. Follicle-cell fate markers are unaffected in lgl germline clones. (A-H′) Cut is an immature cell fate marker that is downregulated by Notch signaling at stage 6. Hindsight (Hnt) is expressed after stage 6 and Eyes absent (Eya) is normally expressed in anterior follicle cells and immature follicle cells. Pointed-lacZ is a posterior follicle-cell fate marker. No differences in expression of these markers between lgl⁴ germline clones (B,D,F,H) and the wild type (A,C,E,G) were detected. Mutant germline clones were marked by the absence of GFP (green) in the nuclei of the nurse cells.

Lgl phosphorylation can regulate oocyte polarity. Stau (A-C), osk (D-F), the quantification of Stau and osk mRNA localization (G-I), Grk (J-L), Kin:β-Gal (M-O),α -Tubulin (P-R) in the wild type (driver only) (A,D,G,J,M,P), Lgl-overexpressing (B,E,H,K,N,Q) and Lgl-3A-overexpressing (C,F,I,L,O,R) egg chambers are shown. In egg chambers with Lgl overexpression, Stau (B,H), osk (E,H) and Kin:β-gal (N) were partially mislocalized, but Grk (K) showed normal localization, and α-Tubulin (Q) displayed a weaker anterior-posterior gradient than in the wild type (P). In egg chambers with Lgl-3A overexpression, Stau (C,I), osk (F,I) and Kin:β-gal (O) were frequently mislocalized to the center of the oocyte, Grk and the oocyte nucleus (L) were mislocalized in some egg chambers, and α-Tubulin showed higher concentrations along the cortex and lower concentration at the center of the oocyte (R).

The genetic interaction between Lgl and aPKC shows that Lgl phosphorylation by aPKC can regulate oocyte polarity. (A,B) Stau was normally localized in the apkc^k06403 heterozygous mutant (A), but mislocalized in apkc^k06403 mutant germline clones (B); mutant clones were marked by the absence of nuclear GFP (green). (C,D) Loss of one copy of apkc exaggerated the Stau mislocalization phenotype stemming from Lgl overexpression. Stau localization in these backgrounds was quantified (D). (E) Stau is normally localized in aPKC-GFP overexpression. (F) Overexpression of aPKC-GFP can rescue the Stau mislocalization phenotype caused by the Lgl overexpression. (G) Stau is mislocalized in the Lgl-overexpressing egg chambers. (H) Stau localization in these backgrounds was quantified.

aPKC phosphorylation of Lgl restricts Lgl localization to the oocyte posterior. (A,B) Lgl was localized at the oocyte posterior in Lgl-overexpressing egg chambers at stage 7 (A) and stage 9/10 (B). (C,D) Lgl was localized at the oocyte posterior in Lgl-GFP-overexpressing egg chambers at stage 7 (C) and stage 9/10 (D). (E,F) Lgl was localized throughout the cortex in Lgl-3A-overexpressing egg chambers at stage 7 (E) and stage 9/10 (F). (G,H) Lgl was localized throughout the cortex in Lgl-3A-GFP-overexpressing egg chambers at stage 7 (G) and stage 9/10 (H). (I,J) When Lgl or Lgl-GFP was co-overexpressed with aPKC^ΔN-GFP (GFP is not shown here) or aPKC^ΔN, Lgl or Lgl-GFP was excluded from the cortex.

Lgl interacts with Par-1 and is required for Par-1 localization. (A,B) Both the GFP-Par-1(N1S) (A) and GFP-Par-1(N1S) K^* (B) were enriched at the posterior at stage 10. (C,C′) The GFP-Par-1(N1S) K^* lost the posterior enrichment in lgl⁴ germline cells. Mutant germline clones were marked by the absence of β-gal staining (red) in the nuclei of the nurse cells. (D-D”) GFP-Par-1(N1S) colocalized with Lgl and was detected throughout the oocyte cortex in egg chambers with co-overexpression of Lgl-3A and GFP-Par-1(N1S). Arrows indicate the strong GFP-Par-1 and arrowheads indicate the weak GFP-Par-1. (E) Immunoblots of anti-Lgl or anti-GFP immunoprecipitation from ovaries with GFP-Par-1 (N1S) overexpression (mat-Gal4 driver) or the wild-type control (mat-Gal4 driver only). aPKC and GFP-Par-1 (N1S) proteins were precipitated by the anti-Lgl antibody from GFP-Par-1 (N1S) overexpression, and aPKC and Lgl proteins were precipitated by an anti-GFP antibody from GFP-Par-1 (N1S) overexpression. Input was one-fifth of the total ovarian extract. In anti-Lgl immunoprecipitates, controls were beads (without added antibody) and peptide blocks (+pep); in anti-GFP immunoprecipitates, controls were beads (without added antibody) and ovarian extract from wild-type flies.