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First published online 2 March 2005
doi: 10.1242/dev.01720

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Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

¹ Abteilung Stammzellbiologie, CMPB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
² Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
³ Institut für Neurobiologie, Universität Münster, Badestr. 9, 48149 Münster, Germany

View larger version (20K): [in a new window]   Fig. 1. PDZ domains 2 and 3 of Baz bind directly to the C-terminal PDZ-binding motif of PTEN2 in the yeast 2-hybrid system. (A) In the yeast 2-hybrid screen using the three PDZ domains of Baz as bait, we isolated three independent clones corresponding to the C terminus of PTEN2. One clone contains amino acids 316-511 and two clones contain amino acids 297-511 of PTEN2. A deletion mutant lacking the three C-terminal amino acid residues of PTEN2 did not bind to the PDZ domains of Baz, nor did PTEN isoform 3, which differs only at the C terminus from PTEN2 and lacks the PDZ-binding motif. (B) Different regions of Baz were tested for interaction with the C terminus of PTEN2 and with the C-terminal deletion mutant of PTEN2 lacking the PDZ-binding motif. The smallest fragment that interacted with PTEN2, but not with the mutant lacking the PDZ-binding motif, contained PDZ domains 2 and 3.

View larger version (36K): [in a new window]   Fig. 2. PTEN2, Baz and aPKC are associated in a protein complex. (A) Extracts of untransfected S2 cells and S2 cells transfected with PTEN2 alone or with PTEN2 and Baz together were subjected to western blot analysis with an antibody directed against the C terminus of PTEN2. The antibody recognizes a 65 kDa protein that is strongly increased in S2 cells transfected with PTEN2. A band of the same size is detected when PTEN2 is translated in vitro in a reticulocyte lysate system (PTEN2 ivt). An additional band of 85 kDa (asterisk) is probably a crossreacting protein unrelated to PTEN. (B) Extracts from wild-type embryos were subjected to immunoprecipitation with an antibody directed against Baz or with the corresponding preimmune serum. Immunoprecipitates were probed on western blots with antibodies against PTEN (top panel) and Baz (bottom panel). (C) Extracts of untransfected S2 cells and S2 cells co-transfected with PTEN2 or PTEN3 together with Baz were subjected to immunoprecipitation with either PTEN antibody or with the corresponding pre-immune serum. Immunoprecipitates were analyzed by western blot with Baz antibody (top panel), PTEN antibody (middle panel) and aPKC antibody (bottom panel). Specific co-immunoprecipitation of Baz was detected only with PTEN2 but not with PTEN3. Endogenous aPKC co-immunoprecipitates with PTEN2 but not with PTEN3 in the presence of Baz. Western blots of the cell extracts used for immunoprecipitation are shown on the right (Input).

View larger version (105K): [in a new window]   Fig. 3. Colocalization of PTEN2 and Baz in embryonic epithelia and neuroblasts is dependent on the PDZ-binding motif of PTEN2. (A) An embryo expressing PTEN2 ubiquitously under control of a maternal GAL4 driver was stained with antibodies against PTEN and Baz. Both proteins colocalize in the apical cortex of the epidermis (arrows) and in the apical cortex of neuroblasts (asterisks). (B) At later developmental stages, apical colocalization of PTEN2 and Baz in epithelia is even more pronounced. Neurotactin (blue, right panel) was used as a marker for the basolateral membrane. No overlap between PTEN and Neurotactin is visible. (C) The PTEN3 isoform, which lacks the PDZ-binding motif at the C terminus but is otherwise identical to PTEN2 is localized on the whole plasma membrane and in the cytoplasm. PTEN3 staining clearly overlaps with Neurotactin on the basolateral membrane (purple, right panel). Apical localization of Baz is unaffected by overexpression of PTEN3. (D) PtdIns(4,5)P2 colocalizes with Baz in the embryonic epidermis. We expressed a PLC-PH-GFP fusion protein under control of a ubiquitous maternal promoter and stained these embryos with an antibody against Baz. The PLC-PH-GFP fusion protein is present on the whole plasma membrane except for the free apical surface and is strongly enriched in the most apical region of the lateral plasma membrane where it colocalizes with Baz. The embryos shown are at stage 10 (A) and at stage 13 (B-D) (Campos-Ortega and Hartenstein, 1997). Genotypes are: P{w +mC=matalpha4-GAL-VP16}V67;UAS Pten2 (A); P{w +mC=Act5C-GAL4}17bF01;UAS Pten2 (B); P{w +mC=Act5C-GAL4}17bF01/UAS Pten3 (C); and P{w +mC=matalpha4-GAL-VP16}V67/UAS PLC-PH-GFP (D). Scale bar: 20 µm.

View larger version (68K): [in a new window]   Fig. 6. PTEN is required for organization of the actin cytoskeleton during oogenesis. (A) Eggs derived from Pten germ-line clones are generally smaller than wild-type eggs. The number of eggs counted for each genotype was plotted against egg size (in µm). Fifty eggs were counted for each genotype. (B) Wild-type eggs are long and slender, whereas eggs derived from Pten germ-line clones (C,D) are shorter and more rounded. (E) In a wild-type egg chamber at stage 10, actin (E') is localized along the cell borders of the nurse cells, in ring canals (arrowheads) and underlying the plasma membrane of the oocyte. The Staufen protein (E'') is localized to the anterior and posterior pole of the oocyte. (F) In an egg chamber at stage 10 in which the germline is mutant for Pten, actin (F') is disorganized but the localization of Staufen (F'') is normal. (G) In a wild-type egg chamber at stage 10, the nurse cell nuclei (G', visualized with DAPI) do not penetrate into the oocyte. (H) By contrast, in many stage 10 egg chambers with Pten germ-line clones, nurse cell nuclei are found within the oocyte. Clones of homozygous Pten mutant cells were marked by absence of GFP fluorescence. The weak green fluorescence signal in H is bleed through from the follicle cells, as images in G and H were taken with a conventional epifluorescence microscope in order to visualize DAPI. The oocyte is circled with a broken white line in G and H. In all panels, anterior is towards the left. nc, nurse cell; oo, oocyte. Scale bar: 100 µm.

View larger version (76K): [in a new window]   Fig. 4. Pten mutant embryos fail to localize germ plasm determinants and do not form pole cells. (A) In freshly laid wild-type eggs, oskar mRNA is strongly enriched at the posterior pole. (B,C) in eggs derived from Pten germline clones, oskar mRNA is detectable at the posterior pole only at a low level (B) or not at all (C). (D,D') In wild-type embryos at nuclear division cycle 6 (64 nuclei), the nuclei have spread into an ellipsoid cloud in the yolk (dashed ellipse) and the germ plasm component Vasa is enriched at the posterior pole (arrowhead). (E,E') In Pten mutant embryos derived from germ-line clones at nuclear division cycle 6 the nuclei stay together in an almost circular cloud (broken circle) and Vasa is only slightly enriched at the posterior pole. (F,F') Vasa staining is restricted to pole cells (arrowhead) in wild-type embryos at late syncytial blastoderm stage. (G,G') In Pten mutants, pole cells do not form and Vasa staining is absent from the posterior pole (arrowhead). Arrows indicate borders between nuclei in different stages of the cell cycle. (H,H') In a wild-type embryo at the extended germ band stage, Vasa staining is restricted to germ cells (arrowhead). (I,I') In a Pten mutant embryo derived from a germ-line clone, germ cells are absent and no Vasa staining can be detected (arrowheads indicate the position where germ cells are found in wild type). DNA was stained with YOYO-1. In all images, anterior is towards the left. Scale bar: 100 µm.

View larger version (84K): [in a new window]   Fig. 5. Pten mutant embryos show defects in nuclear migration, cell cycle regulation and in organization of the actin cytoskeleton during cellularization. (A,A',C,C') Cortical nuclei in wild-type embryos are evenly spaced and divide in a nearly synchronous pattern. (B,B') In Pten mutant embryos, nuclear density in the posterior region of the embryo is much lower than in the anterior region because of a defect in nuclear migration in pre-blastoderm stages. (D,D') At syncytial blastoderm, nuclei are still unevenly spaced and divide asynchronously. (A-D) Whole embryos stained for DNA; (A'-D') higher-magnification images of the same embryos as shown in A-D, stained for DNA (green) and -tubulin (blue). The arrowheads in B,B',D,D' are in corresponding positions to illustrate which region of the embryo is shown at higher magnification. (E,E',G,G') During cellularization of wild-type embryos, a regular network of actin filaments (red) forms at the front of the ingrowing plasma membrane. (F,F',H,H') In Pten mutant embryos, the cellularization front is very uneven. Cellularization is particularly slow at the posterior pole where the pole cells have failed to form (F',H'). Arrows in F,F',H,H' indicate borders between nuclei at different cell cycle stages. In all images, anterior is towards the left. Scale bar: 100 µm in A-H; 20 µm in A'-H'.

View larger version (30K): [in a new window]   Fig. 7. A model for the activities of PTEN after recruitment to the PAR/aPKC complex. Recruitment of PTEN by Baz probably leads to local reduction of PtdIns(3,4,5)P3 and a local increase of PtdIns(4,5)P2 in the plasma membrane at the site where recruitment occurs. This should result in downregulation of the activities of aPKC and Cdc42, because PDK1, the kinase that activates aPKC, and guanine nucleotide exchange factors (GEFs) that activate Cdc42 are recruited to the plasma membrane via their PH domains by PtdIns(3,4,5)P3. How exactly recruitment of PTEN to the PAR/aPKC complex would affect actin organization is difficult to predict, as both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 are important effectors of actin dynamics. Proteins that bind to phosphoinositide lipids via PH domains are highlighted in red. Direct protein-protein interactions within the PAR/aPKC complex are indicated by double bars. ERM, ezrin, radixin, moesin; WASP, Wiskott-Aldrich syndrome protein; Arp2/3, actin related protein 2/3 (for reviews, see Pollard et al., 2000; Millard et al., 2004).