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First published online 23 October 2008
doi: 10.1242/dev.029009
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1 Institut Jacques Monod, Unité Mixte de Recherche 7592, CNRS,
Universités Paris 7, 2 place Jussieu, F-75251, Paris Cedex 05,
France.
2 Cell Division Group, ICREA and IRB, Parc Cientific de Barcelona, c/Baldiri
Reixac 10-12, 08028 Barcelona, Spain.
3 Institut für Entwicklungsbiologie, Universität zu Köln,
Gyrhofstrasse 17, 50923, Köln, Germany.
* Author for correspondence (e-mail: guichet{at}ijm.jussieu.fr)
Accepted 28 September 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Microtubules, PAR proteins, PIP2, PIP5K, Polarity
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Drosophila oocyte, in which axis-determining mRNAs are
localized to various cortical regions, has been successfully used to study the
specification of cell polarity and the establishment of polarized transport
(St Johnston, 2005
). During
mid-oogenesis, at least two perpendicular subsets of MTs are formed,
reflecting the dorsoventral (DV) and anteroposterior (AP) axes of the oocyte
(Januschke et al., 2006;
MacDougall et al., 2003). The
axis-determining mRNAs are localized in a MT-dependent fashion
(Riechmann and Ephrussi,
2001): bicoid (bcd) mRNA is localized to the
anterior cortex of the oocyte, oskar (osk) mRNA to the
posterior pole, and gurken (grk) mRNA to an anterodorsal cap
near the oocyte nucleus. In order to identify new factors involved in the
polarized transport of mRNAs in the Drosophila oocyte, we carried out
a germline mosaic screen for mutations on chromosome arm 2R that disrupt AP
and/or DV axis formation. In the course of this screen, we identified a type I
phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles (Sktl), as an
essential factor for oocyte polarization.
Type I PIP5K synthesizes phosphatidylinositol 4,5 bisphosphate (PIP2) from
phosphatidylinositol 4-phosphate. The membrane phospholipid PIP2 is involved
in the control of cell polarity and plays a role in various cellular
activities (Doughman et al.,
2003). PIP2 regulates the membrane localization and activity of
many cellular proteins via its specific interaction with
phosphoinositide-binding domains (Downes
et al., 2005). Although accumulated data suggest that PIP2 is an
important regulator of actin-based cellular processes, in vivo analysis of
type I PIP5K
during development has been lacking. In
Drosophila, Sktl has been shown to be required for chromatin-mediated
gene regulation (Cheng and Shearn,
2004) and is important in germline development
(Hassan et al., 1998), but its
cytoplasmic function remains to be determined.
In this work, we demonstrate that the PIP5K Sktl controls the PIP2 level at the plasma membrane. We show that sktl mutations disrupt the maintenance of oocyte polarity and cause defects in actin and MT organization. We provide evidence that PIP2 synthesis by Sktl is required to activate the actin-associated protein Moesin at the cortex. Moreover, our observations indicate that Sktl activity is required for cortical recruitment of the PAR proteins Bazooka (Baz), Par-1, Lethal (2) giant larvae [Lgl; L(2)gl - FlyBase] to the cell membrane. This study suggests that PIP2, by regulating several proteins, could mediate interactions between the plasma membrane, PAR proteins and the cytoskeleton that are essential for cell polarization.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A lethal complementation analysis revealed that
Df(2R)Exel6070 fails to complement l(2)2.3. Overlapping deficiencies,
Df(2R)Exel7164 and Df(2R)Exel7166, complement the allele. This enabled us to
locate the mutation within a 150 kb fragment containing thirteen genes.
l(2)2.3 is lethal over sktl
20.
sktl20,
sktl5 and
sktl15 have been described
(Hassan et al., 1998).
Germline clones were generated by the FLP/FRT technique
(Chou et al., 1993). nanosGAL4
VP16 (nosgal4) (Van Doren et al.,
1998) and 4tubulin67c GAL4 (tubgal4)
(Januschke et al., 2002) were
used to express the transgenes during oogenesis. Other fly lines: GFP-Stau
(Schuldt et al., 1998),
Kin-lacZ (Clark et al., 1994),
Dmn-GFP (Januschke et al.,
2002), UAS-Baz-GFP and UAS-BazS151A,S1085A-GFP
(Benton and St Johnston,
2003b), UAS-GFP-Par-1 (N1S)
(Doerflinger et al., 2006),
UAS-GFP-Lkb1 (Martin and St Johnston,
2003) and UAS-Lgl-GFP (Tian
and Deng, 2008).
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-GFP construct was cloned from the
pEGFPN1-PH-PLC plasmid into a pUbi-mod poly vector (a gift from Y.
Belaiche, Curie Institute, Paris, France). The cDNA of sktl from DGRC
(LP03320) was amplified, sequenced and subcloned into pUASP, pUASP:GFP,
pUASP:RFP and pTub (a gift from P. Roth, Temasek Life Sciences Laboratory,
Singapore) vectors to prepare Drosophila transgenes.
Immunohistochemistry and in situ hybridization
Immunolocalization and in situ hybridization were performed using standard
protocols (Tautz and Pfeifle,
1989; Wilkie and Davis,
2001). Antibodies were used at the following dilutions:
anti-β-galactosidase (Promega), 1:200; anti-Osk
(Hachet and Ephrussi, 2001),
1:2000; anti-Grk (DSHB), 1:200; anti-Stau
(St Johnston et al., 1991),
1:5000; anti-phospho-ERM (Cell Signaling Technology), 1:100; anti-Spn-F
(CG12114) (Abdu et al., 2006),
1:10; anti-DPLP (Cp309) (Kawaguchi and
Zheng, 2004), 1:500; and anti-Baz
(Wodarz et al., 2000), 1:
2000. Western blots were probed with anti-phospho-ERM at 1:5000, anti-Moesin
at 1:30,000, and anti--Tubulin at 1:2500. F-actin was visualized after
staining with Alexa Fluor 680-phalloidin (Molecular Probes). DAPI and
propidium iodide were used for DNA detection. Texas Red labeled
Lycopersicon esculentum (LE) lectin (Vector Laboratories) was used at
150 µg/ml and Wheat germ agglutinin (WGA) at 1/100 (Molecular Probes).
Anti-Khc (AKIN02-A, Cytoskeleton) was used for MT detection. Cold-shock
experiments were performed as described
(Januschke et al., 2006).
Images were obtained with a Leica SP2 AOBS microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the
protein Staufen (Stau) tagged with green fluorescent protein (GFP), which
moves to the posterior of the oocyte with osk mRNA
(Fig. 1A)
(Bolivar et al., 2001;
St Johnston and Nusslein-Volhard,
1992). To visualize DV axis formation, we scored the positioning
of the nucleus relative to the anterodorsal corner of the oocyte
(Fig. 1A, asterisk). One
homozygous mutant line affecting both nuclear positioning and Stau
localization was identified in the screen and named l(2)2.3. In 57% of such
egg chambers, Stau accumulated in the center of the oocyte
(Fig. 1B), whereas in the
remainder Stau was diffusely distributed in the cytoplasm (not shown)
(n=118). The nucleus was randomly positioned within the oocyte at
stages 9-10 (Fig. 1B,
asterisk). Eggs deposited by l(2)2.3 mutant females were ventralized [loss of
dorsal appendages 51%, fused appendages 43%, wild type (WT) 6%;
n=116] (Fig. 1D), but
always presented an aeropyle at the posterior
(Fig. 1D, inset). Hence, these
results indicate that l(2)2.3 affects AP and DV axis formation without
impairing the early signaling step from the germline to the posterior follicle
cells (Gonzalez-Reyes et al.,
1995; Roth et al.,
1995). Finally, posterior follicle cell clones mutant for l(2)2.3,
marked by the loss of GFP, did not affect oocyte polarization, as revealed by
the posterior localization of Stau (Fig.
1E), whereas mutant clones restricted to the germline impaired
both nucleus positioning (Fig.
1F, asterisk) and Stau localization
(Fig. 1F). This demonstrates
that the l(2)2.3 phenotype is strictly germline dependent.
With the help of three overlapping deficiencies, we mapped l(2)2.3 to a 150
kb interval (see Materials and methods). We tested a series of lethal
mutations in the genes of this region and found that l(2)2.3 is an allele of
sktl. Whereas sktl20,
which is reported to be a null allele
(Hassan et al., 1998), is
lethal in trans to l(2)2.3,
sktl5 and
sktl15 are female-sterile in
trans to l(2)2.3. sktl5/l(2)2.3
females laid ventralized eggs and their oocytes had defects in the
localization of both the nucleus and Stau (data not shown).
Molecular characterization revealed that the l(2)2.3 mutation comprises a
small deletion (276 bp) in the unique intron of sktl, as do
sktl5 and
sktl15. l(2)2.3 results in a
large reduction in sktl transcript levels in nurse cells and oocytes
(Fig. 1, compare H with G),
confirming that l(2)2.3 is a hypomorphic allele of sktl similar to
sktl5 and
sktl15. Finally, the lethality
and oogenesis phenotypes were rescued by ubiquitous expression of a
sktl cDNA transgene, confirming that these defects are caused by
mutations in this gene. Germline clones, mutant for
sktl20 and
sktlM5 (see Materials and methods), arrested oogenesis at
stage 4 (see Fig. S1A in the supplementary material).
|
). The early arrest found in null alleles of
sktl is not due to oocyte determination defects. Indeed, Orb, a
suitable marker for oocyte determination, was restricted as normal to one cell
in the cyst (see Fig. S1I in the supplementary material). Furthermore, Orb,
and the centrosomes as revealed by 
-Tubulin, were correctly
translocated to the posterior of the oocyte suggesting that early AP
polarization takes place normally in the mutant egg chambers (see Fig. S1G,I
in the supplementary material). However, starting from stage 2, posterior
components such as Orb started to lose their posterior restriction (see Fig.
S1I, white arrowhead, in the supplementary material) and were eventually found
mislocalized throughout the cyst. Thus, Sktl is not required for the
determination of the oocyte during its polarization in region 3 of the
germarium. However, Sktl appears to be required for the maintenance of oocyte
polarity. We therefore conducted our analysis with the hypomorphic
sktl alleles from which we were able to obtain vitellogenic oocytes,
enabling us to study the function of Sktl during the later stages of oocyte
polarization (mid-oogenesis).
We investigated whether Sktl is required for polarized transport, i.e. for
asymmetric mRNA localization. To score for mRNA localization defects along the
DV axis, we analyzed grk mRNA. In sktl2.3
germline clones, up until stage 8, grk mRNA accumulated above the
nucleus in the anterodorsal corner of the oocyte
(Fig. 2C), as in the WT
(Fig. 2A). However, during
stage 9, grk mRNA became mislocalized into the cytoplasm, where it
accumulated around the mispositioned nucleus
(Fig. 2D) (85%, n=64).
Likewise, when analyzing mRNA localization along the AP axis, we found that
bcd mRNA was localized correctly to the anterior margin up until
stage 8 (Fig. 2G), but became
mislocalized in the oocyte during stage 9, forming a ring around the
mispositioned nucleus (Fig. 2H)
(88%, n=43). In the WT, osk mRNA transport toward the
posterior pole of the oocyte is achieved during stage 9, and Osk is
subsequently translated at the posterior
(Markussen et al., 1995;
Rongo et al., 1995). During
stage 8, when bcd and grk have reached their final location
(Fig. 2A,E), osk mRNA
accumulates temporarily in the center of the WT oocyte
(Fig. 2I), before it reaches
the posterior pole (Fig. 2J).
In sktl mutant oocytes, osk mRNA localized normally to the
center of the cytoplasm during stage 8
(Fig. 2K). However, during the
subsequent stages, it failed to reach the posterior and remained in the middle
of the oocyte (Fig. 2L) (98%,
n=57). Moreover, Osk protein was never translated in these mutants
(see Fig. S2B in the supplementary material). These results indicate that Sktl
is required for mRNA transport along the AP and DV axes of the oocyte.
Sktl controls nuclear anchoring and microtubule network organization
In the oocyte, the anterodorsal positioning of the nucleus involves at
least two distinct steps. First, during the transition from stage 6 to 7, the
oocyte nucleus migrates from a posterior to an anterodorsal position in the
cytoplasm. Then, the nucleus is anchored at the anterodorsal cortex. This
attachment is a MT-based process involving at least two distinct mechanisms
for anchoring to the anterior and the lateral cortex, respectively
(Guichet et al., 2001;
Januschke et al., 2002;
Koch and Spitzer, 1983). In
sktl2.3 mutant oocytes, there was no defect in oocyte
nucleus migration during stage 7 (data not shown), and at stage 8 the nucleus
was always correctly localized (Fig.
2C,K, asterisks). However, in 80% of stage 9 (n=54) and
96% of stage 10 (n=48) oocytes, the nucleus was mislocalized
(Fig. 2M). Optical
cross-sections of sktl2.3 mutant oocytes revealed that the
mislocated nucleus was still tightly associated with the lateral cortex
(Fig. 2N). These results
indicate that Sktl is required for the anterodorsal maintenance of the nucleus
and, more specifically, for its anterior anchorage.
|
; Januschke et al.,
2006). We investigated whether the inactivation of Sktl affects MT
organization. In sktl2.3 germline clones, we observed that
up to stage 8, the MT network is organized as in controls
(Fig. 3A,B). However, from
stage 9 onwards, the anterior-to-posterior MT organization seemed to be lost
in the sktl2.3 germline clones. Indeed, the MT array was
organized around the mislocalized nucleus
(Fig. 3D), in contrast to the
MT organization in the WT (Fig.
3C). We next analyzed the distribution of various markers for MT
polarity. MT plus ends can be marked by a fusion of the Kinesin heavy chain
with β-galactosidase (Khc-β-gal)
(Clark et al., 1994). In WT
oocytes, Khc-β-gal localizes to the posterior cortex during stage 9,
revealing that the MT plus ends are pointing towards the posterior
(Fig. 3E). In
sktl2.3 germline clones, Khc-β-gal was never detected
at the posterior and, in 23% of the egg chambers (n=79), it
accumulated in anterior regions of the cytoplasm
(Fig. 3F). Likewise, with a
different marker for MT plus ends, Dynamitin-GFP (Dmn-GFP),
(Duncan and Warrior, 2002;
Januschke et al., 2002), we
found that the posterior localization of Dmn-GFP seen in WT was always lost in
sktl2.3 mutant oocytes, where it accumulated at the
anterior in 33% of the cases studied (n=35)
(Fig. 3, G versus H). This is
in agreement with the results obtained with Khc-β-gal
(Fig. 3F). Hence, it appears
that MT plus ends can be ectopically located close to the anterior of the
oocyte, indicating that sktl mutation prevents the correct
orientation of the MTs.
|
;
Januschke et al., 2002)
(Fig. 3G, red arrowhead). In
sktl2.3 germline clones, Dmn-GFP was found around the
mislocalized nucleus (Fig. 3H,
red arrowhead). In addition, MT minus ends in the WT are also distributed
along the anterior margin of the oocyte
(Clark et al., 1997;
Theurkauf et al., 1993). These
MT minus end extremities are especially highlighted by Spindle-F (Spn-F)
(Fig. 3K)
(Abdu et al., 2006) and can
also be revealed by the localization of bcd mRNA
(Fig. 2E,F)
(Cha et al., 2001). In
sktl2.3 mutant oocytes, Spn-F and bcd mRNA were
distributed along the anterior margin up to stage 8
(Fig. 3L;
Fig. 2G). However, from stage 9
onwards, Spn-F formed a cortical ring at the position of the misplaced nucleus
(Fig. 3M). This localization is
very reminiscent of the localization of bcd mRNA in
sktl2.3 mutant oocytes
(Fig. 2H). Taken together,
these observations suggest that MT minus ends are present as a cortical ring
around the mispositioned nucleus.
Since the nucleus may function as a MT nucleation center in the oocyte
(Januschke et al., 2006), we
investigated whether the misplaced nucleus is still associated, as in WT, with
centrosomal components such as DPLP (Cp309 - FlyBase)
(Januschke et al., 2006). In
both WT (Fig. 3I) and
sktl mutant (Fig. 3J)
oocytes, DPLP was localized around the nucleus, and as a bright dot in the
vicinity of the nucleus possibly corresponding to the centrosome. In order to
test the MT-nucleating capacity of the mispositioned nucleus, we used a
cold-induced MT disassembly assay
(Januschke et al., 2006). As
in the WT, during the initial period of recovery at 25°C after complete
depolymerization through cold-shock treatment
(Fig. 3P), MT polymerization
only took place in the immediate vicinity of the mispositioned oocyte nucleus
(Fig. 3Q). Therefore, as in WT
oocytes, MT nucleation in the sktl mutant is asymmetric and mainly
restricted to the area surrounding the nucleus. However, we cannot exclude the
possibility that we might have missed some MT nucleation activity at the
cortex as a result of the experimental set-up. These results confirm the
previously described role of the nucleus as the main active MTOC in the oocyte
(Januschke et al., 2006). To
conclude, in sktl mutants, the MT array is reorganized around the
delocalized nucleus with a reverse posterior-to-anterior orientation and this
is very likely to be responsible for mRNA mislocalization.
The PIP5K Sktl is cortically localized and controls the level of PIP2 at the plasma membrane
sktl encodes a putative ortholog of a type I PIP5K
(Knirr et al., 1997) that
catalyzes the phosphorylation of phosphatidylinositol 4-phosphate to generate
the phosphatidylinositol 4,5 bisphosphate (PIP2), a major component of the
plasma membrane. We examined the localization of a GFP-tagged Sktl protein by
expressing a UASp-GFP-sktl transgene under the control of the
germline-specific driver nanosGal4 (nosgal4). This
combination rescues the phenotypes of
sktl2.3/sktl2.3 and
sktl5/sktl2.3
mutant oocytes, indicating that the GFP-sktl transgene is functional.
From the early stages of oogenesis until the development of mature egg
chambers, Sktl-GFP accumulated at the cortex just below the plasma membrane of
the oocyte and nurse cells, where it colocalized with the actin cytoskeleton
(Fig. 4A-D). A fraction of
Sktl-GFP was detected as cytoplasmic particles
(Fig. 4A). The localization of
Sktl at the plasma membrane is consistent with its involvement in PIP2
synthesis (Oude Weernink et al.,
2004).
In order to monitor the distribution and the level of PIP2, we generated a
PLCPH-GFP transgene containing GFP fused to the PIP2-specific
pleckstrin-homology domain of phospholipase C
(Balla et al., 1998). During
oogenesis, the PIP2 reporter was specifically distributed along the plasma
membrane in both the germline and follicle cells
(Fig. 4E,I, arrow and
arrowhead). Moreover, PIP2 reporter colocalized with the glycosamyl-modified
proteins present in the oocyte plasma membrane
(Fig. 4L-N) and with the
cortical actin cytoskeleton (Fig.
4E-H). Sktl (Fig.
4J) and PIP2 (Fig.
4I) colocalized along the oocyte cortex
(Fig. 4K, arrow in the
inset).
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Sktl regulates PAR polarity along the AP axis
How might Sktl regulate the positioning of the nucleus and the localization
of mRNAs in the oocyte? The mutually antagonistic interactions between the PAR
proteins Baz and aPKC at the anterior, and between Par-1 and Lgl at the
posterior, are required for mRNA localization and MT organization in the
oocyte during mid-oogenesis (Benton and St
Johnston, 2003a; Martin and St
Johnston, 2003; Shulman et
al., 2000; Tian and Deng,
2008; Tomancak et al.,
2000). Furthermore, some elements of these PAR complexes, such as
Lgl and Par-1, are also involved in the anterodorsal positioning of the
nucleus (Doerflinger et al.,
2006; Tian and Deng,
2008). Interestingly, it has recently been shown that in MDCK
cells, Par-3 recruitment to the plasma membrane requires PIP2
(Wu et al., 2007). Thus, we
examined whether Sktl activity is required for Baz localization. In the
oocyte, Baz-GFP localizes strongly at the anterior and lateral cortex, but is
excluded from the posterior (Fig.
5A) (Benton and St Johnston,
2003a; Benton and St Johnston,
2003b). In
sktl2.3/sktl5 mutant
oocytes, Baz-GFP was less enriched along the anterolateral cortex, but became
distributed in the cytoplasm (Fig.
5B) (52%, n=63). Furthermore, the requirement for Sktl to
control Baz localization is not restricted to the oocyte because in follicle
cell clones mutant for sktlM5, the apical restriction of
Baz was lost and the protein became ectopically distributed in the cytoplasm
(Fig. 5C,D). Thus, Sktl
activity is required to recruit Baz at the membrane in the oocyte and
epithelial follicle cells. Because the deficit of PIP2 might have a global
effect on the oocyte cortex, we tested whether Lkb1 (the Drosophila
PAR-4 homolog), which is known to be bound to the plasma membrane through a
prenylation motif (Martin and St Johnston,
2003), was still localized in the absence of Sktl. We found that
in sktl2.3 germline clones, GFP-Lkb1 localized to the
oocyte cortex as in WT (Fig.
5E,F). This indicates that the delocalization of Baz observed in
sktl mutants is due to the decrease in PIP2 level, rather than to a
complete disorganization of the cortex.
Previous analysis revealed that the restricted localization of Baz is
essential for oocyte polarity. Indeed, overexpression of a mutated form of Baz
that cannot be phosphorylated by Par-1 (BazS151A,S1085A-GFP),
induced a delocalization of Baz all along the cortex and some accumulation in
the cytoplasm associated with penetrant oocyte polarity phenotypes
(Benton and St Johnston,
2003b). Interestingly, we found that the overexpression of this
mutated form of Baz affected the positioning of the nucleus, as in
sktl mutants (Fig.
5G). Thus, we questioned whether Sktl and Baz might act together
to polarize the oocyte. Using a
sktl2.3/sktl15
hypomorphic combination, in which the delocalization of the oocyte nucleus
occurs in only 31% of cases examined (Table
1), we found that the nucleus was mislocalized in 63% when the
gene dosage of baz is lowered
(Table 1). Thus, sktl
and baz interact genetically. Taken together, these results suggest
that the targeting of Baz to the cortex, as mediated by Sktl, could be
required for the positioning of the nucleus and for MT organization.
|
)
(Fig. 5H,J). Thus, we analyzed
the localization of the fusion proteins Lgl-GFP and GFP-Par-1 (N1S isoform) in
sktl2.3/sktl5
oocytes. Interestingly, Lgl and Par-1 were no longer restricted to the
posterior but became distributed uniformly along the cortex and started to
accumulate in the cytoplasm (Fig.
5I,K) (Lgl mislocalization, 100%, n=30; Par-1
mislocalization, 100%, n=8). These results indicate that Sktl is
required for the maintenance of complementary compartments at the oocyte
cortex along the AP axis, and by this means might regulate oocyte polarity
during mid-oogenesis.
Sktl controls the cortical organization of the F-actin cytoskeleton and the activation of Moesin
Interestingly, in a hypomorphic combination of sktl2.3
and sktl15 alleles, the
delocalization of the oocyte nucleus occurred in only 31% of the oocytes, as
compared with 90% in sktl2.3/sktl2.3
(Table 2), and the
mislocalization of bcd and grk mRNAs was similarly
decreased. However, osk mRNA was still found diffusely distributed in
the cytoplasm of 62% of the oocytes (n=47)
(Table 2). This suggests that
Sktl might also control osk mRNA by a mechanism independent of
nucleus positioning. Previous work has shown that the anchorage of
osk mRNA to the posterior oocyte cortex is actin dependent
(St Johnston, 2005). We
addressed the relationship between Sktl and actin organization. In WT oocytes,
the microfilaments form a continuous layer along the entire cortex and the
orientation of the actin bundles is parallel to the plasma membrane
(Robinson and Cooley, 1997)
(Fig. 6C-E). Sktl colocalizes
with actin (Fig. 4C,D). In the
sktl2.3 mutant, the organization of actin filaments
appeared to be normal during early oogenesis (data not shown), but from stage
8 onwards we detected two distinct types of actin defect. In 36% of oocytes
(n=118), the cortical actin network was disrupted at the border
between the anterior margin and the lateral cortex
(Fig. 6H-J). In 15%, the actin
microfilaments in the oocyte were loosely bound to the lateral cortex,
including to the posterior pole, and they delaminated into the cytoplasm
(Fig. 6M-O). It is important to
emphasize that the continuity of the plasma membrane was unaffected in the
sktl mutant, as revealed by LE lectin labeling
(Fig. 6G,L). Thus, impairing
Sktl function leads to disorganization of the microfilament scaffold along the
oocyte cortex and to detachment of cortical actin. Mutations in several
actin-related genes disrupt mRNA localization by inducing premature MT-based
cytoplasmic streaming (Emmons et al.,
1995; Manseau et al.,
1996; Theurkauf,
1994). However, in sktl mutant oocytes, we did not
observe the early movement (stages 7-9) of yolk particles that is
characteristic of premature cytoplasmic streaming (data not shown).
|
). Among
these, Moesin, which links the actin cytoskeleton to the plasma membrane, is
required for osk mRNA localization without affecting cytoplasmic
streaming (Jankovics et al.,
2002; Polesello et al.,
2002). Previous ex-vivo studies have shown that Moesin activation
is promoted by PIP2 binding to its FERM domain
(Barret et al., 2000;
Niggli et al., 1995). We
evaluated whether Sktl function is required for Moesin activation by examining
the level of Moesin phosphorylation in sktl mutants using an antibody
specific to phosphorylated ERM proteins
(Polesello et al., 2002). In
the WT, we detected the phosphorylated form of Moesin (P-Moe) all along the
cortex of the oocyte and nurse cells (Fig.
7A). In sktl2.3 germline clones, P-Moe
distribution at the cortex was lost (Fig.
7B). Accordingly, an immunoblot analysis showed that P-Moe was
dramatically reduced in
sktl2.3/sktl5 egg
chambers (Fig. 7C), whereas the
level of total Moesin remained unchanged
(Fig. 7D). Taken together,
these results indicate that Sktl is required for the activation of Moesin in
the oocyte and could explain the defects in actin organization in the
sktl mutant. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in cell
polarization.
Our results indicate that the type I PIP5K, Sktl, is essential for PIP2
synthesis in the Drosophila oocyte. PIP2 directly controls the
localization and activity of many proteins via its interaction with
phosphoinositide-binding domains. Among them, the activation of ERM proteins
such as Moesin, results in the unmasking of their functional binding sites.
Our study indicates that in vivo, PIP2 provided by a PIP5K is required for
Moesin phosphorylation, supporting results obtained previously with cellular
systems (Fievet et al., 2004;
Lacalle et al., 2007). Since
PIP2 is also required for PIP3 synthesis, it is possible that Sktl also
affects PIP3 production. It will be interesting to investigate the
requirements of PIP3Ks during middle oogenesis and to compare them with those
of Sktl.
|
|
; Januschke et al.,
2006). Since MT minus ends are found as a cortical ring around the
nucleus, we propose a model whereby, in sktl mutants, MTs nucleated
from the nucleus might be translocated to, and anchored at, the surrounding
cortex, as previously proposed for anchoring at the anterior margin in the WT
(Guichet et al., 2001;
Januschke et al., 2008).
How could Sktl control the anchoring of the nucleus? Recent results in the
C. elegans embryo indicate that PPK-1, a PIP5K, controls spindle
movements by regulating the heterotrimeric G proteins GPR-1/2 and LIN-5, which
are similar to Pins (Raps - FlyBase) and Mud (Mushroom body defect),
respectively, in Drosophila
(Panbianco et al., 2008). Pins
requirement has not been reported in the oocyte. However, Mud is distributed
around the nucleus (Yu et al.,
2006), like the Dynein-Dynactin complex with which it has been
reported to control spindle attachment in other systems
(Gonczy, 2008). However,
although the positioning of the nucleus is Dynein dependent
(Januschke et al., 2002), it
does not necessarily require Mud (Yu et
al., 2006). It would however be interesting to investigate whether
Pins and Mud act redundantly with other factors to control the positioning of
the nucleus.
Our results also indicate that Sktl regulates PAR polarity proteins along
the AP axis. In the absence of Sktl function, Baz, Lgl and Par-1 are
mislocalized in the oocyte. In MDCK cells, PIP2 has been shown to bind to
Par-3 and to participate in its recruitment at the plasma membrane. In the
absence of Sktl, Baz localization is affected in both the oocyte and follicle
cells. One hypothesis is that in the absence of a sufficient level of PIP2,
Baz is not recruited at the cortex of the oocyte, compromising the equilibrium
between anterior and posterior PAR complexes and inducing the mislocalization
of Lgl and Par-1. It is also possible that in the absence of Sktl, the
defective actin cytoskeleton in the oocyte compromises the localization of
Lgl, as occurs in neuroblasts (Betschinger
et al., 2005), and of Par-1 (N1S) to the posterior cortex
(Doerflinger et al.,
2006).
PAR proteins are well-known regulators of MT organization and MT-based
transport in polarized cells (Munro,
2006). Furthermore, ectopic expression along the entire cortex of
Par-1 (N1S) (Doerflinger et al.,
2006), Lgl (Tian and Deng,
2008) or Baz (this study), as well inactivation of lgl
(Tian and Deng, 2008), affect
the positioning of the nucleus, as in absence of Sktl. This further suggests
that the correct positioning of the PAR proteins along the AP axis is crucial
for the anterodorsal anchorage of the nucleus. It is however possible that the
defects in the localization of mRNAs observed in sktl mutant oocytes
are not only caused by the mispositioning of the nucleus, but also involve a
more direct effect of the defective distribution of PAR proteins on MT
organization.
It is interesting to note that in the C. elegans embryo, PAR
proteins such as PAR-2 and PAR-3 regulate the activity and the distribution of
the PIP5K, PPK-1, but that PAR-2 and PAR-3 are not regulated by PPK-1
(Panbianco et al., 2008),
whereas in the Drosophila oocyte Sktl controls the distribution of
the PAR proteins. Hence, phosphoinositide regulators and PAR proteins are
closely associated in the control of polarity establishment in different
organisms; however, they can act at different levels relative to each
other.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/23/3829/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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