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First published online 23 October 2008
doi: 10.1242/dev.029009

Development 135, 3829-3838 (2008)
Published by The Company of Biologists 2008
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PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in the Drosophila oocyte

Louis Gervais1, Sandra Claret1, Jens Januschke1,2, Siegfried Roth3 and Antoine Guichet1,*

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.


Figure 1
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  		Fig. 1. sktl affects axis formation in the Drosophila oocyte. (A,B) Egg chambers expressing GFP-Stau. (A) Wild type (WT). (B) l(2)2.3 mutant germline clone. (C,D) Dorsal view of an egg shell. (C) In WT, the two appendages mark the dorsal side of the egg. (D) In the l(2)2.3 mutant germline clone, the egg is ventralized and lacks dorsal appendages, but the posterior aeropyle is normal (compare insets in C and D). (E,F) Homozygous l(2)2.3 mutant clones, marked by the absence of GFP (green). Stau protein localization (red) and nucleus positioning are not affected when the posterior follicle cells are mutant (E), but are lost in mutant germline clones (F). (G,H) sktl mRNA expression. The level of sktl mRNA (blue) is reduced in l(2)2.3 mutant (H) as compared with WT (G) nurse cells. Anterior is to the left and posterior is to the right. Asterisk, nucleus.

 

Figure 2
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  		Fig. 2. mRNA localization and nucleus positioning defects in the sktl mutant oocyte. Wild-type (A,B,E,F,I,J) and sktl2.3 mutant (C,D,G,H,K,L) Drosophila germline clones. (A-D) grk mRNA distribution. In stage 8 sktl2.3 mutants, grk mRNA (blue) localizes as in WT (compare C with A). However, in sktl2.3 stage 10 oocytes, grk mRNA is mislocalized with the nucleus (compare D with B). (E-H) bcd mRNA distribution. In stage 8 sktl2.3 mutant oocytes, bcd mRNA (blue) localizes as in WT (compare G with E). In stage 10 sktl2.3 oocytes, bcd mRNA is mislocalized in the form of a cortical ring close to the nucleus (compare H with F). (I-L) osk mRNA localization. In stage 8 sktl2.3 oocytes, osk mRNA (brown) localizes as in WT (compare K with I). However, during stage 10, osk mRNA does not reach the posterior, unlike in WT (compare L with J), and is mislocalized in the cytoplasm. (M,N) Stage 10 sktl2.3 mutant egg chamber expressing GFP-Stau (green) and stained for DNA (red). (M) The oocyte nucleus is not anchored to the anterior cortex and is mislocalized in the cytoplasm. (N) Optical cross-section (at the level of the opposing arrowheads in M) showing that the anteriorly detached nucleus remains attached to the lateral cortex of the oocyte. Asterisk, nucleus.

 

Figure 3
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  		Fig. 3. Microtubule organization in sktl mutant oocyte. WT (A,C,E,G,I,K,N,O) and sktl2.3 mutant (B,D,J,L,M) Drosophila germline clones and sktl2.3/sktl{Delta}5 mutant egg chambers (F,H,P,Q). (A-D) Microtubule (MT) network detected with Khc antibody. In stage 8 sktl2.3 oocytes, MT network organization is not affected (compare B with A). However, in stage 10 sktl2.3 oocytes, MT organization is disrupted (compare D with C). MT bundles appear to project from the mislocalized nucleus towards the center of the cytoplasm (arrowheads in D). (E,F)Khc-β-gal (red) and DNA (green) staining. In sktl2.3/sktl{Delta}5 oocytes, the posterior pool of Khc-β-gal is lost and it accumulates in the anterior part of the oocyte (F). The inset in F shows Khc-β-gal staining alone. (G,H) Dmn-GFP. In sktl2.3/sktl{Delta}5 oocytes, the posterior pool of Dmn-GFP is lost and an accumulation is found in the most anterior part of the cytoplasm (H). However, cortical- and nuclear-associated localization are unaffected, even when the nucleus is mislocalized. (I,J) In both mutant and WT, the centrosome component DPLP (red) is detected around the oocyte nucleus and as a dot close to it (arrowhead). The dots surrounding the oocyte correspond to the centrosomes of the follicular cells. (K-M) Projections of confocal sections from oocytes immunostained with Spn-F antibody revealing the minus ends of the MTs. Spn-F is found mislocalized in a characteristic cortical ring close to the mislocalized nucleus in stage 10 sktl2.3 oocytes (compare M with K), but not in stage 8 (L). (N-Q)MT detection by anti-Khc. WT (N) and sktl2.3/sktl{Delta}5 (P) oocytes showing complete depolymerization of MTs after 30 minutes of cold shock. (O,Q) WT and mutant oocytes after a 10-minute recovery at 25°C. MTs regrow from the misplaced nucleus (arrowheads in Q). Asterisk, nucleus.

 

Figure 4
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  		Fig. 4. Sktl controls the PIP2 level at the oocyte cortex. (A-D) nosgal4>GFP-Sktl egg chambers. GFP-Sktl (A, green in C,D) colocalizes with actin (B, red in C,D) along nurse cell and oocyte cortex (merge in C,D). (E-H) PH-GFP distribution. PH-GFP (E, green in G,H) localizes at the actin (F, red in G,H) cortex of follicular cells, nurse cells and oocyte (merge in G,H). (I-K) Colocalization between PH-GFP (I, green in K) and nosgal4>RFP-Sktl (J, red in K) along the oocyte cortex and in the cytoplasm. (L-N) Wild-type Drosophila egg chamber expressing PH-GFP (L, green in N), co-stained with the plasma membrane marker LE lectin (M, red in N). PH-GFP localizes all along the plasma membrane of the oocyte (merge in N). Insets in L and N are magnifications of anterior margin and lateral cortex, respectively. (O-Q) sktl2.3 mutant germline clones expressing PH-GFP (O, green in Q) co-stained with LE lectin (P, red in Q). The accumulation of PIP2 marker is greatly reduced all along the oocyte plasma membrane. Insets in O and Q are magnifications of anterior margin and lateral cortex, respectively. Arrows and arrowheads (I,J,K,L,N,O,Q) indicate the plasma membrane of the oocyte and follicle cells, respectively. Asterisk, nucleus.

 

Figure 5
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  		Fig. 5. Sktl is required to properly organize the cortical domains of the oocyte. (A) Baz-GFP localizes strongly at the anterolateral cortex of the WT Drosophila oocyte. (B) In sktl2.3/sktl{Delta}5 oocytes, Baz-GFP has lost most of its cortical localization and is found as puncta in the cytoplasm. (C,D) skltlM5 follicle cell clone (FCC; dashed line) marked by the absence of GFP (green) and stained for Baz (red in C, white in D). (E,F) Cortical localization of Lkb1 (E) is maintained in sktl2.3/sktl{Delta}5 oocytes (F). (G) Baz-GFPS151A,S1085A overexpression induces oocyte polarity defect phenotypes, with nucleus mispositioning and ventralized eggs (inset in G). (H) Lgl-GFP is localized at the posterior of the oocyte. (I) In sktl2.3/sktl{Delta}5 oocytes, Lgl-GFP is no longer enriched at the posterior and is instead detected mainly in the cytoplasm and along the cortex. (J) GFP-Par-1 (N1S) is enriched at the posterior at stage 9 (arrowhead). (K) In sktl2.3/sktl{Delta}5 oocytes, GFP-Par-1 (N1S) is no longer enriched at the posterior and instead has started to accumulate all along the cortex and in the cytoplasm. Asterisk, nucleus.

 

Figure 6
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  		Fig. 6. Sktl is required for actin organization. Wild-type (A-E) and sktl2.3 mutant (F-O) Drosophila germline clones co-stained for plasma membrane (B,G,L, green in merge A,F,K) and F-actin (C-E,H-J,M-O, red in merge A,F,K). Interruption of the actin cortex along the anterior margin is found in 36% of mutant oocytes (arrowheads in I,J). Ectopic F-actin structures are observed in 15% of mutant oocytes. D,I,N are magnifications from C,H,M, respectively. E,J,O are optical cross-sections of C,H,M, respectively, at the level of the red arrowheads. Asterisk, nucleus.

 

Figure 7
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  		Fig. 7. Sktl activity is required for Moesin phosphorylation. (A,B) Immunostaining using an antibody specific for the phosphorylated form of Moesin (P-Moe). P-Moe staining is found at the cortex in WT Drosophila oocytes (red in A) and is abolished in sktl2.3 mutant germline clones marked by the loss of GFP (B). (C,D) Western blot of ovary extracts (1, 2 and 4 mean that the equivalent of 1, 2 and 4 ovaries were loaded) from WT and sktl2.3/sktl{Delta}5 probed with anti-P-Moe (C), anti-Moesin (D) and anti-Tubulin ({alpha}-tub) as a loading control (C,D). (E,F) Immunostaining using an antibody against Moesin. Moesin is localized at the cortex in both WT and sktl2.3/sktl{Delta}5 oocytes (E,F, arrowheads). Asterisk, nucleus.

 

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© The Company of Biologists Ltd 2008