Figure 3.

Asymmetric Localization of Core Proteins in Junctional Puncta

(A) Fmi antibody internalization with chase times up to 30 min in wild-type wings. Quantitation of extracellular Fmi staining in junctional puncta, junctional nonpuncta, or the apical plasma membrane, or total (see Figure 1G). Asterisks indicate p values comparing Fmi levels between junctional puncta and nonpuncta (red) or between junctional nonpuncta and the apical membrane (orange). The mean intensity of Fmi staining in puncta does not change over time nor does the size or number of puncta (not shown). (B and C) Extracellular Fmi staining (green) after a 30-min chase in prepupal wings, colabeled with total Stbm (B) or Dsh (C) staining (red). Arrows point to prominent junctional puncta. (D–G) Twenty-eight hour pupal wings, stained for: (D) total Fmi (green), Fz (red); (E) total Fmi (green), Stbm (red); (F) Stbm (green), Fz (red); and (G) Arm (green), Stbm (red). White arrows indicate strongly colocalizing puncta; yellow arrows indicate Stbm puncta that do not colocalize with Arm. (H and I) fz-EYFP (H) and stbm-EYFP (I) mosaics in 28-hr wings, stained for GFP (green) and Fmi (red). Arrows indicate distal (H) or proximal (I) puncta. (J and K) Quantitation of fluorescence intensity of Fz-EYFP (J) or Stbm-EYFP (K) staining in proximal and distal puncta and nonpuncta in mosaics. Puncta were selected on the basis of Fmi staining. Asterisks are p values comparing proximal and distal puncta or nonpuncta (gray), or as indicated by bars. Weak Fz and Stbm PD asymmetry is seen in nonpuncta (2.0- and 1.5-fold, respectively), but asymmetry is stronger within puncta (9.0- and 2.5-fold, respectively). (L and M) Prepupal (L) or 20-hr (M) pupal wings stained for Fmi (green) and Stbm (red), showing typical puncta (arrows). (N–P) Twenty-eight hour pupal wings containing clones of dgo³⁸⁰ (N), pk^pk-sple13 (O), or dsh^V26 (P), marked by loss of β-gal (red), stained for Stbm (green). Arrows point to puncta in wild-type tissue. Puncta are less prominent in mutant tissue. (Q and R) Quantitation of puncta size (Q) or mean fluorescence intensity (R) in 28- and 20-hr wild-type pupal wings, and in dgo³⁸⁰, pk^pk-sple13, and dsh^V26 mutant clones stained for Stbm. Asterisks are p values compared to 28-hr wild-type wings, or as indicated by bars. (S–S″″) Time-lapse images of 27- to 29-hr pupal wing, imaged over a 2-hr period. Puncta (representative examples circled) persist over time. Scale bars, 2.5 μm (B–I, L, and M) or 5 μm (N–P and S). See also Figure S4 and Movie S1.