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

This Article Summary Full Text Full Text (PDF) 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 Tolwinski, N. S. Articles by Wieschaus, E. Search for Related Content PubMed PubMed Citation Articles by Tolwinski, N. S. Articles by Wieschaus, E. Social Bookmarking                         What's this?

Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear anchor dTCF/Pan

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA

View larger version (100K): [in a new window]   Fig. 1. Overexpression of Arm alleles in embryos using the GAL4/UAS system. The two alleles, Arm and S10, both produce the same activated wg signaling phenotype in first instar larval cuticles, but Arm localizes to the membrane while S10 localizes to the nucleus. (A) Phase contrast images of cuticle preparations of ArmGAL4 crossed to either Arm, or S10. A wild-type cuticle is shown for comparison. The ventral side of all embryos is shown. (B) Confocal microscope images of embryos using the ArmGAL4 driver to drive expression Arm (-HA, red), S10 (-c-Myc, red), and UAS-full-length-Arm (rAb, green). (C) Using the maternal GAL4 driver, 67.15, expression of Arm (-HA, red), endogenous Arm protein (rAb, green), and nuclei (Hoechst, blue) is shown. Lower panel shows a close up of the same embryo. The amino-terminal deletions in S10 and Arm delete the epitope used to make the Arm antibody allowing staining of expressed protein and endogenous protein without cross-reactivity. Overexpressed full-length Arm shows both endogenous and expressed protein.

View larger version (122K): [in a new window]   Fig. 2. Arm is dependent on endogenous Arm to activate Wg signaling, while S10 is not. arm043A01 germline clones made using the FLP recombinase system expressing either Arm or S10 using the GAL4/UAS system. (A) Confocal images of the four embryos obtained when arm043A01; ArmGAL4/+ germline clones were crossed to Arm. En stripes are shown in red. Sxl antibodies were used to detect female arm/+ embryos (blue). Arm expression is shown by -HA staining (green). (B) Confocal images of embryos (arm043A01; ArmGAL4/+ crossed to S10) showing the En stripes (-En, red), and lack of Sxl expression in these male embryos (arm/Y, blue). (C) Phase contrast images of arm043A01; ArmGAL4/+ crossed to either S10 or Arm. The percentages are the approximate frequency with which the phenotype was observed over the course of three independent experiments. Cuticle preparations from the arm043A01/Arm cross show approx. 25% naked (n=43), approx. 25% wild-type (n=36), and approx. 50% severe arm phenotype (n=78), consistent with arm/+; Arm/ArmGAL4 leading to naked cuticle, arm/+; Arm/+ leading to wild-type cuticle, arm/Y; Arm/ArmGAL4 and arm/Y; Arm/+ both leading to the severe arm phenotype. Cuticle preparations from the arm043A01/S10 cross show 50% naked (n=105), 25% wild-type (n=57), and 25% severe arm phenotype (n=51), consistent with arm/+; S10/ArmGAL4 and arm/Y; S10/ArmGAL4 both leading to naked cuticle, arm/+; S10/+ leading to wild-type cuticle, arm/Y; S10/+ leading to the severe arm phenotype.

View larger version (104K): [in a new window]   Fig. 3. Arm expression can activate the transcriptional activity of a moderate loss-of-function allele of arm. armXM19 germline clones made using the FLP recombinase system expressing either Arm or S10 using the GAL4/UAS system. (A) Phase contrast images of (armXM19; ArmGAL4/+ crossed to Arm) first instar larvae show approx. 25% naked (n=32), approx. 25% wild-type (n=26), approx. 25% arm phenotype (n=27), and approx. 25% naked/arm phenotype (n=35), consistent with arm/+; Arm/ArmGAL4 leading to naked cuticle, arm/+; Arm/+ leading to wild-type cuticle, arm/Y; Arm/+ leading to arm cuticle, and arm/Y; Arm/ArmGAL4 leading to the naked/arm cuticle. (B) Confocal images of embryos (armXM19; ArmGAL4/+ crossed to either Arm or S10) showing the En stripes (-En, red). An embryo with wild-type Engrailed stripes is shown for comparison. All panels were also stained with Sxl (not shown) to separate out the armXM19/+ embryos.

View larger version (69K): [in a new window]   Fig. 4. (Top) Protein levels in Arm-expressing embryos are not significantly higher than in wild-type embryos, but much lower than in zw3 or axin germline clones. The zw3 lane contains extracts from four embryos from zw3M11-1; ArmGAL4/+ crossed to wild type. Half the embryos will receive a paternal wild-type copy of zw3, reducing the number of true mutant embryos per lane. The axin lane contains extracts from four embryos from axn germline clone crossed to axn/TM3, therefore half the embryos will receive a zygotic wild-type copy of axn reducing the number of true mutant embryos. However, though the axin and zw3 lanes mix mutant and non-mutant embryos, they still show a significant increase in endogenous Arm protein levels. The Arm lane contains extracts from four embryos where the 67.15 GAL4 driver was crossed to Arm, therefore all embryos express Arm. The wild-type lane contains extracts from four OreR embryos. All embryos were selected to be at similar stages (stage 11 to 12). (Middle) ß-tubulin was used as a loading control, and shows that all lanes were loaded equally. (Bottom) Arm bands were quantitated using NIH Image, and the results graphed. The units are arbitrary.

View larger version (104K): [in a new window]   Fig. 5. Arm acts downstream of zw3, and is independent of the increased levels present in zw3 germline clones. Confocal microscope images of stage 9 embryos showing Arm protein (N2, green) and nuclei (Hoechst, blue). (A) A wild-type embryo, OreR. (B) zw3M11-1;ArmGAL4/+ crossed to wild type. The embryo was also stained with -Sxl to separate out the females (not shown). (C) zw3M11-1; ArmGAL4/+ crossed to Arm. The embryo was also stained with -HA to find embryos expressing Arm, and with -Sxl to separate out the females (not shown).

View larger version (110K): [in a new window]   Fig. 6. Axin functions in retaining Arm in the cytoplasm. (A) Confocal microscope images of embryos showing endogenous Arm protein (N2, green) and nuclei (Hoechst, Blue). (A) axin germline clone embryos show very specific nuclear and plasma membrane localization, and a lack of cytoplasmic staining. (B) Wild-type embryo shown for comparison where diffuse staining is observed throughout the cell. (C) 67.15 crossed to Arm, UAS-Axin. This embryo was also stained with -HA to confirm expression of Arm (not shown). Diffuse staining is observed throughout the cell with no obvious preference for the nucleus. (D) 67.15 crossed to Arm alone shown for comparison. Nuclear localization of endogenous Arm is observed.

View larger version (106K): [in a new window]   Fig. 7. Tcf/pan may function as a nuclear anchor for Arm. Confocal microscope images of embryos showing endogenous Arm protein (N2, green) and nuclei (Hoechst, Blue). (A) 67.15; Arm, dTCFN embryos show a lack of nuclear accumulation of endogenous Arm expected from Arm alone. (B) 67.15; dTCFN embryos also show a lack of nuclear accumulation. (C) 67.15; dTCF (full length) embryos do not show nuclear accumulation of endogenous Arm protein.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter