Redundant regulation of localization and protein stability of DmPar3

Abstract

Apical-basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical-basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical-basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical-basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical-basal cell polarization.

Keywords: Adherens junctions; Cell polarity; Drosophila; Lipid binding; Par3.

Fig. 1

Oligomerization of Baz is dispensable for viability. a Schematic representation of different Baz deletion constructs. All constructs were expressed from the same genomic locus (attP40) with an N-terminal GFP-tag under the control of the ubiquitin promotor. The ∆OD mutation (V14DD68K) prevents oligomerization and the ∆LB mutation (K1173-74A) abolishes membrane binding. The ability to rescue the embryonic lethality of baz^815-8 germ lines clones was quantified and the localization determined, where “+” indicates the wild-type situation, “+/−” indicates a lateral cortical localization and “−” indicates a cytoplasmic localization. b Expression of GFP-Baz in the baz^815-8 mutant background results in a weak overexpression compared towards endogenous Baz. Full-length Baz is indicated with an arrow. c Co-immunoprecipitation of GFP- and Myc-tagged Baz or Baz∆OD in S2R cells. Mutations of the OD domain strongly attenuate the capacity of Baz to self-associate. d In vitro crosslinking of the Baz OD domain (aa 1–83). Mutations of the OD domain (V14DD68K) strongly impair the formation of oligomers in vitro

Fig. 2

Oligomerization and lipid-binding promote Baz localization redundantly. a In immunostainings of the Drosophila embryonic epithelium, endogenous Baz (green) co-localizes with DE Cadherin (DE-Cad, red) at the apical junctions. Disc large (Dlg, blue) was stained as a lateral marker. b Localization of GFP-Baz is indistinguishable from the endogenous protein. c Oligomerization-deficient GFP-Baz∆OD displays an accumulation at the AJ, overlapping with DE-Cad. d The localization of a lipid-binding deficient GFP-Baz∆LB protein is similar to wild-type Baz. e GFP-Baz∆OD∆LB double mutant is absent from the cell cortex and displays a diffuse cytoplasmic localization. All transgenes were expressed in a wild-type background. Scale bars are 10 µm

Fig. 3

Loss of oligomerization and lipid-binding causes embryonic lethality. Baz variants that carry a C-terminal One-Strep (OneS) tag were expressed under the ubiquitin promoter and tested for their capacity to rescue baz^818-8 germ line clones. Baz-OneS efficiently rescues the lethality of the baz⁸¹⁸⁻⁸ allele (10.5 ± 1.8% embryonic lethality). Baz∆OD∆LB-OneS failed to rescue the embryonic lethality. Fusion of the oligomerization domain of the human TEL protein (aa 45–115) to the N-terminus of Baz restores its function and rescues embryonic lethality of baz^815-8 to a large extent (50.0 ± 6.1% embryonic lethality). Similarly, wild-type Baz-GFP overexpressed with mat-Tub::Gal4 using the Gal4/UAS-system had comparable efficiencies as the constitutively expressed variant (12.7 ± 8.5%), whereas overexpression of Baz∆OD∆LB-GFP failed to rescue (100% embryonic lethality). Bars represent the mean ± SD, n = 300 each

Fig. 4

Epithelial polarization requires the functional redundancy of the OD and LB domains. a Immunostaining of endogenous Baz (green), aPKC (red), and Sxl (blue) in the embryonic epidermis. b–e Immunostaining of Baz variants and endogenous aPKC in the embryonic epidermis of baz^818-8 germ line clones. Hemizygous mutant embryos were identified by the absence of Sxl staining. b Loss of Baz in the embryonic epidermis of disrupts epithelial polarization and aPKC accumulates in the cytoplasm. c Baz-OneS efficiently recruits aPKC to the apical junctions, such as endogenous Baz, whereas Baz∆OD∆LB-OneS displays a cytoplasmic mislocalization. d Baz∆OD∆LB-OneS rescues targets some aPKC to the apical junctions, but the majority of the protein still accumulates in the cytoplasm. e TEL-Baz∆OD∆LB-OneS localizes at the apical junctions and is capable of recruiting aPKC to rescue the defects of Baz∆OD∆LB-OneS. f–j Immunostaining of Baz (green), DE-Cad (red), and Sxl (blue) in the embryonic epidermis demonstrates a loss of AJ in baz^815-8 germ line clones, which can be rescued by wild-type Baz (h) and TEL-Baz∆OD∆LB-OneS (j) but not by Baz∆OD∆LB-OneS (i). Scale bars are 10 µm

Fig. 5

Neuroblast polarity requires Baz’s capacity to either self-associate or to bind lipids. a Immunostaining of endogenous Baz (green), Mir (red), and Sxl (blue) in embryonic metaphase NBs. Baz localizes at the apical cortex, whereas Mir accumulates basally. b–e Immunostaining of Baz variants (green) and endogenous Mir (red) in embryonic NBs of baz^818-8 germ line clones during metaphase. Hemizygous mutant embryos were identified by the absence of Sxl staining. b Loss of Baz in baz^818-8 germ line clones disrupts NB polarity and Mir localization. c Baz-OneS rescues NB polarity, whereas Baz∆OD∆LB-OneS does not localize at the cortex and fails to polarize NBs (d). e TEL-Baz∆OD∆LB-OneS restores apical–basal polarity in metaphase NBs, such as wild-type Baz. f Immunostaining of endogenous Baz (green), aPKC (red), and Sxl (blue) in embryonic metaphase NBs. Baz recruits aPKC to the apical pole of metaphase NBs. g aPKC accumulates in the cytoplasm in metaphase NBs of baz^818-8 germ line clones. h, j Baz-OneS and TEL-Baz∆OD∆LB-OneS both show a comparable localization and recruit aPKC to the apical pole. i In contrast, Baz∆OD∆LB-OneS localizes in the cytoplasm, such as endogenous aPKC. Scale bars are 5 µm

Fig. 6

Recruitment of Baz by DE-Cad, Ed, and Stan does not depend on self-association or lipid-binding of Baz. a Wild-type Baz-GFP was expressed with the ubiquitin promoter in S2R cells and localizes at the plasma membrane. b Baz∆LB-GFP accumulates in cytoplasmic aggregates. c RFP displays a diffuse cytoplasmic localization and cannot recruit Baz∆LB-GFP to the cortex. d, e DE-Cad-RFP and Ed-RFP recruit Baz∆LB-GFP to artificial cell–cell contacts. f Similar, the intracellular domain of Stan fused to the extracellular and transmembrane domain of DE-Cad (DE-Cad∆intra-Stan∆extra) targets Baz∆LB-GFP to cell–cell contacts. DE-Cad∆intra-Stan∆extra was detected with an anti DE-Cad antibody, which recognizes the extracellular domain of DE-Cad. g–k In the same experimental setup with Baz∆OD∆LB-GFP, the double mutant was efficiently recruited by DE-Cad, Ed, and Stan without apparent differences. l Deletion of all three PDZ domains in Baz∆LB does not affect the localization of the mutant protein at the apical junctions (green = GFP-Baz∆PDZ1-3 ∆LB, red = DE-Cad, blue = aPKC). DIC differential interference contrast. Scale bars are 5 μm in a–k and 10 μm in l

Fig. 7

Oligomerization and lipid binding are crucial for Baz’ stabilization. a Lysates of baz^818-8 germ line clones that express different Baz variants were blotted against Baz. Actin was used as loading control. Full-length Baz is indicated with an arrow. Loss of Baz oligomerization and lipid binding (Baz∆OD∆LB) strongly decreases the amount of Baz protein. TEL-Baz∆OD∆LB-OneS rescues the degradation of Baz∆OD∆LB-OneS. b Quantification of Baz-OneS variants in baz^818-8 germ line clones. The whole Baz lanes were quantified and normalized towards actin from three biological replicates. c qPCR of total RNA from the baz^818-8 germ line clones shows that all transgenes were expressed without significant differences. d Embryonic lysates of different GFP-Baz variants in a baz^818-8 genetic background were blotted against GFP. Full-length Baz is indicated with an arrow. Actin was used as loading control. Loss of oligomerization reduces the stability of GFP-Baz∆OD, which is drastically enhanced upon the mutation of the lipid-binding motif in GFP-Baz∆OD∆LB. However, mutation of the lipid-binding motif alone (GFP-Baz∆LB) does not affect the protein stability. e Quantification of GFP-Baz variants. The whole GFP lanes were quantified and normalized towards actin from three biological replicates. f Scheme of the functional redundancy between the OD and LB domains. Bars represent the mean ± SD. Statistics were one-way ANOVA followed by Tukey’s post hoc test, n.s. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001