Electrostatic plasma membrane targeting contributes to Dlg function in cell polarity and tumorigenesis

Abstract

Discs large (Dlg) is an essential polarity protein and a tumor suppressor originally characterized in Drosophila but also well conserved in vertebrates. Like the majority of polarity proteins, plasma membrane (PM)/cortical localization of Dlg is required for its function in polarity and tumorigenesis, but the exact mechanisms targeting Dlg to the PM remain to be fully elucidated. Here, we show that, similar to recently discovered polybasic polarity proteins such as Lgl and aPKC, Dlg also contains a positively charged polybasic domain that electrostatically binds the PM phosphoinositides PI4P and PI(4,5)P₂ Electrostatic targeting by the polybasic domain contributes significantly to the PM localization of Dlg in follicular and early embryonic epithelial cells, and is crucial for Dlg to regulate both polarity and tumorigenesis. The electrostatic PM targeting of Dlg is controlled by a potential phosphorylation-dependent allosteric regulation of its polybasic domain, and is specifically enhanced by the interactions between Dlg and another basolateral polarity protein and tumor suppressor, Scrib. Our studies highlight an increasingly significant role of electrostatic PM targeting of polarity proteins in regulating cell polarity.

Keywords: Cell polarity; Dlg; Drosophila; Lgl; PI(45)P2; PI4P; Par-6; Phosphoinositides; Polybasic domain; Scrib; Tumorigenesis; aPKC.

Fig. 1.

A polybasic domain in Dlg mediates its electrostatic PM targeting. (A) Polybasic domains in human DLG1 (hDlg1, NP_004078.2) and Drosophila Dlg (NP_996405). Mutations in Dlg^KR6Q, Dlg^KR15Q, Dlg^S4A and Dlg^S4D, as well as the point mutation of dlg^m30 (“m30”) allele, are also shown. Deletions in Dlg^ΔPDZ and Dlg^ΔGUK are shown beneath. Bold black amino acids are conserved Ser/Thr residues; bold colored amino acids are point mutations introduced in mutants. (B) Western blot using GST antibody showing that GST-PB, but not GST or GST-PB-KR6Q or GST-PB-KR15Q, co-sedimented with PI4P- and PIP2-containing liposomes. (C) PM localization of wild-type and mutant Dlg::GFP in follicular cells. PM Index: values above 1 (dashed line) indicate predominant PM localization, whereas those below 1 indicate cytosolic localization. Sample numbers (n) are shown in parentheses. ****P<0.00001; ns, P>0.05. Error bars represent s.d. (D,E) Follicular cells expressing Dlg::GFP^KI and Lgl::mCherry (D, Movie 1), or Dlg^ΔPDZ::GFP and Lgl::mCherry (E, Movie 3), undergoing hypoxia followed by reoxygenation. (D′,E′) Kymographs (top) were sampled at the areas indicated by the boxes in D and E. Arrowheads in kymographs highlight the persistent residual PM localization of Dlg under hypoxia. Graphs show quantification of Dlg and Lgl PM localization (n=20). Time stamps ae shown in hh:mm:ss format. Error bars represent s.d. Scale bars: 5 µm.

Fig. 2.

Electrostatic PM targeting of Dlg depends on PI4P and PIP2. (A) dlg^KI::GFP or dlg^ΔPDZ::GFP follicular epithelia were treated with either DMSO (control) or rapamycin [rapa(+)] and imaged live. Cells expressing Lck-FRB-CFP (not imaged) and RFP-FKBP-INPP5E (INPP5E) were labeled by nuclear RFP. (A′) Quantification of PM localization of Dlg::GFP or Dlg^ΔPDZ::GFP in wild-type (green dots) and INPP5E⁺ (red dots) cells. Sample numbers are given in parentheses. (B,C) Follicular cells expressing Dlg^KI::GFP (B, Movie 4) or Dlg^ΔPDZ::GFP (C, Movie 5) undergoing hypoxia and reoxygenation. PI4KIIIα-RNAi cells were labeled by RFP. (B′,C′) Quantification of Dlg::GFP (B′) or Dlg^ΔPDZ::GFP (C′) PM localization in both wild-type (green lines) and PI4KIIIα-RNAi (red lines) cells (n=20). Error bars represent s.d. Asterisks in image panels indicate RNAi cells. Scale bars: 5 µm.

Fig. 3.

Scrib enhances the electrostatic PM targeting of Dlg. (A) Follicular cells expressing te Dlg::GFP or mutants as indicated. (A′) Quantification of the PM localization of each mutant. (B,C) Follicular cells expressing Dlg::GFP or mutants as indicated. scrib-RNAi (B) or lgl-RNAi (C) cells were labeled by RFP. (B′,C′) Quantification of wild-type and mutant Dlg::GFP PM localizations in wild-type (green dots) and RNAi (red dots) cells. Error bars represent s.d. Sample numbers (n) are shown in parentheses. ****P<0.001; ns, P>0.05. Asterisks in image panels indicate RNAi cells. Scale bars: 5 µm.

Fig. 4.

Electrostatic PM targeting of Dlg regulates cell polarity and tumorigenesis. (A) dlg^−/− follicular cells were marked by the loss of RFP (blue in all merged images), except for ‘dlg^−/− wt’ in which the dlg^−/− clones were marked by the absence of Dlg (green). Samples expressed wild-type or mutant Dlg::GFP as indicated, and were stained for GFP (green) and aPKC (red). Note that dlg^−/−, dlg^m30-ΔGUK::GFP clones showed roughly equal frequency of rescued and non-rescued polarity defects. Asterisks highlight dlg^−/− mutant cells. (B) Eyes from adult flies of Ras^V12, or dlg-RNAi or Ras^V12/dlg-RNAi combined with additional expression of wild-type and mutant Dlg as indicated. ctrl, Ras^V12/dlg-RNAi only. (C) Quantification of the rescue of Ras^V12/dlg-RNAi lethality by wild-type and mutant Dlg as indicated. (D) A hypothetical model on the potential phosphorylation-dependent allosteric control of the Dlg polybasic domain and its regulation by Scrib. Potential regulatory defects in Dlg^m30, Dlg^S4A and Dlg^ΔUGK are also shown. Kinases phosphorylating the polybasic domain of Dlg remain to be characterized but could include aPKC and PKCα. The sequential order between Scrib binding to Dlg and its phosphorylation is postulated. Scale bars: 5 µm.