QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online May 23, 2006
doi: 10.1242/10.1242/dev.02398

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McCartney, B. M. Articles by Peifer, M. Search for Related Content PubMed PubMed Citation Articles by McCartney, B. M. Articles by Peifer, M.

Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila

Brooke M. McCartney^1,, Meredith H. Price^2,*, Rebecca L. Webb^1,*, Melissa A. Hayden³, Lesley M. Holot¹, Mengning Zhou¹, Amy Bejsovec⁴ and Mark Peifer^2,3,5,

¹ Department of Biological Sciences, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA 15213, USA.
² Department of Biology, University of North Carolina at Chapel Hill, CB# 3280 Coker Hall, Chapel Hill, NC 27599, USA.
³ Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, CB# 3280 Coker Hall, Chapel Hill, NC 27599, USA.
⁴ Department of Biology, Duke University, Durham, NC 27710, USA.
⁵ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, CB# 3280 Coker Hall, Chapel Hill, NC 27599, USA.

View larger version (24K): [in a new window]   Fig. 1. Human and fly APC1 and APC2. (A-C) There are nine mutant alleles of fly APC2: five point mutants in the Arm repeat region (black arrows) and four truncations (blue arrows). We used a null allele of APC1 (APC1Q8) resulting from a nonsense mutation in Arm repeat 4 (Ahmed et al., 1998).

View larger version (35K): [in a new window]   Fig. 2. Protocol used for screening new APC2 alleles. See Materials and methods for details.

View larger version (33K): [in a new window]   Fig. 3. APC2 mutant proteins. (A-C) Protein samples derived from ovaries of APC2 allele/deficiency females, or from embryos whose mothers and fathers were APC2 allele/deficiency, so that only mutant protein was present (see Materials and methods), were immunoblotted with anti-APC2-CT (A,B) or anti-APC2-NT (C). Arrows indicate mutant proteins; arrowheads indicate background bands.

View larger version (191K): [in a new window]   Fig. 4. APC2 mutant proteins lose cortical association. Germ-band-extended embryos stained for phosphotyrosine labeling the cortex (green), and APC2 (red). (A,A') Wild-type APC2 localizes to the cortex (arrow) and in the cytoplasm of the ectoderm. (B,C) Mutant proteins fall into two categories: (C) those with no detectable cortical localization (APC2g41 shown); and (B) those with some residual cortical localization (arrow; APC2N175K shown). Scale bar: 10 µm.

View larger version (106K): [in a new window]   Fig. 5. APC proteins are not essential for cell adhesion in ovaries. (A-D',F,F') Wild-type (A,C) and APC2g10 APC1Q8/APC2g10 (B,D,F) stage 7-8 egg chambers labeled for actin and Arm as indicated. fc, follicle cells; rc, ring canals; o, oocytes. (E) Frequency of mispositioned oocytes. Scale bars: 10 µm in A,B; 5 µm in C,D,F.

View larger version (163K): [in a new window]   Fig. 6. APC proteins are not essential for embryonic cell adhesion. (A-F) Stage-matched stage 9-10 wild-type and mutant embryos labeled for Arm, DE-cadherin, -catenin and Dlg (a basolateral marker), as indicated. To select M/Z APC2g10 APC1Q8 embryos, females carrying germline clones were crossed to moeGFP APC2g10 APC1Q8/+ males. Wild-type, his-GFP-marked embryos stained in the same tubes. (E'',F'') z-axis cross-sections of E,F. Arrows indicate adherens junctions. Scale bar: 10 µm for A-F'; 2.5 µm for E'',F''.

View larger version (113K): [in a new window]   Fig. 7. Nuclear loss, spindle morphology and spindle orientation. (A-C,H-J) Syncytial embryos in nuclear cycle 13 (A-C) and dividing ectoderm in germband-extended embryos (H-J); actin, red; microtubules, green; DNA, blue; lower rows show microtubules alone. (A-C) APC2g10 APC1Q8 syncytial embryos (B,C) do not have significant defects in spindle morphology. Note weak actin rings (arrow, B) and nuclear loss evidenced by empty actin rings (arrow, C). (D,E) Syncytial wild type (D) and APC2g10 (E). DNA is stained with DAPI. Arrowheads indicate out-of-focus yolk nuclei; arrows indicate nuclei lost from surface. (F) Quantification of nuclear loss in APC2 and APC2 APC1 mutant syncytial embryos. Bars show the percentage embryos with 2% of cortical nuclei lost (see Materials and methods). APC2c9, purple; APC2N175K, yellow; APC2S, red; APC2d40, green; APC2g10, blue. (G) Quantification of syncytial spindle length (pole-to-pole). (H-J) Wild-type spindles are parallel to the epithelium (arrowheads, H) and divisions are symmetric (arrow, H). Spindle orientation (arrowheads, I,J) and division plane (arrows, I,J) are normal in APC2g10 APC1Q8 M/Z mutants (J) and embryos maternally APC2d40 and zygotically APC2d40/+ (I). (K) Quantification of these phenotypes. Scale bars: 10 µm.

View larger version (142K): [in a new window]   Fig. 8. APC2 mutant alleles vary widely in their effect on Wg signaling. Embryonic cuticles scored using phenotypic criteria in Table 1. (A-J) Representative pictures of each class are presented.

View larger version (145K): [in a new window]   Fig. 9. Effects on Arm stability match effects on cuticle phenotype. Arm levels, stage 9. (A-H) Ventral views, anterior to the left. Wild type (A); APC2 M/Z mutants (B-H). Confocal settings were normalized using his-GFP wild-type controls. (I,J) Wild type, APC2g10 APC1Q8 M/Z mutants, paternally-rescued embryos, and zw3 M/Z mutants as indicated. Scale bars: 20 µm.

View larger version (19K): [in a new window]   Fig. 10. Structure/function of APC2. Wild-type APC2 is shown in the middle. (Above) Domains defined by mutations (black bars) important for cortical localization or nuclear retention. (Below) Truncated proteins that are null or have reduced function, domains important for Wnt signaling, and a region conferring a more severe phenotype on APC2g41.