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Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP163, 1, rue Laurent Fries, 67404 Illkirch, France

View larger version (88K): [in a new window]   Fig. 1. dlg-1(RNAi) embryos show abnormal morphogenesis and epithelial defects. (A-D) Nomarski interference microscopy of wild-type and dlg-1(RNAi) embryos. (E-H) Confocal images showing immunostaining with the monoclonal antibody MH27, which recognises CeAJs in the epidermis, pharynx (arrows) and intestine (arrowheads). (C,E,G) External focal plane showing the epidermis. (A,B,D,F,H) Internal focal plane showing the pharynx and intestine. (A,E,F) 1.5-fold stage wild-type embryos (6-7 hours development). (B) Wild-type pretzel stage embryo (9-10 hours development). (C,G,H) dlg-1(RNAi) embryos at 6-7 hours development. (D) dlg-1(RNAi) embryos at 9-10 hours development. In dlg-1(RNAi) embryos, CeAJ staining is punctate and worsens with age; epidermal CeAJs are more abnormal than in the pharynx or intestine. In C and D, white arrows indicate epithelial vacuoles. Also in C a black arrowhead indicates abnormal bulges at the embryo surface and in D a white arrowhead indicates loose cells that have leaked out of the embryo. In this and all following figures unless otherwise stated, the embryos are orientated with the anterior to the left and dorsal to the top of the image. Scale bar,10 µm.

View larger version (59K): [in a new window]   Fig. 2. DLG-1, a member of the MAGUK family of proteins, is localised at the subapical membrane of epithelial cells. (Ai) The genomic structure of the dlg-1 gene is shown underneath the scale bar, which corresponds to the coordinates in cosmid C25F6. White boxes correspond to the 12 coding exons and grey boxes at either end of the gene to the 5' and 3' untranslated regions (UTR), respectively. Note that the ATG is 291 nucleotides downstream of the previous ACeDB prediction, that exon 12 is 86 nucleotides longer at the 5'end than the previous ACeDB prediction, and that the stop site is 766 nucleotides upstream of the previous ACeDB prediction. Large black arrows show the localisation of primers used to generate a long-range PCR product that was tagged with a green fluorescent protein (GFP) cDNA. Arrowheads and small arrows show the localisation of T3-tagged primers used to generate dsRNA, against coding and 3'UTR sequence, respectively, for RNA interference (RNAi). (Aii) Schematic structure of the C. elegans DLG-1 protein and homologous proteins, Drosophila Lethal(1)Discs Large (accession no: P31007) and human presynaptic protein hDlg/SAP97 (accession no: NP_004078). Each protein possesses three PDZ domains (red boxes), 1 SH3 (green box) and 1 GUK (blue box) domain; percentages of amino acid identity/similarity between the individual domains of DLG-1 and its homologues are shown above each domain. The vertical black arrowhead indicates the position of GFP fusion. (B,C) Fluorescence and Nomarski views of uninjected and dlg-1(RNAi) dlg-1::gfp transgenic embryos demonstrating the strong reduction in DLG-1::GFP expression after injection with dsRNA against dlg-1. Both fluorescent images were taken using the same exposure time (1 second) after 8 hours of development. Confocal images showing an external (D) and internal (E) focal plane of a 1.5-fold dlg-1::gfp transgenic embryo after immunostaining with anti-GFP antibody. Weak background fluorescence could often be seen around the pharyngeal lumen and rectum (arrowheads in E; these expressing cells have not been identified). (F-H) GFP fluorescence of a young dlg-1::gfp transgenic adult showing expression in epithelial cells of the pharynx (arrow in F), intestine (arrowhead in F and large arrow in H), vulva (large arrow in G), uterus (arrowheads in G), spermatheca (long arrows in G), rectum (long arrow in H) and epidermis (arrowhead in H). Scale bars, 50 µm (B,C,F-H); 10 µm (D,E).

View larger version (35K): [in a new window]   Fig. 3. Apico-basal polarity is affected in let-413(RNAi) but not dlg-1(RNAi) embryos. (A-F) Confocal analysis showing the internal focal plane of embryos after immunostaining with anti-PAR-3 (A-C) or anti-PAR-6 (D-F) antibodies alone, or along with anti-HMP-1 antibodies (A'-C' and D'-F', respectively). (A'-F') Regions within the dotted box in A-F, respectively, at over 2x magnification. (G,H) Most external focal plane, (I) more internal focal plane within epidermis, showing GFP fluorescence of che-14::gfp transgenic embryos. (A,D,G) Wild-type embryos; (B,E,H) dlg-1(RNAi) embryos; (C,F,I) let-413(RNAi) embryos. In this and all following figures, dlg will refer to dlg-1(RNAi) embryos and let-413 will indicate let-413(RNAi) embryos. Apical markers of the epidermis and intestine are mislocalised in let-413(RNAi) embryos while the same markers remain apical to or coincidental with HMP-1 in dlg-1(RNAi) embryos; note that there are regions of lateral CHE-14::GFP expression in dlg-1(RNAi) embryos (arrows in H) and that CHE-14::GFP was not visible in the most external focal plane of let-413(RNAi) embryos. The intestinal lumen is wider in dlg-1(RNAi) embryos than in wild-type embryos (compare A,D with B,E). (J) Diagram illustrating the general localisation of PAR-3, PAR-6 and CHE-14 (green) in a hypothetical epithelial cell in wild-type, dlg-1(RNAi) and let-413(RNAi) embryos. Scale bars, 10 µm (A-I); 5 µm (A'-F').

View larger version (186K): [in a new window]   Fig. 4. DLG-1 is necessary for the assembly of the CeAJ electron-dense structure. Electron microscopic analysis of CeAJs in the epidermis (A-E) and intestine (F-I) of wild-type (A,F), dlg-1(RNAi) (B,C,G,H), let-413(RNAi) (D,I) and double dlg-1(RNAi) let-413(RNAi) embryos (E). The electron-dense structure of CeAJs (arrowhead in A) was absent in most epidermal cells of dlg-1(RNAi) embryos (see text), while neighbouring cell membranes remained correctly apposed (B); where this structure was present it was slightly more elongated than in wild-type embryos (arrowhead in C). Where epidermal junctions are present in let-413(RNAi) embryos, they are discontinuous and largely extended further basally along the lateral membrane (arrowheads in D). In double dlg-1(RNAi) let-413(RNAi) embryos, the basolaterally mislocalised electron-dense junctions are no longer detectable (E). In the intestine of dlg-1(RNAi) embryo (G,H), CeAJs are also abnormal compared to wild-type (arrowheads in F) but not affected as badly as in the epidermis. In many cases, the electron-dense junctions are clearly visible (arrowhead in G) while others are absent or faint (arrows in G and H). (I) Shows a typical intestinal section in let-413(RNAi) embryos where both CeAJs are present (arrow and arrowhead), although one is elongated with respect to wild-type junctions (arrowhead). Scale bar, 300 nm.

View larger version (69K): [in a new window]   Fig. 5. CeAJ proteins are spread along the lateral membrane in differentiating epidermal cells of wild-type embryos. (A) Diagram of an early wild-type embryo (at the time of epidermal differentiation) showing a dorsal view representing embryos in images B-E. (A') Schematic transverse section across an epidermal cell corresponding to the blue bracket in A and representing images B'-E'. (B-E) Confocal analysis showing external focal plane projections of wild-type (B,D) and dlg-1::gfp transgenic (C,E) embryos at a stage of development prior to (B), and coincidental with (C-E), epidermal cell differentiation, after immunostaining with anti-HMP-1 (B,C), anti-LIN-26 (B), MH27 (anti-JAM-1) (D,E), anti-GFP (C,E) and anti-UNC-70 (D) antibodies and DAPI (D). UNC-70 is used here and elsewhere as a general membrane marker. (B'-E') Z-axis rotations of a single transverse slice through a section of the embryo as denoted by the corresponding lines in embryos B-E, showing CeAJ markers distributed along the lateral membranes. Apical is to the right in all Z-axis rotations here and elsewhere. (C') Reveals the mutually exclusive nature of DLG-1 and HMP-1 localisation and (E') illustrates that DLG-1 and JAM-1 exhibit complete co-localisation. Asterisks in B' and D' indicate the position of epidermal nuclei. (F) A cartoon showing the position of membrane-associated proteins in immature and mature epidermal cells. CeAJ proteins are positioned along the membrane in immature epidermal cells and relocate to a compact subapical junction as the cells mature (see Fig. 6). Scale bars, 10 µm.

View larger version (46K): [in a new window]   Fig. 6. Epidermal CeAJs are heterogeneous structures in wild-type embryos. (A) Schematic diagram of part of an embryo at the beginning of elongation indicating the positions of the projected transverse sections (blue and tan brackets) displayed in (B) and in Fig. 7C. (C,D) Z-axis rotations of transverse sections through the epidermis along the membrane of two connecting epidermal cells where the CeAJ is visible as a subapical belt, as illustrated in B. In B-D, asterisks denote epithelial nuclei on either side of the membrane in question. (C) dlg-1::gfp transgenic embryo after immunostaining with MH27 (anti-JAM-1), anti-GFP and anti-LIN-26 antibodies; (D) jam-1::gfp transgenic embryo after immunostaining with anti-HMP-1, anti-GFP and anti-LIN-26 antibodies. DLG-1 and JAM-1 appear to colocalise completely (C) while HMP-1 and JAM-1 appear to be mutually exclusive in their localisation (D; yellow regions may result from limited resolution). (E-G) Wild-type, dlg-1(RNAi) and let-413(RNAi) embryos, respectively, at 8-11 hours development after immunostaining with MH27. (H-J) Wild-type, dlg-1(RNAi) and let-413(RNAi) embryos, respectively, at 8-11 hours development after immunostaining with anti-HMP-1 antibodies. It can be seen that the JAM-1 belt around epidermal cells can be altered independently of the HMP-1 belt. Scale bars, 5 µm (C,D); 10 µm (E-J).

View larger version (52K): [in a new window]   Fig. 7. CeAJ proteins are mislocalised differently in epidermal cells of let-413(RNAi) and dlg-1(RNAi) embryos. Embryos were stained with anti-HMP-1 or anti-JAM-1 along with anti-UNC-70 antibodies and DAPI. Confocal sections were captured and analysed as described in Materials and Methods. (A) Bar graph showing the spread of CeAJ proteins (JAM-1, pink bars, and HMP-1, orange bars) along the lateral membrane of epidermal cells in mature wild-type (mWT), dlg-1(RNAi), let-413(RNAi) and early wild-type (eWT) embryos. Values are means ± s.e.m. (B) Bar graphs showing the position of the same CeAJ proteins along the lateral membranes of epidermal cells. The graphs show the percentage of membranes where JAM-1 or HMP-1 were detected at the top (T), middle (M) or bottom (B) of the lateral membrane (see C). Proteins were often distributed over more than one membrane section and so the total percentage for each category is greater than 100%. Note that the spread and position of HMP-1 was not determined in early wild-type embryos (ND in graphs). The numbers of lateral membranes scored for each set of embryos were as follows. Wild-type: 71 (JAM-1), 88 (HMP-1); dlg-1(RNAi): 57 (JAM-1), 80 (HMP-1); let-413(RNAi): 78 (JAM-1), 85 (HMP-1); early wild-type: 85 (JAM-1). (C) Schematic diagram of a transverse section through the mature epidermal layer of a wild-type embryo (see tan bracket, Fig. 6A), indicating how a lateral membrane was sectioned to score protein position. Nuclei are shown in blue, CeAJs in pink and lateral membranes in green. (D-I) Z-axis rotations showing transverse sections through the epidermal layer of mature wild-type (D,G), dlg-1(RNAi) (E, H), and let-413(RNAi) (F,I) embryos after immunostaining with MH27 (red) (D-F) or anti-HMP-1 (red) (G-I), along with anti-UNC-70 (green) antibodies and DAPI (blue). The basal extent of UNC-70 denotes the position of the epidermal basal membrane and the nucleus of the cell under investigation is indicated with an asterisk, as in C. (J,K) Internal view of a let-413(RNAi) dlg-1::gfp transgenic embryo after immunostaining with anti-GFP antibody alone (J), and both anti-GFP and MH27 antibodies (K), showing that DLG-1 is mislocalised in the absence of LET-413 but remains colocalised with JAM-1. (L,M) Internal view of a wild-type (L) and a dlg-1(RNAi) (M) let-413::gfp transgenic embryo after immunostaining with an anti-GFP antibody, illustrating that LET-413 is not mislocalised in the absence of DLG-1. Scale bars, 5 µm (D-I); 10 µm (J-M).

View larger version (36K): [in a new window]   Fig. 8. A model for CeAJ assembly and composition in the epidermis. Cartoons showing the localisation of HMP-1 (blue), JAM-1 (green), DLG-1 (red) and LET-413 (purple) proteins in epidermal cells. (A) An immature wild-type cell showing that HMP-1 and DLG-1/JAM-1 exhibit a mutually exclusive distribution along the lateral membrane. (B) A mature wild-type cell illustrating a compact subapical junction consisting of a JAM-1/DLG-1 unit associated with the electron-dense component, and a HMP-1 unit, which is generally localised more apically than JAM-1/DLG-1 and is always distinct. Arrows from the CeAJ domains indicate their possible functions. (C) A LET-413-deficient cell showing mislocalisation of CeAJ proteins. This suggests that LET-413 plays a role in compacting and positioning CeAJ components to form mature CeAJs. (D) A DLG-1-deficient cell, showing that JAM-1 exhibits a discontinuous pattern around the cell and is mislocalised slightly further basally where present. The electron-dense CeAJ component is also missing in these cells, which suggests that DLG-1 aggregates JAM-1 and other proteins at the CeAJ.