Fig 1.
Magi localizes to the apicolateral region and adherens junctions in epithelia.
(A-C). Enface view of the cellularizing embryo showing Magi (green) localization to the furrow canal where Baz (red) is present. (D-F). Enface view of stage 11 embryo showing Magi (green) present in the same membrane domain as Baz (red). (G-I). Enface view of stage 13 embryo showing Magi (green) is present in the same membrane domain as Baz (red) in both epithelial and amnioserosa cells. (J-L). Side view of the salivary gland showing Magi (green) and Baz (red) colocalizing in the same membrane region at the apicolateral domain. (M-O). Side view of the hindgut showing Magi (green) and Baz (red) colocalization at the apicolateral membrane. (P-R). Side view of the salivary gland showing Magi (green) is below the apical membrane domain where Crumbs (Crb, red) is present. (S-V’). Enface and side views of wing imaginal disc epithelia showing that Magi (green) predominantly colocalizes with Baz (Baz::GFP, red) but not with Dlg (blue). (W-Y’). Enface and side view of the wing imaginal disc epithelia showing that Magi (green) is present at the adherens junction, marked with Ecad (red) immunolabeling. Scale bars indicate 10μm. Each enface view represents a single Z slice.
Fig 2.
Magi associated with Baz and aPKC at the adherens junctions.
(A, B) Immunoprecipitation of embryonic extracts with endogenous Baz tagged with GFP. Proteins from the embryonic extracts (Input), supernatant (Supe) and immunoprecipitation (IP) were loaded for Western analysis. The Magi antibody (A) and the Baz antibody (B) were used for immunoprecipitation and in both experiments Magi is pulled down with Baz and aPKC but not with the basolateral protein Dlg. (C) Diagram of the wing imaginal disc from a third instar larvae with the expression pattern of apterous-GAL4 shown in red and bisecting the wing pouch. A side view shows the wing disc within the columnar epithelia of the wing pouch labeled in red. (D-T) Proximity Ligation Assays were performed on wing imaginal discs with Bazooka endogenously tagged with GFP (Baz::GFP). (C-E) Baz::GFP (green) with PLA (red) using anti-GFP and anti-Magi antibodies. (F) Magi overexpressed with apterous-GAL4 with Baz::GFP with PLA using anti-GFP and anti-Magi antibodies shows greater levels of PLA on the apterous (left) side of the wing disc. (G) aPKC::GFP overexpressed with apterous-GAL4 with anti-GFP and anti-Magi antibodies shows greater levels of PLA on the apterous side of the wing disc. (H-K) Controls with Baz::GFP and PLA (red) with only anti-GFP (G-H) and Baz::GFP and PLA (red) with anti-Magi alone (I-J). PLA levels are reduced compared to panel D. (L-N) Side projection of Baz::GFP (green) with PLA (red) with anti-GFP and anti-aPKC antibodies with the PLA signal concentrated at the apical domain of the columnar epithelium (arrow). Nuclei were labeled with DAPI (blue). (O-Q) Side projection of Baz::GFP (green) with PLA (red) with anti-GFP and anti-Magi antibodies with the PLA signal concentrated at the apical domain of the columnar epithelium (arrow). Nuclei were labeled with DAPI (blue). (R-T) Controls showing a side projection of Baz::GFP discs with PLA using only anti-GFP (R), only anti-Magi (S) or only anti-aPKC (T).
Fig 3.
Somatic null clones of Magi are viable with no loss of polarity or AJ.
Mutant clones of Magibst in the wing imaginal disc were generated by FRT mediated recombination. Nuclear-localized GFP driven by the ubiquitin promoter (Ubi-nlGFP, green) labeled wildtype and heterozygous cells, and Magibst clones are indicated by the lack of GFP. The large GFP positive nuclei are from the overlying peripodial cells. Magibst clones lack Magi immunolabeling (A-D) but had no effect on the levels and localization of Baz (E-H, red), aPKC (I-L, red) or the adherens junction protein Armadillo (Arm, red) (M-P). There was no effect on cell survival, which is shown by lack of apoptotic marker cleaved Caspase-3 (Cas3, red) (Q-S). Panels D,H,L,P,T were digitally magnified 200% and the clonal boundaries are marked with white lines. Scale bars indicate 10μm.
Fig 4.
Magi localization requires aPKC but not the other polarity proteins, Baz, Sdt or Crb.
(A-I) Wing imaginal discs showing mitotic clones mutant for apical polarity protein genes, baz, sdt and crb (clone boundaries are indicated by white lines). Mutant clones for baz, sdt and crb survive and no polarity defects are seen. Somatic clones are marked by the loss of a cellular marker (GFP or RFP). Loss of these polarity proteins had no effect on membrane recruitment of Magi (red). In crb mutant clones, the continuous localization of Magi on the plasma membrane was altered. (J-L) Wing imaginal disc showing mitotic clones mutant for aPKC. Only small aPKC mutant clones were recovered and frequently twin spots were not associated with mutant clones. Magi (red) did not appear to be affected in the small aPKC mutant clones. (M-O) Wing imaginal discs co-expressing aPKC-RNAi and p35 to block cell death (ap>aPKC-RNAi, p35). Temporal control of expression was controlled using Gal80ts. Blocking cell death resulted in tissue overgrowth and loss of apicobasal polarity as Ecad (green) localization was impaired. Magi (red) localization to the plasma membrane was also disrupted. Scale bars indicate 5μm in A-L and 10μm in M-O.
Fig 5.
Overexpression of Magi reduces Baz at the plasma membrane.
Cherry-tagged Magi was overexpressed in the wing imaginal disc using apterous-GAL4 (ap>Magi::Cherry). The average membrane intensity of Baz, and aPKC was measured and compared between the apterous (ap-GAL4, black bars) and non-apterous side (WT, white bars) of the wing imaginal disc (X). The apterous boundary is indicated with a white line and in all panels the apterous side is to the left. All panels represent a single Z slice within the apical or basal domain of the columnar epithelia (H). (A-D) Wing imaginal disc overexpressing Magi::Cherry (red) immunolabeled for Baz (green). High levels of Magi result in mislocalization of Baz to the cytoplasm and a significant reduction on the plasma membrane (D). (E-G) At the basolateral region of the epithelia (H), Baz (green) was detected in large puncta (arrows) with Magi::Cherry (red). (I-L) Wing imaginal discs overexpressing Magi::Cherry (red) immunolabeled for aPKC (green). aPKC was significantly reduced at the plasma membrane (I) and present in small cytosolic puncta. (M-P) Wing imaginal disc expressing a Magi transgene lacking the PDZ domains (MagiΔPDZ) tagged with the FLAG epitope (red). Expression of this transgene had no effect on the membrane localization of Baz (green)(P) and the Magi protein appeared to be cytosolic. (Q-T) Wing imaginal disc expressing a Magi transgene lacking the two WW domains (MagiΔWW) tagged with the FLAG epitope (red). Expression of this transgene resulted in a significant reduction in Baz (green)(T). (U-W) MagiΔWW was found at the membrane but did not form large intracellular accumulations in basal regions. *** p<0.001; ns p>0.05. n = 5 discs for each experiment and error bars indicate SEM. Panels (E-G, U-W) were digitally magnified 200%. Scale bars indicate 5μm.
Fig 6.
Magi is associated with lipid subdomains and alters PIP3 levels.
(A-I) Cherry-tagged Magi (red) was expressed using apterous-GAL4 and accumulations within the basolateral region. (A-C) Magi::Cherry accumulations were positive for the ER marker Calnexin (green), arrows. (D-F) PIP3 (PH::GFP; green) was co-localized in the large accumulations (arrows) with Magi::Cherry. (G-I) Magi::Cherry accumulations were positive for Cholera toxin B (CTB, green)(arrows). (J-L) Endogenous Magi (red) colocalized with cholera toxin B (CTB, green) in wildtype discs at the plasma membrane. (M-S) Cherry-tagged Magi was overexpressed using apterous-GAL4 (ap>Magi::Cherry). The apterous boundary is indicated with a white line. (M-P) Wing imaginal discs overexpressing Magi::Cherry (red) with a PIP3 indicator (PH::GFP, green) under the control of the tubulin promoter. High levels of Magi resulted in an increase in of PIP3 at the plasma membrane. *** p<0.001; n = 5 discs. Error bars indicate SEM. (Q-S) Side projections showing the increase in PIP3 is within the apical domain (arrow) and within the large Magi accumulations (arrowhead). (T-Y) PTEN was expressed using apterous-GAL4 with Magi::Cherry (T-V) or without (W-Y). (T-V) Baz (green) was still displaced from the membrane when PTEN (blue) and Magi::Cherry (red) were coexpressed. (W-Y) Expression of PTEN alone had no effect on Magi recruitment to the membrane (arrowhead). Panels (A-L) were digitally magnified 200%. Scale bars indicate 5μm
Fig 7.
Overexpression of Baz results in loss of Magi from the plasma membrane.
GFP-tagged Baz was overexpressed in the wing imaginal disc using apterous-GAL4 (ap>Baz::GFP). The average membrane intensity of each protein was measured and compared between the apterous (black bars) and non-apterous side (white bars) of the wing imaginal disc. The white lines mark the apterous dorsal/ventral boundary. All images showing the apical region of the epithelia. (A-D) Overexpression of Baz::GFP (green) immunolabeled for Magi (red). Cells containing high levels of Baz show a significant reduction in the membrane levels of Magi. (E-P) Overexpression of Baz::GFP (green) resulted in a significant increase in the membrane levels of aPKC (red) (E-H), Crb (red) (I-L) and Par-6 (red) (M-P). (Q-V) Overexpression of Baz::GFP (green) lead to membrane ruffling (arrows) but not the accumulation of Magi (red) or aPKC (red) in intracellular puncta. *** p<0.001; ** p<0.01. n = 5 discs for each experiment and error bars indicate SEM. Panels (Q-V) were digitally magnified 200%. Scale bars indicate 5μm
Fig 8.
A balance between the levels of Magi and Baz regulates aPKC membrane levels.
apterous-GAL4 was used to overexpress different combinations of tagged proteins in the wing imaginal disc. The average membrane intensity of each protein was measured and compared between the apterous (black bars) and non-apterous (white bars) side of the wing imaginal disc. The white lines mark the apterous dorsal/ventral boundary. (A-H) Overexpression of aPKC::GFP (green) had no effect on the membrane localization or levels of Magi (red) (A-D) or Baz (red) (E-H). (I-L) Co-expression of Magi::Cherry (blue) and Baz::GFP (green) attenuated the changes in aPKC (red) caused by high levels of Baz or Magi alone. aPKC levels were not increased by overexpression of Baz::GFP when Magi::Cherry was co-expressed. (M-P) Coexpression of Magi::Cherry (blue) and aPKC::GFP (green) lead to a reduction in the Baz membrane levels (red) and increased accumulation in the Magi vesicles. Overexpression of wildtype aPKC did not block the Magi induced reduction in Baz. (Q-X) Higher resolution image of the large accumulations seen with Magi::Cherry expression at the basolateral region of the epithelial cells. Each panel was digitally magnified 200%. (Q-T) Co-expression of Magi::Cherry (blue) and Baz::GFP (green) lead to the accumulation of Baz and Magi within the large internal accumulations. aPKC (red) was only weakly recruited (arrows). (U-X) Co-expression of Magi::Cherry (blue) and aPKC::GFP (green) lead to an increased accumulation of Magi vesicles that were also positive for Baz (red) (arrows). *** p<0.001; * p<0.05; ns not significant. Error bars indicate SEM. n = 5 discs for each experiment. Scale bars indicate 5μm for A-O and 2μm for Q-X.