Figure 1.

Vps35 Regulates Levels of Fmi and Stbm at Apical Junctions Independently of the Wash Complex

(A) Diagram illustrating asymmetric localization of the core planar polarity proteins in the pupal wing. Two cells are shown, with Fmi, Fz, Dsh, and Dgo localizing on distal cell edges. This forms an intercellular complex with Fmi, Stbm, and Pk on proximal edges of the neighboring cell.

(B) During polarization, complexes sort from a uniform distribution (left), and all the complexes become oriented in the same direction (right). This specifies positioning of trichomes (black in right diagram) to distal cell edges.

(C, F, G, and H) 28-hr after puparium formation (APF) (C, G, and H) or 32-hr APF (F) pupal wings carrying clones of Vps35 (C and F), Fam21 (G), or wash (H), marked by loss of β-gal staining (blue in C, G, and H and green in F). Wings are immunolabeled for Fmi in green and Stbm in red (C, G, and H) or Fmi in blue and phalloidin in red (F). The reduced phalloidin staining in Vps35 mutant tissue (F) indicates a delay in trichome initiation. In older wings, phalloidin-stained trichomes are visible in Vps35 clones (not shown). Scale bar 10 μm.

(C’) High magnification image of wild-type and mutant regions immunolabeled with Fmi and used to quantitate polarity.

(C’’) Polarity nematic showing the magnitude and angle of polarization for each cell.

(D) Quantitation of mean intensity of Fmi (red dots) or Stbm (orange dots) membrane labeling in pupal wing clones. Intensity is shown as a ratio of signal in mutant compared to wild-type in each wing; error bars are SD. One-sample t tests were used to determine whether the ratio differed from 1.0.

(E) Mean polarity and variation in polarity angle of wings immunolabeled for Fmi in wild-type and Vps35 mutant tissue (see C’ and C’’). Values from the same wing are linked by black bars; mean and SD are listed. Paired t tests were used to compare values in the same wing. ^∗∗∗p < 0.001.

See also Figure S1 and Data S1 for all statistical comparisons.