclei were reproducibly contained within two open syncytia, the first comprising eight, and the second four, nuclei (see Fig. S4A in the supplementary material).
Lack of Rab6 does not seem to affect oocyte fate, because the enrichment
and restriction of BicD, Par-1 and Staufen observed from the germarium stage
until mid-oogenesis (Shulman et al.,
2000; St Johnston et al.,
1991; Suter and Steward,
1991; Tomancak et al.,
2000) occurred in rab6D23D oocytes the same as
in wild-type (see Fig. S2A'' in the supplementary material, and data not
shown). In addition, no egg chamber with 16 nurse cells and no oocyte, which
would indicate a lack of oocyte specification or a loss of oocyte fate, was
observed in rab6-null mutants. Finally, in strongly affected
rab6D23D egg chambers in which no oocyte was observed, the
oocyte-specific markers BicD and Par-1 were detected around the central
cluster of ring canals (see Fig. S3B'' in the supplementary material, and
data not shown). This ectopic enrichment suggests the prior existence of a
correctly specified oocyte whose membrane integrity was impaired. Thus
rab6 function is dispensable for oocyte specification, but is
required to maintain the organization of egg chambers and of their internal
plasma membranes.
rab6-null egg chambers display defects in Yolkless and Gurken trafficking
The loss of plasma membrane in rab6D23D germline
clones, together with the known role of Rab6 homologs in vesicular traffic
through the Golgi, led us to examine whether membrane trafficking is affected
in rab6D23D egg chambers. As a readout of this process, we
first made use of the vitellogenin receptor Yolkless, which is translated in
the oocyte and secreted before being actively recycled for re-exocytosis
(Schonbaum et al., 2000).
In young wild-type egg chambers, Yolkless is uniformly distributed
throughout the ooplasm (Fig.
3A, arrowhead). At stage 8, with the onset of vitellogenesis,
Yolkless switches from a cytoplasmic to a cortical localization. By stage 10,
Yolkless is essentially restricted to the oocyte membrane
(Fig. 3A). Yolkless goes
through cycles of endocytosis, recycling and exocytosis to import vitellogenin
from the perivitelline space into the oocyte
(Schonbaum et al., 2000). In
young affected rab6D23D egg chambers, Yolkless
localization in the oocyte appeared normal
(Fig. 3B, arrowhead). At
mid-oogenesis, however, although some Yolkless was detected at the oocyte
cortex, a significant proportion of the protein remained cytoplasmic
(Fig. 3B). Thus, Rab6 is
required for correct Yolkless trafficking at mid-oogenesis, but not for its
early enrichment in the oocyte.
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). At this location, the mRNA is associated with local
antero-dorsal ER-Golgi units and is translated; subsequently, Gurken protein
is processed for secretion (Herpers and
Rabouille, 2004). In wild-type egg chambers at stage 10, Gurken is
detected as a sharp signal between the oocyte nucleus and the proximal plasma
membrane (Fig. 3C)
(Neuman-Silberberg and Schupbach,
1996), as well as in the cytoplasm of the overlying follicle cells
(Fig. 3C'', arrow)
(Peri et al., 1999). By
contrast, the perinuclear signal appeared diffuse and most Gurken protein was
detected in droplet-like structures dispersed throughout the ooplasm in the
majority of rab6D23D egg chambers
(Fig. 3D,E). In some egg
chambers (29.8%), Gurken was nevertheless detected in the cytoplasm of the
apposed follicle cells (Fig.
3D'', arrow). However, in others (25.7%), the protein was not
detected beyond the oocyte cytoplasm (Fig.
3E''). Remarkably, no ventralized eggs were observed,
indicating that affected rab6D23D egg chambers degenerate
before egg-laying. As gurken mRNA localization and translation are
normal in rab6D23D egg chambers (data not shown), Gurken
mislocalization is most likely the consequence of impaired exocytic
trafficking of the protein.
Sec5 and Syx1A membrane localization is differentially impaired in rab6D23D egg chambers
To characterize further the exocytosis defects observed in rab6
egg chambers, we examined the localization of different proteins involved in
this process. In eukaryotic cells, proteins of the SNARE family mediate the
fusion of intracellular membranes (for a review, see
Chen and Scheller, 2001).
Syntaxin-1A (Syx1A), a t-SNARE that is found uniformly distributed on target
membranes, promotes the fusion of docked vesicles of the regulated secretory
pathway (Schulze and Bellen,
1996; Sollner et al.,
1993).
In young wild-type egg chambers, Syx1A is detected on all membranes of the germline cyst (Fig. 4A') and, at mid-oogenesis, predominantly localizes to nurse cell membranes (Fig. 4A, arrow). In strongly affected rab6D23D egg chambers at early stages, and in mildly affected rab6D23D egg chambers at mid-oogenesis, Syx1A is detected around the cluster of ring canals and actin debris (Fig. 4B, arrowheads). However, in these two classes of egg chambers, it is also detected on the remaining membranes that individualize nurse cell syncytia (Fig. 4B, arrows). These results indicate that Syx1A transport, delivery and/or recycling to the remaining plasma membranes of the egg chamber are not abolished by the lack of Rab6.
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; Murthy and Schwarz,
2004). The exocyst is a conserved complex of proteins implicated
in exocytosis and vesicle trafficking (reviewed in
Lipschutz and Mostov, 2002).
It plays a crucial role in new membrane addition and marks regions of active
secretion and cell growth in yeast and mammalian cells. During
Drosophila oogenesis, Sec5 enrichment correlates with the zone of
active growth, where activity of the protein is required
(Murthy and Schwarz, 2004). In
amorphic sec5E10 mutants, oogenesis arrests early and
clusters of ring canals are found in the cytoplasm of those egg chambers.
Hypomorphic sec5E13 mutant egg chambers develop further
and present a variety of actin phenotypes ranging from wild-type-like, to a
lack of cortical actin associated with cytoplasmic ring canal clusters. In
those egg chambers, the distribution of Yolkless and Gurken proteins is
abnormal (Murthy and Schwarz,
2004).
The striking similarity of the sec5 and rab6 mutant
phenotypes led us to examine Sec5 distribution in rab6D23D
egg chambers. Sec5 was present on nurse cell and oocyte plasma membranes in
wild-type egg chambers (Fig.
4C,C') (Murthy and
Schwarz, 2004), where it appeared to colocalize with the actin
cytoskeleton when examined by confocal microscopy
(Fig. 4C, arrow). By contrast,
Sec5 was absent from the residual membranes separating the open nurse cell
syncytia of rab6D23D egg chambers
(Fig. 4D, arrow), suggesting
that the trafficking and/or membrane maintenance of Sec5 was impaired.
Remarkably, Sec5 was readily detected on the oocyte plasma membrane of these
rab6 egg chambers (Fig.
4D'). Hence, there is an important, yet differential,
requirement for Rab6 in Sec5 localization to membranes within the germline
cyst.
Our finding that loss of rab6 in the germline does not hamper localization of the t-SNARE protein Syx1A, but affects plasma membrane localization of exocyst component Sec5, suggests that some of the rab6 phenotypes we observe might be due to Sec5 mislocalization. However, the observed defects in Gurken secretion by rab6D23D oocytes, in which Sec5 is still correctly localized, suggest that Rab6 might act directly, independent of Sec5, in Gurken exocytosis.
Furthermore, investigation of the distribution of Golgi and ER markers by
immunofluorescence and electron microscopy revealed an enlargement of the
Golgi, but not of the ER compartment, indicating an impairment of exocytosis
at the Golgi level in rab6D23D egg chambers (see Fig. S5
and Fig. S6 in the supplementary material, and data not shown). These results
are consistent with the characterized role of mammalian Rab6 in the
trafficking of Golgi-derived membranes
(Antony et al., 1992;
Martinez et al., 1994;
Matanis et al., 2002;
Short et al., 2002;
White et al., 1999;
Young et al., 2005).
|
; Jankovics et al.,
2001; Ruden et al.,
2000). We therefore wished to determine if rab6 might
also play a role in this process. To this end, we assessed the distribution of
oskar mRNA either directly or by using the RNA-binding protein
Staufen as a reporter (Ephrussi et al.,
1991; Kim-Ha et al.,
1991; St Johnston et al.,
1991). During early oogenesis, oskar mRNA concentrated in
rab6D23D oocytes as it did in wild type (data not shown).
At mid-oogenesis, Staufen/oskar localized to the posterior pole of
wild-type oocytes, where they form a tight crescent from stage 9 onwards
(Fig. 5A,E)
(Ephrussi et al., 1991;
Kim-Ha et al., 1991). By
contrast, at stages 9 and 10, approximately 70% of affected
rab6D23D egg chambers showed mislocalized
Staufen/oskar (n=268,
Fig. 5C,D,G-H).
As posterior localization of oskar mRNA requires a correctly
polarized MT cytoskeleton, we assessed this polarity in
rab6D23D egg chambers using the Kin:ß-gal reporter, a
fusion of ß-galactosidase to the motor domain of Kinesin
(Clark et al., 1994). In
contrast to wild-type stage 9-10 oocytes, a large proportion of
rab6D23D oocytes displayed an abnormal distribution of
Kin:ß-gal and Staufen. To ensure that these phenotypes were not the
consequence of defective egg-chamber organization (actin cytoskeleton and
membranes), we restricted our evaluation to egg chambers presenting the
stereotypical pattern of two major open syncytia and an individualized oocyte,
described above.
In close to one third of affected rab6D23D egg chambers, Kin:ß-gal and Staufen localized at the posterior of the oocyte (Fig. 5B-B''), as in wild-type (Fig. 5A-A''). In the remaining two-thirds, the crescent of Kin:ß-gal and Staufen was either dramatically reduced (Fig. 5C) or entirely absent (Fig. 5D), and the two proteins were detected as a blob away from the oocyte cortex (Fig. 5C,D; see Fig. S7 in the supplementary material for detailed percentages). Thus, nearly 70% of affected rab6D23D egg chambers displayed a mislocalization of Staufen that correlated with mispolarization of the MT network.
|
;
Kim-Ha et al., 1995;
Markussen et al., 1995;
Rongo et al., 1995).
Consistent with the distribution of Staufen, in close to 30% of affected
rab6D23D egg chambers, oskar mRNA and Oskar
protein formed a tight crescent, as occurred in wild type
(Fig. 5E,F). In the remaining
70%, oskar mRNA was either partially or completely mislocalized and
was detected away from the cortex (Fig.
5G',H'). No ectopic Oskar protein was associated with
the unlocalized mRNA in these egg chambers
(Fig. 5G'',H''),
indicating that the unlocalized oskar mRNA remained translationally
repressed. Taken together, these results strongly suggest that the
oskar mRNA mislocalization defect in rab6D23D egg
chambers results from a failure in mRNA localization, rather than in its
anchoring.
Our analysis of egg-chamber organization suggests that Sec5 might be a
downstream functional effector of Rab6 in that process. We therefore assessed
whether the localization of oskar mRNA is normal in
sec5E13 egg chambers. Whereas rab6D23D
oocytes displayed a mis-polarization of the MT cytoskeleton
(Fig. 5C,D),
sec5E13 oocytes did not
(Murthy and Schwarz, 2004).
Despite the disorganization of sec5E13 egg chambers,
Staufen localization appeared in sec5E13 as it did in
wild-type oocytes (Fig. 6B).
Therefore, although both Sec5 and Rab6 are required for egg-chamber
organization, Rab6 is specifically required for MT organization and
oskar mRNA localization at mid-oogenesis.
Rab6 and BicD are in a complex and act together in oskar mRNA localization
In mammalian cells, BicD homologs localize to the TGN, where they interact
with Rab6 and the Dynein-Dynactin complex, creating a link between Golgi
vesicles and MT tracks and motors
(Hoogenraad et al., 2001;
Matanis et al., 2002). To test
whether Rab6 and BicD are present in a complex in Drosophila ovaries,
we performed co-immunoprecipitation experiments using ovarian extracts of
transgenic flies expressing Myc-tagged Rab fusion proteins. In addition to
flies expressing Myc-Rab6, we included flies expressing Myc-Rab7 as a control,
because these two Rab proteins are 71.4% identical but perform different
functions. As shown in Fig. 7A,
BicD co-immunoprecipitates with Myc-Rab6 but not with Myc-Rab7. This
demonstrates that Rab6 and BicD interact specifically in vivo.
We next tested whether the depletion of Rab6 might affect BicD protein
function or localization in the egg chamber. At mid-oogenesis, BicD activity
is required for cortical anchoring of the oocyte nucleus at the antero-dorsal
corner of this cell (Swan and Suter,
1996). Although BicD localizes normally in
sec5E13 oocytes, the nucleus is occasionally displaced
(Murthy and Schwarz, 2004). By
contrast, within rab6D23D oocytes, no mislocalization of
the nucleus was observed, and BicD was detected between the oocyte nucleus and
the cortex, as it was in wild type (Fig.
7C',D'). However, within rab6D23D
nurse cell syncytia, BicD protein was detected ectopically around the cluster
of ring canals (Fig. 7D,
arrowhead). This might reflect the abnormal distribution of Golgi material in
rab6D23D egg chambers. Thus, the functions of BicD in
oocyte specification in early oogenesis and later, in the anchoring of the
oocyte nucleus, do not appear to be affected by a lack of Rab6 and might
therefore involve interactions with other proteins.
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;
Hoogenraad et al., 2003;
Matanis et al., 2002;
Short et al., 2002), was
originally identified in Drosophila for its role in embryonic
patterning and in mRNA localization in the oocyte and embryo
(Bullock and Ish-Horowicz,
2001; Oh and Steward,
2001; Suter and Steward,
1991). We therefore wished to test whether Rab6 might cooperate
with BicD in oskar mRNA localization at mid-oogenesis. To this end,
we examined Staufen localization at mid-oogenesis in various combinations of
rab6 and BicD mutant alleles. In addition to the genomic
null allele rab6D23D, we also made use of
rab6wrt and BicDR5. As a control, we
included par-19A, a mutant allele of par-1, whose
product, the serine/threonine kinase Par-1, is a known regulator of the oocyte
MT cytoskeleton, required for its reorganization at mid-oogenesis and for the
concomitant localization of oskar mRNA
(Shulman et al., 2000;
Tomancak et al., 2000). Flies
homozygous for the hypomorphic allele rab6wrt are viable,
fertile (Purcell and Artavanis-Tsakonas,
1999) and did not show any oogenesis phenotype (data not shown).
Flies homozygous for the strong hypomorphic allele par-19A
are viable, but 70% of the egg chambers show full or partial mislocalization
of oskar mRNA in the middle of the oocyte at mid-oogenesis
(Tomancak et al., 1998).
BicDR5 is homozygous lethal and does not support
oogenesis: BicDR5 egg chambers contain 16 nurse cells and
no oocyte (Oh and Steward,
2001). Staufen localization at mid-oogenesis appeared to be only very mildly affected, and to a similar degree, in BicDR5 heterozygous females, par-19A heterozygous females and in females bearing the strongest viable combination of rab6 alleles (rab6wrt/rab6D23D) (Fig. 7B). An additive effect of the rab6 and par-1 mutations on the penetrance of Staufen mislocalization was observed in rab6D23D, par-19A/rab6wrt double-mutant egg chambers (5.8%, Fig. 7B). By contrast, a synergistic effect of the simultaneous mutation of rab6 and BicD was observed in rab6D23D,BicDR5/rab6wrt double-mutant egg chambers (20%, Fig. 7B). This synergy between the rab6 and BicD mutant alleles in Staufen mislocalization, together with the presence of Rab6 and BicD proteins in a complex, suggests that they cooperate to promote oskar mRNA localization. They might act directly in this process, or might do so indirectly by affecting the organization of the oocyte MT and actin cytoskeletons.
Taken together, our findings indicate that Drosophila Rab6 exerts dual functions during oogenesis. First, Rab6 mediates membrane trafficking that is important for growth and organization of the egg chamber, possibly acting upstream of exocyst component Sec5 in this process. Second, Rab6, in conjunction with BicD but independently of Sec5, has a novel and specific role in the polarization of the oocyte MT cytoskeleton and in oskar mRNA localization.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During polarized exocytosis, secretory vesicles emerging from the TGN are
targeted via molecular motors and cytoskeletal tracks to the plasma membrane,
where they are tethered. Subsequently, their fusion with the plasma membrane
permits the secretion of the vesicle contents, as well as the incorporation of
vesicular lipids and proteins into the plasma membrane, allowing membrane
growth and the establishment of specific domains. The exocyst complex plays a
crucial role in the incorporation of particular membranes and membrane
proteins at specific sites or in active domains of the plasma membrane
(reviewed in Hsu et al., 2004;
Lipschutz and Mostov, 2002).
Consistent with this, Drosophila sec5 mutant egg chambers display
mislocalization of other exocyst components, cytoplasmic clusters of actin and
a loss of plasma membranes (Murthy et al.,
2005; Murthy and Schwarz,
2004). Thus, Sec5 protein is at the core of the exocyst complex in
Drosophila, as is the case in yeast and in mammals
(Guo et al., 1999;
Matern et al., 2001).
Both sec5 null (sec5E10) and strongly affected
rab6D23D egg chambers display actin and general
organization defects, and arrest development during early oogenesis
(Murthy and Schwarz, 2004)
(our observations). Similarly, sec5 hypomorphic
(sec5E13) and rab6D23D egg chambers
that develop past stage 7 display phenotypes ranging from wild type to a loss
of nurse cell cortical actin and the concomitant presence of ring canal
clusters in the nurse cell cytoplasm
(Murthy and Schwarz, 2004)
(this study). The striking parallel between the rab6 and
sec5 phenotypes, together with our finding that a loss of Rab6
affects Sec5 localization, suggests that the varying degrees of membrane loss
observed in rab6D23D egg chambers reflects the relative
reduction of exocyst-complex function in the egg chamber. Thus, during
Drosophila oogenesis, Rab6 promotes Sec5 localization and therefore
appears to be important for exocyst-complex organization and function.
However, consequent to loss of rab6 function, we observed a striking
difference between nurse cells and oocyte in the severity of plasma membrane
collapse and Sec5 mislocalization. We hypothesize that the oocyte acts as a
major source of membrane in rab6D23D egg chambers and/or
that multiple exocytic pathways cooperate within the germline cyst to promote
cyst development.
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; Lin et al.,
1994). It is thought that the oocyte is the four-ring-canal cell
that retains the greater part of fusome during the first division
(de Cuevas and Spradling,
1998; Huynh and St Johnston,
2004). Furthermore, a Drosophila Balbiani body has
recently been discovered, which, together with the fusome, organizes the
specific enrichment of organelles in the oocyte throughout oogenesis
(Cox and Spradling, 2003). It
is therefore possible that, in rab6 clones, in which the fusome
appears normal (data not shown), such a mechanism of enrichment of organelles
in the oocyte concomitantly ensures that the concentration in the oocyte of
any perduring Rab6 protein, thus privileging the growth of the plasma membrane
of the oocyte over that of the nurse cells. Supporting this notion is the
observation that GFP-tagged Rab6 expressed in the germline is enriched in the
oocyte from the early stages of oogenesis (germarium region 2) onwards (see
Fig. S2 in the supplementary material). Together, the combined actions of a
residual Rab6-dependent and of additional Rab6-independent pathways might also
permit most rab6D23D oocytes to maintain sufficient
vesicular trafficking to develop past stage 7. The stereotypic organization of affected rab6D23D egg chambers at mid-oogenesis is striking (see Fig. S4A in the supplementary material). The oocyte is connected to open syncytia via its four ring canals, suggesting that the membranes linking nurse cells and oocyte are the most resistant (see Fig. S4B in the supplementary material). Furthermore, the growth of the remaining membranes indicates that additional vesicular material is delivered and incorporated into these plasma membranes. This suggests that, in these rab6D23D egg chambers, sustained vesicle trafficking in the oocyte causes new membrane addition to the oocyte plasma membrane. We hypothesize that, due to the continuity of the plasma membrane defining the cyst, the oocyte acts as a source of membrane that spreads by lateral diffusion throughout the plasma membrane of the cyst, allowing its growth.
Multiple exocytic pathways within the germline cyst
It appears that Rab6-independent exocytic pathways also contribute to the
delivery of vesicular material to the plasma membrane in the
Drosophila egg chamber. Indeed, Syx1A is detected on the remaining
plasma membrane of both rab6-null
(Fig. 4) and sec5 egg
chambers (Murthy and Schwarz,
2004; Sommer et al.,
2005), supporting the existence of a Rab6- and Sec5-independent
exocytic pathway mediating protein export. This selective loss of Sec5 from
nurse cell membranes in rab6 open syncytia, together with the known
functions of the exocyst, suggest a simple explanation (depicted in Fig. S4 in
the supplementary material) for the defects caused by a lack of Rab6 function
in oogenesis. We hypothesize that Rab6-dependent and -independent pathways
might differ qualitatively in the proteins whose traffic they mediate, or
quantitatively in their relative contributions to the delivery of the same
cargo between nurse cells and oocyte. These differences may account for the
observed differential requirement for Rab6 in the localization of Sec5 in
nurse cell, versus oocyte, plasma membranes (see Fig. S4C,C' in the
supplementary material).
Oocyte polarization and vesicular trafficking
Our analysis has revealed two separate functions of Rab6: one is a general
role in the organization and growth of the egg chamber, and the other is its
specialized role in MT cytoskeleton polarization and oskar mRNA
localization (Fig. 5). This
second function appears specific to Rab6 because, in sec5 mutant egg
chambers, Staufen localization is normal
(Fig. 6) and the MT
cytoskeleton is correctly organized
(Murthy and Schwarz, 2004).
Only oskar mRNA, and not Oskar protein, is ectopically detected in
rab6D23D egg chambers
(Fig. 5). This suggests an
impairment of oskar mRNA localization, rather than a defect in its
anchoring, in which case Oskar protein would be detected with the detached RNA
(Vanzo and Ephrussi, 2002).
Defects in oskar mRNA localization, which relies on MT polarity,
could be due to a failure in the focusing of the MT cytoskeleton that is
observed in rab6 egg chambers
(Fig. 5).
In Drosophila and mammalian cells, BicD is known to regulate MT
organization (Oh and Steward,
2001; Fumoto et al.,
2006; Claussen and Suter,
2005). At mid-oogenesis, Rab6 and BicD cooperation could direct MT
organization and/or promote the vesicular transport necessary for oocyte
polarization and oskar mRNA localization. Given the implication of
membrane trafficking in the asymmetric localization of mRNAs (reviewed in
Cohen, 2005), it also possible
that polarized membrane transport along the oocyte MT network directs
oskar mRNA to the posterior of the oocyte, by hitch-hiking along
trafficking vesicles.
In MDCK cells, definition of apical and basolateral plasma membrane domains
is required during polarization for the arrangement of MT along an
apical-basal axis (Bacallao et al.,
1989). Vesicular trafficking is crucial to establish, specify and
maintain these membrane domains (reviewed in
van der Wouden et al., 2003;
van Ijzendoorn et al., 2003).
By analogy, at stage 7, the polarizing signal from the posterior follicular
cells to the Drosophila oocyte that causes repolarization of the MT
cytoskeleton might do so by inducing the definition of anterior-lateral and
posterior membrane domains. It is therefore possible that, in
rab6D23D oocytes, as in epithelia, defects in vesicular
trafficking and TGN sorting underlie the observed defects in MT-network
organization. Consistent with this idea, a mispolarized MT cytoskeleton is
also observed in oocytes lacking Rab11
(Dollar et al., 2002;
Hoekstra et al., 2004;
Jankovics et al., 2001). Thus,
vesicular trafficking and the specification of membrane domains may be
required for repolarization of the MT network and for the localization of
molecular determinants in the Drosophila oocyte at mid-oogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/1419/DC1
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ACKNOWLEDGMENTS |
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