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doi: 10.1242/10.1242/dev.00526

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Strabismus is asymmetrically localised and binds to Prickle and Dishevelled during Drosophila planar polarity patterning

Rebecca Bastock*, Helen Strutt* and David Strutt{dagger}

Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK



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  		Fig. 1. Localisation of Stbm in the developing wing. Confocal images of third instar wing disc (A) or pupal wings (B-K), or images of dorsal surface of adult wings between vein 3 and vein 4 (L-N). Distal is rightwards and anterior is upwards. (A-D) Stbm-YFP is apicolateral and progressively becomes distributed to proximodistal cell boundaries. (E) Staining with an antibody against Stbm shows the protein to be apicolateral and at proximodistal cell boundaries. (F) No Stbm staining (red) is seen in a stbm6 mutant clone (labelled by absence of green lacZ staining). Note that Stbm is ectopically localised in cells on edge of clone, owing to the non-autonomous phenotype of stbm clones in the wing (Taylor et al., 1998Go). (G-I) Stbm-YFP (green) colocalises with Dsh (red, G) and Fmi (red, H) but not Dlg (red, I). (J) Mosaic expression of Stbm-YFP (white in left panel and green in right panel) reveals it to be preferentially localised to proximal cell boundaries (arrowheads), where it is colocalised with the adherens junction marker Armadillo (white in middle panel and red in right panel). Note in this experiment that all cells have endogenous stbm activity and patterning is normal. A subset of cells also express Stbm-YFP. At the boundaries between cells that express Stbm-YFP and those that do not, it is possible to see at which cell boundary Stbm-YFP preferentially localises. (K) Stbm-YFP (green) localises normally in a stbm6 mutant clone (labelled by absence of red lacZ staining). (L) stbm6/Df(2R)45-30n. (M) stbm6/Df(2R)4530n; P[w+; Act-Stbm-YFP]/+. (N) stbm6 P[w+; Act-Stbm- {Delta}PBM]/stbmVang-A3.

 


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  		Fig. 2. stbm, fz and fmi are required for apicolateral asymmetric localisation of polarity proteins. Confocal images of pupal wings at about 28 hours. Distal is right and anterior is upwards. (A-F) Loss-of-function clones; mutant tissue indicated by absence of lacZ staining (red, left panels). (G-J) Clones of overexpression indicated by lacZ staining (red, left panels). Note that in all genotypes except fmi, the clones also have significant non-autonomous effects on planar polarity that leads to ectopic localisation of polarity proteins in cells surrounding the clone. (A) Fz-GFP in stbmVang-A3. (B) Stbm-YFP in fz25. (C) Fmi in stbmVang-A3. (D) Stbm-YFP in fmiE59. (E) Dsh in stbm6. (F) Pk in stbm6. (G) Stbm-YFP in Act-GAL4/UAS-Fz overexpression clone. (H) Stbm-YFP in Act-GAL4/UAS-Fmi overexpression clone. (I) Fz-GFP in Act-GAL4/UAS-Stbm overexpresssion clone. (J) Pk in Act-GAL4/UAS-Stbm overexpression clone.

 


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  		Fig. 3. pk and dsh are required for asymmetric localisation and accumulation of polarity proteins. Confocal images of pupal wings at about 28 hours. Distal is rightwards and anterior is upwards. (A-F) Loss-of-function clones, mutant tissue indicated by absence of lacZ staining (red, left panels). (G-J) Clones of overexpression indicated by lacZ staining (red, left panels, H,I) or Pk staining (red, left panels, G,J). (A) Stbm-YFP in dsh3. (B) Fmi in dsh3. (C) Fz-GFP in pk-sple13. (D) Stbm-YFP in pk-sple13. (E) Fmi in pk-sple13. (F) Fmi in pk-sple13 stbm6 double mutant clone. Note that Fmi is ectopically localised in cells surrounding clone, owing to the non-autonomous phenotype of stbm clones in the wing (Taylor et al., 1998Go). (G) Fmi in Ptc-GAL4/UAS-Pk wing at compartment boundary. (H) Stbm-YFP in Act-GAL4/UAS-Pk overexpression clone. (I) Stbm-YFP in Act-GAL4/UAS-Dsh overexpression clone. (J) Fmi in Ptc-GAL4/UAS-Pk; fz15/fz23 wing at compartment boundary.

 


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  		Fig. 4. Stbm co-immunoprecipitates Dsh and Pk. Immunoprecipitations from COS7 cells transfected with the plasmids indicated. Proteins in the lysate prior to immunoprecipitation are shown on the left (input), and either five times (B-D) or 10 times (A,E,F) equivalent immunoprecipitated material is on the right. (A) Dsh-GFP co-immunoprecipitated with FLAG-Stbm. (B) Myc-Pk co-immunoprecipitated with FLAG-Stbm. (C,D) Lysate containing FLAG-Stbm, Dsh-GFP and Myc-Pk immunoprecipitated with anti-FLAG co-immunoprecipitates both Dsh-GFP (C) and Myc-Pk (D). (E) Weak co-immunoprecipitation of Dsh-GFP by Myc-Pk. (F) Myc-Pk co-immunoprecipates a fusion of CD2 extracellular and transmembrane regions to Stbm C-terminal intracellular domain lacking the putative PBM. Arrows indicate specific bands of expected molecular weights detected by western blotting for co-immunoprecipitated proteins.

 


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  		Fig. 5. Stbm recruits Dsh and Pk to membranes. Confocal images of COS7 cells transfected with the plasmids indicated (A-Q), or third instar wing disc expressing Stbm-YFP (R). Distribution of proteins expressed singly is shown in white. Stbm (detected with anti-Stbm), FLAG-Stbm (detected with anti-FLAG) and Stbm-YFP (detected by YFP fluorescence) is always shown in green in multiply labelled panels. Dsh (detected with anti-Dsh) is always shown in red in multiply labelled panels. Pk (detected with anti-Pk) and Myc-Pk (detected with anti-Myc) are always shown in blue in multiply labelled panels. Stbm-{Delta}PBM (K-M) lacks the last three amino acids of the ORF. CD2-Stbm-Cterm-{Delta}PBM (N-Q) consists of the extracellular and transmembrane regions of rat CD2, fused to the C-terminal intracellular tail of Stbm with the last three amino acids deleted. (A-C) Stbm (white in A, green in C) can recruit Dsh (white in B, red in C) from cytoplasmic vesicles to Stbm-expressing membranes (largely the Golgi membranes but partly the outer cell membrane). (D-F) FLAG-Stbm (white in D, green in F) can recruit Pk (white in E, blue in F) to Stbm-expressing membranes. (G) FLAG-Stbm (green), Dsh (red) and Pk (blue) colocalise to Stbm expressing membranes. (H) Dsh (red) and Pk (blue) co-expressed together do not colocalise. (I,J) Recruitment of Dsh (white in I, red in J) to the outer membrane by Fz (I) is not disrupted by co-expression of Pk (blue in J). (K-Q) Stbm-{Delta}PBM (white in K, green in L,M) or CD2-Stbm-Cterm-{Delta}PBM (white in N, green in O,Q) can recruit Dsh (red in L,O) and Pk (white in P, blue in M,Q). (R) Stbm-YFP and Dsh colocalise apicolaterally in the wing pouch of a third instar wing disc.

 


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  		Fig. 6. Models for asymmetric localisation of planar polarity proteins. (A) We propose that initial apicolateral recruitment of planar polarity proteins occurs according to the hierarchy shown (see text for details). (B) During the early stages of asymmetric complex formation, planar polarity proteins are apicolaterally localised but not obviously asymmetrically distributed on the proximodistal axis. At this stage, they may either be in symmetric complexes in which the same proteins are present on both sides of the cell-cell boundaries (left) or asymmetric complexes that are randomly orientated relative to the proximodistal axis of the wing (middle). Ultimately, the pattern resolves to that shown (right). Proteins represented are Fmi (red), Fz (green), Dsh (blue circles), Stbm (yellow) and Pk (purple circles).

 

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© The Company of Biologists Ltd 2003