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First published online 28 August 2007
doi: 10.1242/jcs.014902

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Cdc42 acts downstream of Bazooka to regulate neuroblast polarity through Par-6–aPKC

¹ Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA
² Institute of Molecular Biology, Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA

View larger version (77K): [in this window] [in a new window]   Fig. 1. Cdc42 is enriched at the apical neuroblast cortex. (A) Wild-type central brain neuroblasts 120 hours after larval hatching (ALH). Normal apical and basal protein localization and phosphorylated histone H3 (PH3) are shown with background Myc staining. (B-E) cdc42-3 central brain neuroblasts at 96 hours ALH expressing Cdc42-Myc under its native promoter (cdc42-3; cdc42:myc). All stages of mitosis are represented. Arrowheads delineate extent of aPKC (aPKC PH3) and Cdc42-Myc (Cdc42:myc) apical crescents.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Cdc42 is required for neuroblast polarity. (A) Wild-type embryonic neuroblasts at stages 11 to 13 stained for aPKC, Baz, Mira, Par-6 and PH3. (B-E) Embryonic neuroblasts at stages 11 to 13 expressing Cdc42-DN (N17) driven by worniu-Gal4. aPKC displays ectopic cortical staining (B; 82%, n=45) together with Par-6 (C; 76%, n=41) and Mira (B'; 45%, n=67), whereas Baz displays no defects (D; 100%, n=26). (F) Divisions are asymmetric (100%, n=23). (F-J) Embryonic neuroblasts stages 11-13 expressing Myc–Cdc42-CA (V12) as in (B-E). aPKC displays cortical, with some cytoplasmic, staining (F; 94%, n=50) along with Par-6 (G; 90%, n=29) and Myc–Cdc42-CA (H; 89%, n=19), whereas Mira is cytoplasmic (F'; 94%, n=50). Baz displays no defects (I; 100%, n=13). (J) Neuroblast division becomes symmetric upon overexpression of Cdc42-CA (88%, n=9). (K) Wild-type central brain neuroblasts 120 hours ALH stained for aPKC, Par-6, Baz, and Mira. (L-N) cdc42-3 central brain neuroblasts 96 hours ALH. These neuroblasts show cytoplamsic staining of aPKC (L; 84%, n=19) and Par-6 (M; 100%, n=11), whereas Mira is uniformly cortical (L'-N'; 100%, n=46). Baz displays no defects (N; 100%, n=16). (O) Cdc42 is mislocalized in zygotic baz-4 mutant neuroblasts. Embryonic neuroblasts at stages 13 to 14 expressing Cdc42-Myc in a baz-4 background exhibit loss of Cdc42 apical enrichment. Cdc42-Myc is weakly cortical with some cytoplasmic staining and no apical enrichment (O'), whereas aPKC is cytoplasmic (O) and Mira is uniformally cortical (O"; 100%, n=21). (P) Quantification of the Cdc42 requirement for neuroblast polarity in embryonic and larval neuroblasts.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Cdc42–Par-6 interaction is necessary for neuroblast polarity. (A) Alignment of the Par-6 semi-CRIB domain with CRIB domains from other proteins. Mutated residues are boxed and the residues mutated in the Par-6ISAA transgene are boxed in red. (B) The ISAA mutation disrupts Cdc42 binding to the Par-6 CRIB-PDZ domain. The extent of binding between a glutathione-S-transferase (GST) fusion of [35S]GTP-loaded Cdc42 and 55 µM wild-type and mutant Par-6 CRIB-PDZ domains is shown, as determined using a qualitative pull-down assay stained with Coomassie brilliant blue. (C,D) Zygotic par6226 central brain neuroblasts 24 hours ALH expressing par-6 transgenes. HA–Par-6 (HA:Par-6) localizes to the apical cortex of dividing neuroblasts and rescues Mira phenotype (C). HA–Par-6ISAA (HA:Par-6ISAA) is cytoplasmic and is unable to rescue cortical Mira (D). (E) Zygotic par6226 central brain neuroblasts 24 hours ALH expressing Cdc42-Myc. Arrowhead delineates weak apical enrichment of Cdc42-Myc (92%, n=12), whereas Mira is uniformally cortical (100%, n=12).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Par-6 represses whereas Cdc42 partially relieves aPKC kinase activity. (A) Kinase activity of aPKC, Par-6–aPKC, and Cdc42–Par-6–aPKC complexes. The high intrinsic kinase activity of aPKC, expressed and purified from HEK 293 cells, is efficiently repressed by addition of full-length Par-6. Par-6 has no effect on PKC (right panel). Cdc42 partially restores aPKC activity. The signal is from a rhodamine-labeled peptide corresponding to a PKC consensus substrate (sequence shown on left). (B) aPKC fractionates predominantly with Par-6. Fractions of Drosophila embryonic lysate from stages 8 to 14 embryos from a calibrated gel filtration column are shown western blotted with both anti-aPKC and anti-Par-6 antibodies. Very little aPKC fractionates at its native molecular mass (80 kD) but, instead, co-fractionates with Par-6. (C) Pathway for regulation of apical complex activity in neuroblasts.