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First published online 13 February 2008
doi: 10.1242/dev.017293
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Laboratory for Germline Development, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.
* Author for correspondence (e-mail: akiran{at}cdb.riken.jp)
Accepted 7 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Germ plasm, Cell polarity, Intracellular RNA localization, oskar, Microtubules, Actin, Endosomes, Endocytic pathway
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Germ plasm contains germ granules, which are electron-dense, and
non-membranous structures consisting of maternal RNAs and proteins required
for the formation of germ cells. Drosophila germ plasm, also called
pole plasm, forms at the posterior pole of the embryo and is inherited by the
germline precursors, or pole cells
(Mahowald, 2001). Because the
cytoplasmic transplantation of the pole plasm into recipient embryos causes
the ectopic formation of pole cells
(Mahowald, 2001), the pole
plasm contains sufficient factors for germ-cell formation. This observation
also highlights the importance of retaining the pole plasm at the posterior
cortex of the embryo to ensure the germ cells form at the appropriate
location.
In Drosophila, the pole plasm is assembled during oogenesis, which
is divided into 14 morphologically distinct stages of egg chamber development
(Spradling, 1993). The egg
chamber is composed of a single oocyte and 15 nurse cells, surrounded by a
monolayer of somatic follicle cells. During oogenesis, most components of pole
plasm are synthesized in the nurse cells and transported into the oocyte via
ring canals, which are cytoplasmic bridges interconnecting the oocyte with
nurse cells. Within the oocyte, these factors become concentrated at the
posterior pole and are assembled into the polar (germ) granules. These factors
are transported by a polarized microtubule (MT) array that is initially
nucleated at the oocyte posterior and extends into the nurse cells through the
ring canals (Steinhauer and Kalderon,
2006). During stages 6-7, the MT array is reorganized by the
transforming growth factor 
-like Gurken (Grk) signal
(Steinhauer and Kalderon,
2006). In the stage-6 oocyte, posteriorly restricted Grk induces
neighboring follicle cells to adopt the posterior fate. These cells send back
as-yet unknown signals to the oocyte to trigger the reorganization of the MT
cytoskeleton. Consequently, the MT array within the oocyte becomes polarized
along the anteroposterior (AP) axis, with the minus ends abundant at the
anterior of the oocyte and the plus ends extending toward the posterior. This
MT organization promotes the migration of the oocyte nucleus and associated
grk RNA to the future anterior-dorsal corner, where Grk signals the
follicle cells to define the dorsoventral axis. The polarized MT array also
directs the localization of bicoid (bcd) RNA to the anterior
and oskar (osk) RNA to the posterior within the oocyte. The
anterior accumulation of bcd RNA is required for the proper
development of the embryonic head and thoracic structures
(Ephrussi and St Johnston,
2004). The posterior localization of osk RNA is essential
for the formation of the germ cells and abdomen
(Mahowald, 2001).
osk RNA localization is tightly coupled to translational control:
only the posteriorly localized osk message is translated
(Wilhelm and Smibert, 2005).
The localized Osk protein, in turn, recruits downstream components of the pole
plasm, such as Vasa (Vas) and Tudor (Tud) proteins, and the nanos, germ
cell-less and polar granule component RNAs
(Mahowald, 2001).
Misexpression of Osk at the anterior of the oocyte causes ectopic pole plasm
assembly and the formation of germ cells at the new site
(Ephrussi and Lehmann, 1992),
indicating that Osk organizes pole plasm assembly.
Although osk has no known alternatively spliced variants, the
osk message produces two protein isoforms, long and short Osk, by
translation from in-frame alternative start codons
(Markussen et al., 1995).
Short Osk shares its entire sequence with the long isoform. Nevertheless,
genetic evidence shows that the two Osk isoforms have distinct functions in
the assembly of the pole plasm (Markussen
et al., 1995; Breitwieser et
al., 1996; Vanzo and Ephrussi,
2002). Short Osk assembles the pole plasm
(Markussen et al., 1995) and
is likely to recruit Vas directly
(Breitwieser et al., 1996).
Long Osk is required for all the components of the pole plasm, including Osk
itself, to be anchored to the posterior cortex, preventing their diffusion
into the cytoplasm (Vanzo and Ephrussi,
2002). However, the mechanism by which long Osk retains pole plasm
components at the posterior cortex remains unknown.
A recent immuno-electron microscopic study revealed that the two Osk
isoforms localize to distinct organelles in the oocyte posterior: long Osk
associates with endosomes and short Osk is concentrated in the polar granules
(Vanzo et al., 2007). Long Osk
also upregulates endocytosis, which occurs preferentially at the oocyte
posterior (Vanzo et al.,
2007). Therefore, the endocytic pathway may be involved in pole
plasm assembly downstream of long Osk, although data are lacking to show that
the association between long Osk and endosomes is functionally significant.
Several reports have suggested that vesicular trafficking is involved in pole
plasm assembly and germ cell formation
(Ruden et al., 2000;
Jankovics et al., 2001;
Dollar et al., 2002;
Coutelis and Ephrussi, 2007;
Januschke et al., 2007;
Swanson and Poodry, 1980). For
example, in mutants for Rab11, which encodes a small GTPase involved
in the recycling of endosomes, osk RNA fails to be transported to the
oocyte posterior, instead forming aggregates close to the posterior
(Jankovics et al., 2001;
Dollar et al., 2002). However,
the defects in osk RNA localization in Rab11 mutants are
thought to be an indirect consequence of the disrupted MT polarization
(Jankovics et al., 2001;
Dollar et al., 2002).
Here, we show that Drosophila Rabenosyn-5 (Rbsn-5), a Rab5 effector protein involved in the early endocytic pathway, is required for osk RNA localization and pole plasm assembly. Although the primary defect of the rbsn-5 mutation is, as in the Rab11 mutant, caused by the failure to maintain MT polarity, which secondarily affects osk RNA localization, we provide evidence that the endocytic pathway also functions downstream of Osk to anchor the pole plasm components to the oocyte cortex.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
grkHK36/Df(2L)ED623
(Neuman-Silberberg and Schüpbach,
1993). To assess the specificity of newly raised antibody against
Rab5, a Rab5-null allele, Rab52
(Wucherpfennig et al., 2003)
was used. To analyze MT polarity, yw; Pin/CyO;
KZ503 (Clark et al.,
1994) was used. A germline-specific
mat-Gal4VP16 driver was used to express UASp-based
transgenes in oogenesis.
Mutant screen
Isogenized w; P{neoFRT}40A males were starved for 12
hours in an empty vial. These flies were transferred into a vial containing
filter paper soaked with 1% sucrose solution containing 25 mM ethyl
methanesulfonate (EMS) (Sigma) and kept for 16-20 hours at 25°C. We
obtained an average of 1.4 lethal hits per chromosome 2. EMS-exposed flies
were mated with yw hs-FLP; Gla/CyO females. Single male
progenies were crossed to w P{w+; Pvas-egfp::vas};
P{w+; Ubi-GFP(S65T)nls}2L P{neoFRT}40A
females. To obtain germline clones (GLCs), third-instar larvae from this cross
were heat-shocked at 37°C for 2 hours on two successive days.
Subsequently, we hand-dissected ovaries from three to five females of genotype
y w hs-FLP/w P{w+; Pvas-egfp::vas};
* P{neoFRT}40A/P{w+;
Ubi-GFP(S65T)nls}2L P{neoFRT}40A (where the asterisk
represents the mutation) and tested them for GFP-Vas localization in the GLCs
that were marked by the lack of nuclear GFP. The lines that showed defects in
GFP-Vas localization were recovered by crossing males in the same vials to
w; Gla/CyO females to obtain balanced stocks (w;
* P{neoFRT}40A/CyO).
Chromosomal mapping
Chromosomal mapping for mutations was carried out as described
(Berger et al., 2001), using
PCR product length polymorphisms and restriction fragment length polymorphisms
between the P{neoFRT}40A and P{EP} chromosomes. After
defining the mutated position within several hundred kb by deficiency mapping,
the annotated genes in the region were PCR amplified from w;
* P{neoFRT}40A/P{neoFRT}40A flies, and the products were
direct-sequenced.
Transgenic flies
To rescue the rbsn-5C241 mutant, a 4.5 kb genomic
fragment containing the rbsn-5 transcription unit and 2.3 and 0.3 kb,
respectively, of the 5' and 3' genomic flanking regions was cloned
into pCaSpeR4. To express Osk at the anterior pole of the oocyte, the entire
osk ORF sequence was joined to a modified bcd 3' UTR,
in which the putative NRE sequence (a 45 bp HpaI-EcoRV
fragment) was replaced by a 10 bp SpeI linker
(Wharton and Struhl, 1991) and
cloned into the pUASp vector. To express the long or short Osk isoform
specifically, an ATG triplet corresponding to the start codon for the unwanted
isoform was changed to CTG by site-directed mutagenesis [M1L for short Osk and
M139L for long Osk (Markussen et al.,
1995)]. P-element-mediated germline transformation was
carried out by standard methods.
Antibody generation
A full-length rbsn-5 ORF was cloned into pProExHTa (Gibco) to
produce a 6xHis-tagged protein. The fusion proteins were purified with
Ni-NTA agarose (Qiagen) followed by a disc preparative gel electrophoresis and
used to raise rabbit and rat antibodies. The polyclonal rabbit antibodies
against Rab5, Rab11 and Rab7 were generated and purified using the synthetic
peptides SGTGTAQRPNGTSQNKSC (amino acid residues 11-28 of Rab5),
CSQKQIRDPPEGDVIR (amino acid residues 177-191 of Rab11) and CNDFPDQITLGSQNNRPG
(amino acid residues 184-200 of Rab7), respectively. Specificities of these
antibodies were evaluated by immunoblotting and immunostaining
(Fig. 1D and see Fig. S1 in the
supplementary material).
Immunoblotting
Hand-dissected ovaries from 50 females were homogenized in ice-cold lysis
buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.5).
Total ovarian protein (50 µg) was run on SDS-PAGE and transferred onto an
Immobilon-P membrane (Millipore). The signals were detected using the ECL
system (GE Healthcare).
GST pull-down assay
The full-length rbsn-5 ORF was PCR-amplified and cloned into pTNT
(Promega). Recombinant proteins were synthesized in vitro using the TNT Quick
Coupled Transcription/Translation system (Promega) in the presence of cold
amino acids. The full-length Rab5, Rab7 and Rab11 ORFs were
PCR-amplified and cloned into pGEX-5X1 (Pharmacia) to generate glutathione
S-transferase (GST) fusion proteins, which were expressed in Escherichia
coli and purified with glutathione resin (Clontech). Four micrograms of
each purified GST-Rab protein was mixed with 40 µl of 50% glutathione resin
and incubated for 1 hour at room temperature in PBS containing 0.1% Tween 20.
The GDP or GTP-bound form of each GST-Rab protein was prepared as described
(Christoforidis and Zerial,
2000). In-vitro translated Rbsn-5 protein (2 µl) was incubated
with GDP- or GTP-bound GST-Rab resins for 1 hour at 4°C under rotation in
nucleotide stabilization (NS) buffer
(Christoforidis and Zerial,
2000) containing 10 µM GDP or GTP
S. The resins were
washed extensively with NS buffer. Rbsn-5 protein bound to the resins was
detected by immunoblotting.
Immunostaining
Immunostaining of ovaries was performed by standard procedures. The
following primary antibodies were used: rabbit anti-Stau (1:3000; a gift of D.
St Johnston, The Gurdon Institute, Cambridge, UK), guinea pig anti-Osk
(1:3000; laboratory stock), rabbit anti-Osk (1:8000; a gift of A. Ephrussi,
EMBL, Heidelberg, Germany), rabbit and rat anti-Rbsn-5 (1:5000 and 1:2000,
respectively), rabbit anti-Rab5 (1:1000), rabbit anti-Rab11 (1:8000), rabbit
anti-Rab7 (1:3000), mouse anti-KDEL (1:25; StressGen), mouse anti-Golgi
(1:100; Calbiochem), mouse anti-β-gal (1:5000; Promega), rabbit and rat
anti-Vas (1:2500 and 1:1500, respectively; laboratory stock), and rabbit
anti-Tud (1:2000) (Amikura et al.,
2001). Alexa-conjugated secondary antibodies (1:1000; Invitrogen)
were used. Ovaries were counterstained with 4 U/ml Alexa-660-conjugated
phalloidin to label F-actin and/or with 1 µg/ml DAPI to label nuclei. All
images were captured by a laser confocal microscope (Leica TCS SP2 AOBS) using
a 63x PL APO water-immersion lens (N.A. 1.2) and processed with Adobe
Photoshop.
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FM4-64 incorporation assay
The FM4-64 incorporation assay was performed as described
(Sommer et al., 2005).
Briefly, ovaries were dissected in Drosophila-SFM (Invitrogen) containing 10
µM FM4-64 dye (Invitrogen) and kept for 30 minutes at 25°C. Ovaries
that incorporated the dye were back-extracted by washing them twice for 15
minutes each in Drosophila-SFM. Immediately after being mounted on slide
glass, the ovaries were examined under a confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
A GFP-Vas fusion protein enables the direct visualization of the pole plasm
and germ cells in the living organism
(Sano et al., 2002). During
oogenesis, GFP-Vas accumulates at the oocyte posterior from stage 9 onward.
Using GFP-Vas as a marker, we performed a germline clonal screen targeting
chromosome 2L for mutations that disrupted pole plasm assembly (our
unpublished data). From 5122 lines mutagenized with EMS, we isolated 66
mutants defective in GFP-Vas localization. Twenty-seven of these were alleles
of cappuccino, spire or profilin (chickadee), three
genes on 2L that are known to be involved in osk RNA localization
(Steinhauer and Kalderon,
2006), which validated our screening strategy. Among the other mutants recovered (Fig. 1A,B) was a recessive lethal mutation, C241, that mapped to 28C2-29E2. Subsequent deficiency mapping and sequencing of the mutant chromosome revealed that the C241 mutation was a single nucleotide substitution in the CG8506 gene, which resulted in a premature stop codon at position 315 of the 505 amino acid open reading frame (ORF) (Fig. 1C, asterisk). The introduction of a transgene containing a genomic DNA fragment with the CG8506 transcriptional unit rescued the C241 mutant phenotypes (described below). These data show that CG8506 corresponds to the gene that was mutated at the C241 locus. Rabbit and rat polyclonal antisera raised against full-length CG8506 did not detect a truncated form of CG8506 in ovarian extracts from C241 heterozygotes (Fig. 1D). Furthermore, neither antibody showed immunoreactivity in C241 homozygous clones (see Fig. S1A-D in the supplementary material), suggesting that the truncated protein was not expressed at detectable levels and/or was unstable. Therefore, C241 appeared to be a strong loss-of-function, presumably a protein-null, allele of CG8506.
CG8506 encodes a Drosophila Rabenosyn-5 homolog required for the early endocytic pathway
CG8506 (Rabenosyn - FlyBase) encodes a protein homologous
to Rabenosyn-5 (Rbsn-5) (Fig.
1C) (Nielsen et al.,
2000). Rbsn-5 interacts with several Rab proteins, including Rab5,
which functions in early endosomal transport
(de Renzis et al., 2002;
Eathiraj et al., 2005).
Several Rbsn-5 protein domains are conserved across species, including the
FYVE domain, which binds phosphatidylinositol-3-phosphate
(Nielsen et al., 2000).
However, invertebrate Rbsn-5 homologs lack the C-terminal domain common to the
mammalian homologs of this protein (Fig.
1C). As the C-terminal domain of mammalian Rbsn-5 is responsible
for its interaction with Rab5 (de Renzis
et al., 2002; Eathiraj et al.,
2005), we examined whether CG8506 interacted with Rab5. Pull-down
assays showed that GST-Rab5 efficiently pulled down in-vitro-synthesized
CG8506 protein in the presence of a GTP analog, GTP-S, but
inefficiently in the presence of GDP (Fig.
1E). The interaction between CG8506 and Rab5-GTP was specific,
because the interactions of CG8506 with Rab11 and Rab7 were at background
levels (Fig. 1E). Consistent
with a physical interaction between CG8506 and Rab5 in vitro, in
CG8506C241 GLCs, neither auto-fluorescent granules derived
from endocytosed yolk proteins nor the incorporation of a fluorescent marker
for endocytosis, FM4-64, were observed in the oocytes
(Fig. 1F-I), suggesting that
CG8506 functions cooperatively with Rab5 in the early endocytic pathway. Thus,
CG8506 is the Drosophila ortholog of Rbsn-5 and has an evolutionarily
conserved function in the endocytic pathway.
Rbsn-5 is required for osk RNA localization and the maintenance of MT polarity
In rbsn-5- GLCs, little or no GFP-Vas was detected at
the posterior pole of the stage-10 oocyte
(Fig. 1B). The posterior
accumulation of Vas depends on the proper localization and translation of
osk RNA (Mahowald,
2001). We therefore examined whether osk RNA localization
was affected in the rbsn-5- oocyte. In wild-type oocytes,
the Staufen (Stau) protein (St Johnston et
al., 1991), a marker for osk RNP, accumulates at the
posterior region of the oocyte until stage 6
(Fig. 2A). During stages 7-8,
Stau becomes highly concentrated in the center of the oocyte. Stau is then
transported to, and remains at, the posterior pole of the oocyte until
oogenesis completes. The distribution patterns of Stau in
rbsn-5- GLCs were normal until stage 8
(Fig. 2C). However, Stau was
not tightly localized to the posterior cortex in the stage-9
rbsn-5- oocyte, resulting in its ectopic aggregation in
late-stage oocytes (Fig. 2C).
As a consequence, Osk was translated very weakly at the posterior pole in
rbsn-5- oocytes (Fig.
2D). These defects were fully rescued by the introduction of the
rbsn-5 genomic rescue construct
(Fig. 2E,F). The defects in
osk RNP localization in the rbsn-5- GLCs were
confirmed by in situ hybridization analysis of osk RNA
(Fig. 2G-J). By contrast, the
rbsn-5- oocytes showed normal localization of both
bcd and grk RNAs at the anterior and anterior-dorsal corner,
respectively (Fig. 2G-J,
arrowheads). These results indicate that rbsn-5 is specifically
required for the stable accumulation of osk RNP at the posterior
cortex of the oocyte.
The transport of osk RNA to the oocyte posterior depends on the MT
cytoskeleton, which is thought to align along the AP axis during stages 8-10
(Steinhauer and Kalderon,
2006). We therefore examined whether MT polarity was affected by
the loss of Rbsn-5 function. For this purpose, we used an MT plus-end marker,
Kinesin-β-galactosidase (Kin-βgal) fusion protein
(Clark et al., 1994), which is
normally concentrated at the posterior of stage 8-10 oocytes
(Fig. 2K,L). At stage 8, no
difference in the Kin-βgal distribution was observed between
rbsn-5- GLC and wild-type oocytes
(Fig. 2K,M), indicating that
the MT polarity was initially established in the absence of Rbsn-5. However,
in the rbsn-5- oocyte, the posterior localization of
Kin-βgal was lost from stage 9 onward. Instead, Kin-βgal, like Stau,
formed aggregates in the oocyte cytoplasm
(Fig. 2N). These results
indicate that rbsn-5 is required for the maintenance of the MT
polarity.
Posterior concentration of Rbsn-5 and endosomal proteins within the oocyte
Although the above results indicated that defects in osk RNA
localization in rbsn-5- oocytes are primarily attributable
to the failure to maintain MT polarity, the distribution of Rbsn-5 within
developing oocytes prompted us to investigate additional roles for Rbsn-5 in
pole plasm assembly. Double staining for Rbsn-5 and Stau revealed that the
distribution patterns of Rbsn-5 were very similar to those of osk RNP
(Fig. 3A,B). Although
immunolabeling for Rbsn-5 was observed on the entire oocyte cortex, it was
concentrated at the center of the oocytes at stage 7, when Stau is also
enriched in this region (Fig.
3A). Subsequently, Rbsn-5 became highly concentrated at the
posterior pole from stage 8 onward (Fig.
3B). At the posterior pole of the oocyte, Rbsn-5 signals showed
nearly complete co-localization with Stau.
To characterize the Rbsn-5-positive structures in detail, the localization
of Rbsn-5 was compared with several marker proteins for specific types of
endosomes. We found that all the endosomes that were labeled for Rab5 (an
early endosome marker), Rab11 (recycling endosomes) or Rab7 (late endosomes)
showed polarized distributions that were very similar to those of Rbsn-5 and
Stau. Although all the endosomal proteins analyzed were distributed along the
entire cortex within the oocyte, they transiently accumulated at the center of
stage 7 oocytes and later at the posterior pole of the oocyte
(Fig. 3C-H). At the center of
stage 7 oocytes, Rbsn-5 was co-localized with Rab5
(Fig. 3C), consistent with
their interaction in vitro (Fig.
1E). These polarized distributions were specific to endosomal
proteins, because other membranous organelles, such as the endoplasmic
reticulum (ER), marked by the KDEL epitope, and the Golgi apparatus, marked by
gp120 (Stanley et al., 1997),
did not show polarized distribution in the oocytes
(Fig. 3I-L)
(Lee and Cooley, 2007). The
posterior accumulation of endosomal proteins correlated well with the
preferential uptake of FM4-64 dye at the oocyte posterior
(Fig. 1H)
(Vanzo et al., 2007). These
results demonstrate that endosomal proteins and endocytic activity are highly
polarized toward the posterior pole of the oocyte.
Asymmetric endocytic activity within the oocyte depends on Grk and Osk
To test whether the posterior accumulation of endosomal proteins is
governed by osk activity, we examined the localization of Rbsn-5 and
Rab11 in osk mutant oocytes. Wild-type oocytes exhibited a clear
posterior accumulation of Rbsn-5 and Rab11 during stages 8-10
(Fig. 4A,D). In the
osk protein-null mutant, Rbsn-5 and Rab11 were initially concentrated
at the oocyte posterior, and FM4-64 was preferentially endocytosed at the
oocyte posterior at stage 8 (Fig.
4B). However, in agreement with published observations
(Dollar et al., 2002;
Vanzo et al., 2007), Rbsn-5,
Rab11 and FM4-64 uptake lost their polarized distribution in stage 10 oocytes
and were instead distributed uniformly on the oocyte cortex
(Fig. 4B,E). The same defects
were observed in an osk mis-sense mutant (data not shown). These
results demonstrate that Osk activity is required for the maintenance, but not
the establishment, of polarized endocytosis.
|
). In grk mutants, about 90% of stage 8-10 oocytes
showed no posterior accumulation of Rbsn-5 or Rab11, and the remaining 10%
showed faint posterior signals for these proteins
(Fig. 4F). The polarized
incorporation of FM4-64 was also lost in grk mutant oocytes
(Fig. 4C). These results
indicate that the posterior accumulation of endosomal proteins and asymmetric
endocytic activity require the determination of oocyte polarity.
Our observations apparently contradict the previous finding that Rab11
remains accumulated at the posterior in grk mutant oocytes
(Dollar et al., 2002).
However, we noticed that signals for endosomal proteins at the anterior cortex
in wild-type oocytes were somewhat higher than those at the lateral cortex
(e.g. Fig. 4A), suggesting that
endosomal proteins may accumulate, to some extent, at the MT minus ends as
well as the MT plus ends. In grk mutant oocytes, MT minus ends are
found at both poles (Steinhauer and
Kalderon, 2006). It is therefore likely that the weak posterior
accumulation of Rab11 in 10% of the grk mutant oocytes is due to
accumulation at the MT minus ends.
|
).
In oocytes expressing the osk-bcd 3' UTR
transgene, we found that all the endosomal proteins analyzed were recruited to
the oocyte anterior (Fig. 5A,B
and see Fig. S2A,C,E in the supplementary material). Consistent with the
ectopic localization of endosomal proteins, the anterior misexpression of Osk
promoted increased uptake of FM4-64 at the oocyte anterior (see Fig. S2G in
the supplementary material). These results indicate that the ectopic Osk can
recruit endosomal proteins and stimulate endocytosis. We next examined whether one of the Osk isoforms or both were required for the recruitment of endosomal proteins and the stimulation of endocytosis. When short Osk alone was misexpressed at the oocyte anterior, endosomal proteins did not accumulate there (Fig. 5C and see Fig. S2B,D,F in the supplementary material). By contrast, oocytes expressing a mutant version of the osk-bcd 3' UTR transgene that produces long Osk alone exhibited obvious accumulation of endosomal proteins at the anterior pole (Fig. 5D-J). Increased FM4-64 uptake at the anterior region of the oocyte was also observed when the long Osk was ectopically expressed (Fig. 5L). The anterior accumulation of endosomal protein caused by ectopic long Osk was not due to the alternation of the MT polarity within the oocyte, as Kin-βgal never concentrated at the anterior pole in the osk-bcd 3' UTR oocytes (Fig. 5N and see Fig. S2I,J in the supplementary material). These results demonstrate that long Osk alone is sufficient for recruiting endosomal proteins and stimulating endocytosis, even ectopically, without altering the MT polarity.
|
), we
supposed that the endocytic pathway might play a role in the anchoring
process. However, rbsn-5 is essential for the maintenance of MT
polarity (Fig. 2N), making it
impossible to examine the involvement of rbsn-5 in the anchoring
process during normal pole plasm assembly, which depends on the polarized MT
array. By contrast, the anterior localization of bcd RNA was
unaffected in rbsn-5- oocytes
(Fig. 2J). We therefore used
the osk-bcd 3' UTR transgene to direct ectopic pole
plasm assembly at the anterior pole of the oocyte, and examined the effects in
the rbsn-5- oocytes. Anteriorly misexpressed Osk was tightly anchored at the cortex in otherwise wild-type oocytes (Fig. 6A,B). However, in the rbsn-5- oocytes expressing the osk-bcd 3' UTR transgene, the anterior Osk was not tightly associated with the cortex, but diffused into the cytoplasm, where it formed aggregates (Fig. 6D, arrowheads). Other pole plasm components, such as Vas and Tud, also dispersed into the cytoplasm of the rbsn-5- oocyte (Fig. 6E-H and data not shown). These results demonstrate that Rbsn-5 plays a crucial role in retaining pole plasm components at the oocyte cortex.
The anchoring of pole plasm components to the posterior cortex depends on
the actin cytoskeleton (Jankovics et al.,
2002; Polesello et al.,
2002; Babu et al.,
2004). We next examined whether the F-actin organization was
affected in rbsn-5- oocytes. Osk promotes long F-actin
projections from the posterior pole of the oocyte
(Vanzo et al., 2007). We found
that anterior misexpression of Osk also induced long F-actin projections
emanating from cortical actin bundles (Fig.
6A,B). However, in the rbsn-5- oocytes, the
anterior misexpression of Osk induced large aberrant F-actin aggregates, in
which pole plasm components, such as Osk, Vas and Tud, were often engulfed
(Fig. 6D,H and data not shown).
We never observed these F-actin aggregates in rbsn-5-
oocytes that did not express osk-bcd 3' UTR
(Fig. 6C). These results
indicate that Rbsn-5 is involved in the Osk-mediated F-actin
reorganization.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
the underlying mechanism has been elusive. A recent report showed that long
Osk promotes polarized endocytosis within the oocyte
(Vanzo et al., 2007). Here we
show that Osk maintains, but does not establish, the posterior accumulation of
endosomal proteins and asymmetric endocytosis
(Fig. 4), and that Osk can
recruit endosomal proteins and stimulate endocytosis even at an ectopic site
(Fig. 5). We further show that
the anchoring of the pole plasm components to the oocyte cortex requires the
Osk-dependent stimulation of endocytic activity
(Fig. 6). These data reveal an
interdependent relationship between Osk anchoring and localized endocytic
activity at the oocyte posterior (Fig.
7).
|
;
Polesello et al., 2002;
Babu et al., 2004). As
endosomal proteins are recruited by long Osk
(Fig. 5A-J), we favor the idea
that the endocytic pathway functions downstream of long Osk to anchor the pole
plasm components at the cortex by regulating F-actin dynamics. Supporting this
idea, in addition to its roles in early endosomal sorting, Rab5 acts as a
signaling molecule that remodels F-actin networks
(Lanzetti et al., 2004).
Rab11, which regulates the recycling of endosomes, is also involved in F-actin
organization during cellularization in Drosophila blastoderm embryos
(Riggs et al., 2003).
Intriguingly, the recruitment of endosomal proteins by Osk is not sufficient
for proper F-actin reorganization to anchor the pole plasm components at the
cortex, because their recruitment occurs even in oocytes lacking Rbsn-5, in
which cortical anchoring fails (Fig.
6). We therefore propose that the continuous cycling of endosomes
is required for pole plasm components to be anchored to the oocyte cortex.
This scenario is compatible with a model in yeasts, which use endocytic
cycling coupled with localized exocytosis to maintain their polarity
(Valdez-Taubas and Pelham,
2003), although it is unclear if F-actin reorganization is
involved in this process.
Rbsn-5 is primarily required for the maintenance of MT polarity that
directs posterior localization of osk RNA
(Fig. 2). Rab11 is also
required for MT polarization in the oocyte
(Jankovics et al., 2001;
Dollar et al., 2002). However,
the accumulation of endosomal proteins and upregulation of endocytosis at the
oocyte posterior require the oocyte polarization, which promotes the
reorganization of the MT array (Fig.
4). Thus, MT polarization and asymmetric activation of the
endocytic pathway are probably interdependent as well. Furthermore,
maintenance of polarized endocytic activity depends on Osk (Figs
4 and
5). Intriguingly, Osk is also
thought to maintain MT polarity, as posterior accumulation of Kin-βgal is
partially defective in the absence of Osk
(Zimyanin et al., 2007). It is
therefore likely that the endocytic pathway and Osk form a positive-feedback
loop that maintains oocyte polarity: Osk may maintain MT polarity through
recruiting endosomal proteins. Based on these results, we propose a model, in
which the endocytic pathway is involved in several distinct steps in pole
plasm assembly (Fig. 7).
|
). Furthermore, Vps36p, a component of the ESCRT-II
complex, binds bcd 3' UTR in vitro and co-localizes
with bcd RNA at the oocyte anterior, suggesting the direct
involvement of ESCRT-II in bcd RNA localization. osk RNA,
however, appears to use another mechanism for its posterior localization, as
its localization is unaffected in the absence of ESCRT-II function
(Irion and St Johnston, 2007).
Several lines of evidence suggest that ER organization and RNA localization
are linked (Schmid et al.,
2006; Deshler et al.,
1997; Chang et al.,
2004; Wickham et al.,
1999; Gautrey et al.,
2005). However, we consider it unlikely that the ER directs the
posterior localization of osk RNA, because ER components and
osk RNP distributed differentially in developing oocytes
(Fig. 3I,J). Interestingly, the
osk RNP and the endosomal proteins were in close proximity during
their transport to the oocyte posterior
(Fig. 3A-H). Although their
close association may simply be owing to the dynamic rearrangements of the MT
array during stages 7-8 (Steinhauer and
Kalderon, 2006), these findings suggest that the endocytic pathway
may also play a role in the targeting of osk RNP to the posterior
pole of the oocyte (Fig. 7).
Retroviral genomic RNAs are known to hitchhike on endosomal vesicles to reach
the plasma membrane (Basyuk et al.,
2003). Therefore, it will be interesting to learn if osk
RNA is also transported to the posterior pole of the oocyte along with the
endosomes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1107/DC1
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ACKNOWLEDGMENTS |
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