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Inscuteable-dependent apical localization of the microtubule-binding protein Cornetto suggests a role in asymmetric cell division

Research Institute of Molecular Pathology (I.M.P.), Dr Bohr Gasse 7, A-1030 Vienna, Austria

View larger version (33K): [in a new window]   Fig. 1. The Cornetto protein. (A) Predicted amino acid sequence of the Cornetto protein. The part of Cornetto that was found to interact with Inscuteable is underlined. (B) Domain structure of Cornetto. Three predicted coiled-coil domains are indicated by red boxes; the black bar shows the extent of the two-hybrid clone. (C) In vitro translated Cornetto protein binds to a GST fusion of the Inscuteable asymmetry domain (GST-insc-cen; lane 3), but not to GST alone (GST; lane 2). One-tenth of the input used in the in vitro binding assays is shown in lane 1.

View larger version (94K): [in a new window]   Fig. 2. RNA and protein expression from the cornetto gene. (A-D). In situ hybridizations to wild-type Drosophila embryos using the cornetto cDNA fragment identified in the two-hybrid screen as a digoxigenin-labelled probe (Tautz and Pfeifle, 1989). (A) RNA from cornetto is uniformly expressed in embryos during the syncytial blastoderm stage, indicating a strong maternal contribution. (B) Expression becomes progressively restricted to the central nervous system (arrow) in germ-band-extended embryos. (C) After germ-band retraction, cornetto RNA is specifically detected in the central nervous system (arrow) and the developing gut (arrowhead). (D) A high-magnification view of a neuroblast (indicated by bracket) shows the apical asymmetric localization of the cornetto RNA (arrowhead). (E) Antibodies directed against a Cornetto C-terminal peptide detect a single band on immunoblots of Drosophila whole embryonic extracts (-), which can be completely blocked by preincubation of antibodies with excess peptide (+). (F-H) Drosophila embryos at various stages were stained by immunofluorescence using anti-Cornetto-peptide antibodies. (F) Low levels of Cornetto are detected during syncytial blastoderm stages. (G) Cornetto protein is uniformly expressed during germ-band extension. (H) During germ-band retraction, Cornetto protein is detected in the central nervous system (arrow) and the gut (arrowhead). (I) Stage 13 embryos were double stained using anti-Cornetto antibodies (green) and the monoclonal antibody 22C10 (red) to visualize neurons. Cornetto is strongly expressed on axons of the central nervous system (data not shown) and in cap cells of chordotonal organs (arrowhead).

View larger version (109K): [in a new window]   Fig. 3. Inscuteable-dependent asymmetric localization of Cornetto protein. Stage-10 wild-type Drosophila embryos (A-H), embryos homozygous for the inscuteableP72 null allele (I,J) or embryos overexpressing full-length cornetto using the UAS-Gal4 system (K,L) were double stained for Cornetto protein (green in A-J) and DNA (propidium iodide; red in A-F, blue in G,H, not shown in I,J), and for Inscuteable in G and H (red). Optical cross sections through the prospective epidermis and developing nervous system (A,B) or high-magnification views of neuroblasts (C-K, neuroblasts indicated by brackets) are shown. (A,B) In epithelial cells of the developing epidermis, Cornetto protein is concentrated in the apical cytoplasm (arrowhead). (C-F) In wild-type neuroblasts, Cornetto is uniformly distributed in the cytoplasm during metaphase (C). During anaphase (D), the protein starts to accumulate apically. In telophase (E), it localizes into an apical crescent that is maintained until interphase of the next cell cycle (F). (G) In metaphase, Inscuteable (red) localizes into an apical crescent but Cornetto (green) is in the cytoplasm. (H) In telophase, when Inscuteable becomes delocalized, Cornetto localizes apically. The Cornetto crescent forms in the apical cytoplasm in the area of the apical microtubule aster, whereas Inscuteable localizes to the cell cortex. (I,J) No asymmetric localization of Cornetto is detected in most inscuteableP72 (Kraut et al., 1996) mutant neuroblasts during telophase (I) or early interphase (J). (K,L) Upon overexpression of Cornetto, filaments become visible; an optical cross section (K) and a top view onto the apical cortex (L) of telophase neuroblasts are shown. The dashed circles in L indicate the outlines of cells.

View larger version (45K): [in a new window]   Fig. 4. Cornetto is a microtubule-binding protein and colocalizes with microtubules after disruption of the actin cytoskeleton. (A-D) Permeabilized control embryos (A,B) or embryos treated for 30 minutes with 200 µM latrunculin A (C,D) were stained for Cornetto (green) and DNA (propidium iodide, red). Brackets indicate neuroblasts. In control embryos, Cornetto is uniformly distributed in the cytoplasm of neuroblasts in metaphase (A) and forms an apical crescent in telophase (B, arrowhead). In latrunculin-A-treated embryos, the protein colocalizes with microtubules of the mitotic spindle in metaphase (C, arrow) and fails to localize asymmetrically in telophase (D). (E) High-speed supernatants of Drosophila embryo extracts were prepared under microtubule-depolymerizing conditions (see Materials and Methods). Microtubules were repolymerized by incubation with GTP and Taxol and microtubules and microtubule-associated proteins were sedimented by ultracentrifugation. ß-Tubulin and Cornetto are found in the pellet under microtubule-polymerizing conditions (lanes 4 and 5), whereas both proteins remain soluble in a control experiment in the absence of GTP and Taxol (lanes 2 and 3). By contrast, a cytoplasmic protein (Hsp70) remains soluble under both conditions. An amount of extract equivalent to that used in the experiment was loaded in lane 1.