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First published online 18 January 2005
doi: 10.1242/jcs.01619

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Papillote and Piopio: Drosophila ZP-domain proteins required for cell adhesion to the apical extracellular matrix and microtubule organization

Christian Bökel^1,*, Andreas Prokop^2, and Nicholas H. Brown^1,

¹ Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
² Institute of Genetics, University of Mainz, J.-J.-Becherweg 32, 55128 Mainz, Germany

View larger version (39K): [in a new window]   Fig. 1. The wing-blister genes piopio and papillote encode ZP-domain-containing transmembrane proteins. The genes piopio (pio, A) and papillote (pot, B) are shown with their closely flanking genes and their encoded proteins. Untranslated regions are white-filled boxes and coding regions are black. Genomic rescue constructs were generated with the segment of DNA between the restriction sites: BamHI for piopio and SpeI and EclXI for papillote. The position of the 1.7 kb deletion in the pioV132 mutant allele, which removes the signal peptide, is shown in A. The schematic of the proteins show signal peptides (SP) and transmembrane domains (TM) as black boxes, the zona pellucida (ZP) domains as grey boxes, and the insertion points of the fluorescent protein tags (GFP and CFP). For Piopio, the sequence surrounding the potential furin cleavage site (underlined) at the end of the ZP domain is shown, as is the conservation of the short cytoplasmic domain, with identical residues shaded. For Papillote, the position of the frame shift caused by the 13 bp deletion in the potP53 mutant allele, which causes a truncation of the protein after residue 686, is indicated. (C) Pio but not Pot is proteolytically processed. Extracts of pupal wings from wild type (wt) or animals expressing GFP, Pio-GFP or Pot-CFP were analysed by western blotting and probed with an anti-GFP antibody. Two distinct bands were detected for the Pio-GFP fusion protein, with apparent molecular weights as predicted for the full-length fusion protein lacking the signal peptide (76 kDa), and a C-terminal fragment fused to GFP generated by cleavage at the potential furin recognition site (39 kDa), distinct from GFP alone (27 kDa). By contrast, a single band was observed for Pot-CFP of the size expected for the full-length fusion protein (131 kDa).

View larger version (88K): [in a new window]   Fig. 2. Subcellular localization of Pio and Pot. Distribution of fluorescent fusion proteins in late pupal wing (>70 hours after puparium formation), visualized by confocal microscopy. (A) Owing to the folding of the wing, this confocal section shows the localization of Pot-CFP around the circumference of the stellate apical surface (top) and an optical section through the two attached layer of cells (a cell in each layer is marked with a yellow arrow, with the arrowhead basal) (bottom). (bottom) Pot-CFP is uniformly localized along the apical surface (but not including the wing hair). (B,D) By contrast, at the same stage, a Pio-GFP fusion protein (green) is found in discrete dot-like structures, preferentially at the apical side (D) and near the circumference of the wing epithelial cells as visualized by differential interference contrast (right). (C) Tubulin-GFP shows broader but still discrete points of contact with the apical surface, also enriched at the cell periphery. The outline of one cell is drawn in white in B and C. (E) The basal position of integrin adhesive junctions is shown with integrin-linked kinase (ILK-GFP). (F) Schematic diagram showing the sections of the wing epithelia shown in A-E. Bar, 10 µm (applies to A-E).

View larger version (158K): [in a new window]   Fig. 3. Pio, but not other wing-blister gene products, is required for the transalar arrays of microtubule bundles in pupal wings. The distribution of tubulin in late pupal wings (60-70 hours after puparium formation) is shown in white or green in each panel except B, which shows an adult wing. All cells express tubulin-GFP in A,C-E. In a confocal section of a wild-type folded pupal wing (A), the prominent microtubule bundles spanning the wing cells can be seen. Pairs of attached cells are marked with yellow arrows, with the arrowheads basal. (B-E) Clones of mutant cells are marked with a mutation in shavenoid (sha), which disrupts the formation of wing hairs but does not cause blistering, as shown in a bright-field micrograph of the adult wing (B). In the absence of Pio, the microtubule bundles are not seen: (C) a section across the apical surface, combined with differential interference contrast, showing the absence of microtubules and wing hairs in the sha pio mutant clone; (D) the absence of the microtubule bundles in sha pio mutant cells (indicated with a white horizontal bracket); opposing wild-type cells also appear overcontracted. The microtubule-binding protein Shot, which localizes to both ends of the microtubule bundles as detected by antibody staining (F), is not required to maintain the bundles (E); this image combines tubulin-GFP fluorescence and differential interference contrast, and the white horizontal bracket shows the cells lacking Shot (sha shot). (G-I) The MARCM technique was used to express tubulin-GFP in just the mutant clones of cells. A control wild-type clone shows microtubule bundles in one layer of the wing (G). Clones of cells lacking Pot (H) or the integrin ßPS subunit (I, mys) contain microtubule bundles. These bundles are disordered in the absence of integrins, presumably owing to the separation between the two cell layers (the other layer is not visible because it does not contain a clone). Bars, 10 µm.

View larger version (143K): [in a new window]   Fig. 4. The absence of Pio causes cuticle detachment and tracheal defects. (A) In wild-type embryos at the end of embryogenesis, viewed by differential interference contrast, the dorsal trunk of the trachea appears smooth and rather straight. (B) Homozygous pio mutant embryos complete development but the tracheae appear twisted and broken (arrows). (C) In live wild-type embryos, the epidermis (marked with tubulin-GFP) closely follows the outline of the cuticle (viewed by differential interference contrast). (D) In homozygous pio mutant embryos, the cuticle detaches from the GFP-marked epidermis (arrow).

View larger version (101K): [in a new window]   Fig. 5. Ultrastructural analysis of mutant phenotypes. Schematic representations of wild-type cuticle (A) or muscle-attachment sites (B) of late Drosophila embryos (about 21 hours) and micrographs of these structures in wild-type and mutant embryos (C-F"). Symbols and abbreviations are used consistently throughout: aj or white arrow, apical junctions; at or curved white arrows, apical transverse filaments; ch, chitin layer; cu, trilaminar cuticulin layer; dz or black arrowheads, deposition zone; ed, epidermal cell; el, electron-lucent layer; en or asterisk, endocuticle; ep or black dot, epicuticle; es, extracellular space; mt or white arrowhead, microtubules; mtj or curved black arrow, myotendinous junction; mu, muscle; nu, nucleus; pe, fibrillar protein epicuticle; tf or white chevron, tonofilament; tn, tendon cell. [Nomenclature according to Kaznowski et al. (Kaznowski et al., 1985) and Tepass and Hartenstein (Tepass and Hartenstein, 1994).] The characteristic arrangement of the epicuticle (black dots) and the typical occurrence of the darker cuticular deposition zone (dz), which associates with epidermal microvilli (mv), can be seen in wild-type (A-C"), pioV132 (D,D'), potP14 (E,E') and dp1v1 (F,F') mutant embryos alike. Typical features of epidermal tendon cells (tn) are their pronounced basal junctions with muscles (curved black arrows) and apical junctions (white arrows) adhering to tonofilaments (white chevrons) that anchor in the cuticle. Apical and basal junctions of tendon cells are intracellularly connected via microtubules (white arrowheads). These characteristic features of wild-type embryos (B,C") seem unaffected in potP14, pioV132 and dp1v1 mutant embryos (D"-F"), although they are difficult to find and interpret owing to epidermal detachment from the cuticle. The main defect of potP14, pioV132 and dp1v1 mutant embryos consists in a disruption of the chitin layer (ch) of the endocuticle (asterisks), leading to a separation of deposition zone and epicuticle (D-F,D'-F'), and failure of tonofilaments to anchor in the cuticle (D",F", white chevrons). Bar, 0.5 µm (left and right columns, C-F,C"-F"), 0.2 µm (middle column, C'-F').

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