Perlecan is required for cell membrane polarity.trol clones show defects in the distribution of various cell polarity markers (filled arrowheads indicate normal; open arrowheads disrupted protein localization). (A,B) Baz (red) is generally enriched and expanded into the cytoplasm. (C) Patj (red) is not affected by the loss of Pcan. (D) Apical localization of Crb (red) is frequently reduced. (E,F) Arm (red) expression is slightly elevated and expanded to all cell membrane. (G) Dlg (red) is strongly reduced. A'-G' show red channels only. Clones are marked by the loss of GFP and outlined by broken lines. Nuclei of follicle cells in ectopic positions are marked with a circle. Green, GFP; blue, DNA.

Dystroglycan and Perlecan can bind in vitro. Western blots showing co-immunoprecipitation of PcanV with anti-Dg^pep. Embryonic glycoprotein extract was mixed with 5 μg PcanV (R1), 0.5 μg PcanV (R2) or 0.05 μg PcanV (R3), and precipitated with anti-Dg^pep. Controls were: no glycoprotein extract added to reaction mixture (C1), precipitation reaction with boiled Dg antibody (C2), only primary antibody loaded (C3), primary antibody exchanged for rabbit serum (C4). The precipitates were probed with anti-PcanV (upper panel) or anti-Dg^cyto (lower panel). Pure PcanV (PcanV 0.05 μg) and glycoprotein extract (input) were loaded as a positive control. Some of the minor Dg bands in the glycoprotein extract (input) are probably due to protein degradation.

Perlecan is required for basal localization of Dystroglycan. (A,B) Dg (red) is no longer present in the basal cell membrane of trol clones. Occasionally, Dg appears to be redistributed to the apical cell membrane (yellow arrows in B′). (C,D) Pcan localization in the ECM is unchanged in a Dg³²³ clone (C) and orientation of Pcan-stripes appears normal (D). (E) βPS Int (red) is normally expressed in trol clones. Blue indicates DNA. (F) In a FCE entirely composed of mutant cells, gaps in βPS staining can be seen. Green indicates GFP and Dg; arrow indicates gap. (G) βPS Int (red) is still present (arrows) in the basal membrane of a trol clone that has lost Dg (green). (G′) Green channel only, showing both GFP and anti-Dg^cyto staining. (G″) Red channel only. (H) Expression of Lam in a wild-type ovariole. Asterisk indicates muscular sheath. (I) A very large trol clone showing fuzzy Lam (red) distribution. Blue indicates DNA. (J) Lam (red) is still present at the basal side of a medium sized lanA clone (arrow). (K) Two egg chambers whose FCE are entirely composed of lanA mutant cells. Lam (red) is almost completely absent. (L) Three egg chambers whose FCE are entirely composed of lanA mutant cells. Dg (red) is still localized at the basal membrane. Clones are marked by the loss of GFP (green) and indicated by broken lines. (A′-L′) Red channels only.

Perlecan-dependent Dystroglycan lacks the mucin domain. (A) Schematic drawing of the Dg forms A, B and C, which are generated through differential splicing of exon 8 (red box) and exon 9 (black box). The transmembrane-domain is indicated with a yellow line. (B) Graphic showing the potential of mucin type O-linked glycosylation for each position of the Dg-C sequence. A stretch of 80 amino acids (positions 424-503, indicated by the blue bar) contains a cluster of 52 high potential o-glycosylation sites, which constitute the mucin-like domain. The red bar indicates the region encoded by exon 8 (position 243-507). (C) Western blots of embryonic protein extract (0-20 hours) probed with anti-Dg^cyto (cyto), anti-Dg^pep (pep) and anti-Dg^ex8 (ex8). (D-F) Wild-type ovaries stained with anti-Dg^cyto (D,F) and anti-Dg^ex8 (E,G). F and G are higher magnification of D and E, respectively. Dg is strongly concentrated in the basal membrane of the FCE throughout oogenesis (D, arrow in F). Dg-C is expressed in the muscular sheath (yellow asterisks in E and G) but absent from the basal membrane of the FCE (arrow in G). Red, Arm; green, Dg. (H-J) Ectopic expression of Dg-B (H,J) and Dg-C (I) induces ectopic accumulation (arrows) of Lam (H) and Pcan (I,J). Cells expressing the Dg construct are marked by GFP (green). Red indicates Lam (H) and Pcan (I,J). (H′-J′) Red channels only.

Dystroglycan and Dsytrophin are mutually dependent for basal membrane localization. (A) In a wild-type ovariole, Dys is expressed in the muscular sheath (star) and at the basal membrane of the follicle cells. (B) In a Dg³²³ follicle cell clone, Dys (red) is lost from the basal membrane. (C) Expression of dsDys (Dys-hairpin) efficiently reduces Dys expression (red). (D) Reduction of Dys leads to a reduction of Dg (red) in the basal membrane. (E) In a trol clone, Dys is no longer concentrated at the basal membrane but diffusely localized in the cytoplasm. All follicle cell clones are marked with broken lines. Filled arrowheads indicate the wild type; open arrows indicate the mutant expression pattern. (B′-E′) Red channels only. Blue indicates DNA.

Distribution of NrxIV, Con and Nrg in the FCE. All pictures show antibody staining of wild-type ovaries. (A,B) NrxIV (red) gradually concentrates at the basal side of the lateral membrane of follicle cells during development. (C-E) Cont (red) can bee seen in a punctate staining during early and mid-oogenesis (D). In older egg chambers (E), Cont concentrates apicolaterally. (F) Nrg (red) is expressed in the lateral cell membrane. (G) Surface views of the FCE showing NrxIV (green, G′), Cont (red, G″) and Nrg (blue, G‴). All three proteins co-localize at tricell junctions. Green indicates actin in A-D,F. (B′,D′,E′) Red channel only.