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. 2014 Apr 7;9(4):e94038.
doi: 10.1371/journal.pone.0094038. eCollection 2014.

Apical localisation of crumbs in the boundary cells of the Drosophila hindgut is independent of its canonical interaction partner stardust

Alexandra Kumichel  1 , Elisabeth Knust  1
Affiliations

Apical localisation of crumbs in the boundary cells of the Drosophila hindgut is independent of its canonical interaction partner stardust

Alexandra Kumichel et al. PLoS One. .

Abstract

The transmembrane protein Crumbs/Crb is a key regulator of apico-basal epithelial cell polarity, both in Drosophila and in vertebrates. In most cases studied so far, the apical localisation of Drosophila Crumbs depends on the interaction of its C-terminal amino acids with the scaffolding protein Stardust. Consequently, embryos lacking either Crumbs or Stardust develop a very similar phenotype, characterised by the loss of epithelial tissue integrity and cell polarity in many epithelia. An exception is the hindgut, which is not affected by the loss of either gene. The hindgut is a single layered epithelial tube composed of two cell populations, the boundary cells and the principal cells. Here we show that Crumbs localisation in the principal cells depends on Stardust, similarly to other embryonic epithelia. In contrast, localisation of Crumbs in the boundary cells does not require Stardust and is independent of its PDZ domain- and FERM-domain binding motifs. In line with this, the considerable upregulation of Crumbs in boundary cells is not followed by a corresponding upregulation of its canonical binding partners. Our data are the first to suggest a mechanism controlling apical Crumbs localisation, which is independent of its conserved FERM- and PDZ-domain binding motifs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Crb, but not the Crb complex members Sdt and DPATJ, is enriched in the BCs of the embryonic hindgut.
(A–D″) Confocal microscope images of stage 15 wild-type embryos. (A) Dorsal view of a whole mount embryo stained with anti-Crb, showing apical localisation in the PCs (blue arrowhead) and strong enrichment in the BCs (white arrowhead) of the hindgut (hg). Anterior is left. (B) Cross section through the large intestine stained with anti-Crb to show strong apical accumulation of Crb in the BCs (white arrowheads) and SAR localisation in the PCs (blue arrowhead). (C–D″) Confocal microscope images of embryonic hindguts stained with anti-Crb (magenta in C, C″, D and D″), anti-Sdt (grey in C′ and C″) and anti-DPATJ (grey in D′ and D″) as well as anti-Dlg (green in C″ and D″). BCs (white arrowheads) accumulate Crb (C, C″, D, D″) but not Sdt (C′ and C″) or DPATJ (D′, and D″). PCs (C–D″, blue arrowheads) localise Crb (C, C″, D, D″), Sdt (C′ and C″) and DPATJ (D′ and D″) in the SAR. (E) Box Plot showing the fluorescence intensity of anti-Crb staining in PCs and BCs of stage 15 wild-type embryos. The line within the box represents the median value; the whiskers represent the maximum and minimum values; *** indicate p-value <0.001, assessed by two-sided Student's t-test.(F, G) Box Plot showing the Pearson's correlation coefficient of Crb and Sdt, DPATJ, DLin-7, Baz, DPar6, aPKC and Dlg in PCs (F) and BCs (G) of stage 15 wild-type embryos. The line within the box represents the median value; the whiskers represent the maximum and minimum values. Note the difference in the scale of the Pearson's correlation coefficient in F and G.
Figure 2
Figure 2. Localisation of apical and baso-lateral polarity proteins is not altered in BCs.
Confocal microscope images of stage 15 wild-type embryonic hindguts (BCs: white arrowheads, PCs: blue arrowheads). Anterior is left. (A–A″) Hindgut stained for the polarity markers Crb (magenta), Baz (grey) and Dlg (green). Only Crb is upregulated in the BCs (A and A″), while Baz (A′ and A″) and Dlg (A″) show the same amount and localisation as in the PCs. (B–B″) Hindgut stained for the polarity markers Crb (magenta) and DPar6 (green). Crb is enriched in the BCs (B and B″) but DPar6 localises only to the SAR as in the PCs (B′ and B″).
Figure 3
Figure 3. Apical localisation of Crb in BCs is independent of its known interaction partner Sdt.
Confocal microscopy images of a cross section through the large intestine of a stage 15 homozygous sdtK85 mutant embryo stained with anti-Crb (magenta), anti-α-Spectrin (grey) and anti-Sas (green). Crb is upregulated and apically localised in the BCs (white arrowheads), but is not detectable in the SAR of the PCs (A, A″). Sas, an apical marker of the monolayered epithelial tube, is reduced in the BCs (A′, A″).
Figure 4
Figure 4. Localisation of Crb in BCs is independent from its known protein binding motifs.
(A) Schematic representation of the Crb protein and its variants used in this study. Green rectangles: EGF-like repeats, brown hexagons: laminin A G like domains, grey bar: transmembrane domain (TM). (B) Amino acid sequences of the cytoplasmic tails of wild-type and mutant Crb proteins used in this study. Blue: FERM domain-binding motif, brown: PDZ domain-binding motif. Red: point mutations. (C–D″) Confocal microscopy images of cross sections through the large intestine of stage 15 homozygous crb8F105 (C–C″) and foscrbY10A,ΔERLI (D–D″) embryos stained with anti-Crb (magenta), anti-α-Spectrin (grey) and anti-Sas (green). Crb is upregulated and apically localised in the BCs (white arrowheads), but is not detectable in the PCs.
Figure 5
Figure 5. Loss of Crb from the BCs alters the apical membrane structure.
(A–B″) Electron micrographs of cross sections through the large intestines of stage 16 wild-type (A–A″) and homozygous mutant crb11A22 (B–B″) embryos. In A and B, BCs are outlined by red lines, the rectangles indicate areas enlarged in A′, A″, B′ and B″. White arrowheads in A′, A″, B′ and B″ point to the adherens junctions between the BCs and PCs. BCs form longer and more regular microvilli than the PCs in wild-type (A–A″) and homozygous crb11A22 mutant embryos (B–B″). (C) Graph showing the mean length of microvilli in the BCs of stage 16 wild-type and crb11A22 mutant embryos ± standard deviation. s refers to the number of embryos analysed; n refers to the number of microvilli analysed. ***indicate p-value <0.001, assessed by two-sided Student's t-test.
Figure 6
Figure 6. Crb overexpression in PCs leads to an expansion of the apical membrane domain.
(A) Schematic representation of the large intestine with the dorsal domain (dd) in orange, the ventral domain (vd) in blue and the BCs in green. (B–B″) Confocal microscope images of a stage 15 embryonic hindgut expressing UAS-crbfull under the control of en-GAL4 in the PCs of the dorsal domain. The hindgut is stained with anti-Crb (magenta), anti-DPar6 (grey) and anti-Dlg (green). The insets show higher magnifications of the vd (outlined by the grey dotted line), which serves as control tissue and the dd (outlined by the blue dotted line) where the altered apico-basal polarity in cells overexpressing Crb is highlighted (due to the very strong overexpression of Crb in the dd, the gain of the microscope was strongly reduced).
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This work was supported by the Max-Planck Society (MPG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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