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Redundant regulation of localization and protein stability of DmPar3

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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.

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Acknowledgements

We thank the Bloomington Drosophila stock center at the University of Indiana (USA) and the Developmental Studies Hybridoma Bank at the University of Iowa (USA) and Frank Sprenger for providing reagents. This work was supported by Grants of the German Research Foundation (DFG) to M. P. K. (DFG3901/1-2, SFB1348-A05).

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  1. Molecular and Cellular Anatomy, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany

    Lars Kullmann & Michael P. Krahn

  2. Internal Medicine D, University Hospital of Münster, Domagkstr. 3a, 48149, Münster, Germany

    Lars Kullmann & Michael P. Krahn

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Supplemental Fig.

 1: Localization of Baz variants in the embryonic epidermis. (A-D) Immunostainings of different GFP-Baz variants in the embryonic epidermis. All transgenes were expressed with the ubiquitin promotor in a wild-type background. GFP (green), DE-Cad (red), and Dlg (blue) were stained. (A) GFP-Baz1-968 localizes mainly at the apical junctions with some baso-lateral mislocalization. (B) Mutation of the OD domain in this truncated protein causes a cytoplasmic accumulation of GFP-Baz1-968∆OD. (C, D) Chimeric proteins that carry the pleckstrin homology (PH) domains of either human PLCδ or Akt1 fused to the C-terminus GFP-Baz∆OD1-1105 promote a cortical localization in the embryonic epidermis. (E) Western blot of embryonic lysates of the Baz variants (A-D). The Baz variants were detected with a GFP antibody and Actin was used as a loading control. Scale bars are 10 µm (TIFF 857 kb)

Supplemental Fig.

 2: Cuticle phenotypes of Baz variants. (A) Cuticle phenotypes were classified into the four categories wt, shrunken with holes, holes, and cuticle rests. (B) The cuticle phenotypes of baz818−8 germ line clones that express different Baz variants with the ubiquitin promoter were quantified (n = 300 per genotype). Embryos that normally developed and hatched are also included as “hatched”. baz818−8 germ line clones display holes or cuticle rests. Embryos that express Baz-OneS hatch to a large extend (89,5%) or display either wt or shrunken with hole phenotypes. No embryos hatch upon the expression of Baz∆OD∆LB-OneS. Most embryos have either cuticle rests or holes phenotypes (58,1 and 16,3%, respectively). Nevertheless, some embryos develop further and have a wt or shrunken with holes phenotype (2,3 and 23,3%, respectively). The chimeric TEL-Baz∆OD∆LB-OneS protein rescues the Baz∆OD∆LB phenotypes to a large extent as half of the embryos hatch (50,2%) or have either wt or shrunken with holes phenotypes (21,9 and 17,9%, respectively). Scale bars are 200 µm (TIFF 417 kb)

Supplemental Fig.

 3: Quantification of Baz-OneS constructs in the embryonic epithelium. (A) Scheme of the area (33 × 4 µm) of the apical region of all immunostainings in Fig. 4, which has been quantified with FIJI Plot Profile. The gray values of either Baz/aPKC (B-F) or Baz/DE-Cad (G-K) in the apical region were plotted against the distance. (L) The Pearson correlation coefficient of the co-localization of either Baz/aPKC or Baz/DE-Cad has been determined with the Coloc2 Plugin in FIJI for all genotypes in Fig. 4. For the quantification, three different embryos of each genotype have been analyzed. Bars represent the mean ± S.D. Statistics were one-way ANOVA followed by Turkey’s post hoc test, n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (TIFF 1777 kb)

Supplemental Fig.

 4: Expression of Baz variants with the UAS/Gal4-system. (A) Western blot of embryonic lysates from embryos that express Baz-GFP and Baz∆OD∆LB-GFP with the UASp promotor, driven by mat-Tub::Gal4. The Baz variants were detected with a GFP antibody and actin was used as a loading control. Full-length Baz is indicated with an arrow. Note that the pattern of specific bands is different in Baz∆OD∆LB (e.g., the band around 130 kDa is much weaker), which might be due to differences in the posttranslational processing of full-length Baz (presumably by cleavage). (B, C) Immunostaining of Baz variants (green), DE-Cad (red), and aPKC (blue) in the embryonic epidermis. (B) Baz-GFP localizes at the apical junction, (C) whereas Baz∆OD∆LB-GFP displays a diffuse cytoplasmic localization. Scale bars are 10 µm (TIFF 902 kb)

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Kullmann, L., Krahn, M.P. Redundant regulation of localization and protein stability of DmPar3. Cell. Mol. Life Sci. 75, 3269–3282 (2018). https://doi.org/10.1007/s00018-018-2792-1

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