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First published online 17 July 2007
doi: 10.1242/jcs.03474

Journal of Cell Science 120, 2741-2751 (2007)
Published by The Company of Biologists 2007
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C. elegans Disabled is required for cell-type specific endocytosis and is essential in animals lacking the AP-3 adaptor complex

Alex Holmes1, Aileen Flett1, Damien Coudreuse2, Hendrik C. Korswagen2 and Jonathan Pettitt1,*

1 Cell and Developmental Biology Program, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
2 Hubrecht Lab/NIOB, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands


Figure 1
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  		Fig. 1. dab-1 loss-of-function mutations. (A) Schematic representation of the dab-1 gene showing the two isoforms predicted by expressed sequence tag sequences; one of these isoforms is spliced to the SL1 trans-spliced leader RNA. The region deleted by the gk291 mutation is indicated. Exons are represented by numbered boxes, and splicing patterns by diagonal lines. (B) Schematic of the predicted longer DAB-1 isoform (the two isoforms differ only in their extreme N-termini) showing the regions affected by the two dab-1 mutations. The phosphotyrosine binding domain is indicated by the grey box, and the positions of putative adaptor protein and Eps15 homology (EH) domain binding sites are shown: DPF and NPF, respectively. The horizontal line indicates the extent of the isoform-specific region of the N-terminus. (C) Western blot of wild-type (N2), dab-1(gk291)- and dab-1(hu186)-derived lysates showing absence of wild-type DAB-1 immunoreactivity in mutant worm lysates. The antibody recognises several prominent, non-specific bands in both wild-type and mutant worms, one of which is visible here (asterisk). The position of the 50 kDa marker is indicated.

 

Figure 2
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  		Fig. 2. dab-1-null mutants have defects in ecdysis, and display defects in coelomocyte uptake (Cup) and yolk protein endocytosis (Rme). (A) Representative dab-1(hu186) homozygote trapped in unshed cuticle (arrow). (B,C) Adult worms expressing GFP secreted into the pseudocoelom. Images were captured using identical exposure times and camera parameters. GFP accumulates to high levels in the body cavity of dab-1(gk291) homozygotes (B), but is greatly reduced because of uptake by the coelomocytes in dab-1(gk291) homozygotes carrying the dab-1::gfp transgene (C). (D,E) Composite DIC and GFP fluorescence (pseudocoloured green) images showing localisation of YP170::GFP by oocytes in wild-type (D) and dab-1(hu186) (E) hermaphrodites. In the wild type, oocytes accumulate yolk protein (D), whereas in dab-1(hu186) YP170::GFP accumulates in the body cavity (arrows) and is not detectable in oocytes. Asterisks indicate the spermatheca in the two images. Bars, 25 µm (A); 50 µm (B,C); 10 µm (D,E).

 

Figure 3
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  		Fig. 3. Localisation of DAB-1 and RME-2 in developing oocytes. Deconvolved image stacks showing detection of DAB-1 and RME-2 in dissected oocytes. DAB-1 and RME-2 localise to distinct structures associated with the plasma membrane of wild-type oocytes (A-F; A-C, upper focal plane; D-E, middle focal plane). Inset in C shows magnified view of the merged images. dab-1(gk291) oocytes (G-H, upper focal plane; I-J, middle focal plane) lack RME-2 structures seen in wild-type oocytes, although diffuse, membrane-associated RME-2 can still be detected (H,J). Note the lack of immunoreactivity with DAB-1 antibody (G,I). Bar, 10 µm.

 

Figure 4
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  		Fig. 4. DAB-1 localisation in chc-1(RNAi) and apa-2(RNAi) oocytes. Deconvolved image stacks of oocytes derived from DH1033 hermaphrodites, showing localisation of YP170::GFP (A,D,G,J,M,P) and DAB-1 (B,E,H,K,N,Q). Wild-type oocytes (A-F) show the characteristic DAB-1 puncta. Knockdown of chc-1(RNAi) (G-L) leads to the almost complete loss of DAB-1 puncta, however DAB-1 remains associated with the membrane. Similar results were obtained in apa-2(RNAi) oocytes (M-R). All images were collected using identical camera settings. Bar, 5 µm.

 

Figure 5
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  		Fig. 5. DAB-1 and RME-2 localisation in embryos. Deconvolved z-sections showing detection of DAB-1 at the surface (A), and RME-2 localised to internalised puncta (B) of a wild-type one-cell embryo. (C) Merged images. Wide-field images of RME-2 detected in four-cell (upper) and one-cell (lower) embryos derived from wild-type (D) and dab-1 mutant (E) animals. Bar, 10 µm.

 

Figure 6
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  		Fig. 6. DAB-1 colocalises with the clathrin light chain in coelomocytes. Confocal images of a coelomocyte expressing DAB-1::GFP (A,D) and CLIC-1::mDsRed (B,E). Upper panels (A-C) show a focal plane at the level of the coelomocyte cortex; lower panels (D-F) show a focal plane through the middle of the coelomocyte. Bar, 5 µm.

 

Figure 7
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  		Fig. 7. DAB-1::GFP is associated with puncta at the apical membrane of epithelial cells. (A) DAB-1::GFP puncta are associated with the apical membrane of the syncitial epidermal cell. Double-headed arrow indicates a region largely devoid of puncta; this region has a thinner cross-section compared with the regions with high densities of DAB-1::GFP puncta. The lateral epidermis, which is also devoid of DAB-1::GFP puncta, is indicated by a bracket symbol. (B) The intestinal cells show puncta associated with their basolateral surfaces. (C) The apical surface (arrowhead) of the pharynx displays prominent DAB-1::GFP puncta, which are absent from the basolateral surface (arrow). (D) The excretory pore cell, showing enrichment of DAB-1::GFP and associated puncta along its apical surface (which forms part of the lumen of the excretory duct) (arrow). Images are oriented with anterior to the left (A-D). Bar, 10 µm.

 

Figure 8
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  		Fig. 8. DAB-1::GFP puncta are highly dynamic. (A) Images taken from a time-lapse movie (supplementary material Movie 1) of a coelomocyte expressing DAB-1::GFP at two different time points. Overlay of the pseudocoloured images reveals that relatively few of the puncta visible at the two times overlap, indicating that most puncta initially present have disappeared by the time the second image was captured, and that most of the puncta in the second image have formed within the 76-second interval between images. (B) Images of the hypodermis of an adult hermaphrodite expressing DAB-1::GFP captured at the three different time points (see also supplementary material Movie 2). Overlay of the three pseudocoloured images demonstrates the existence of hotspots where puncta tend to form (arrows), although few of the puncta overlap. Time points are given in seconds. Bar, 2.5 µm (A); 5 µm (B).

 

Figure 9
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  		Fig. 9. dab-1 mutant coelomocytes display defects in endosome and lysosomes. Z-projections of deconvolved image stacks showing expression of RME-8::GFP (A,B) and GFP::CUP-5 (C,D) in the coelomocytes of wild-type (A,C) and hu186 (B,D) adult hermaphrodites. Bar, 5 µm.

 

Figure 10
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  		Fig. 10. dab-1 mutants are sensitive to loss of AP-1 and AP-3 function. DIC images showing terminal phenotypes associated with single- and double-mutant combinations. (A) dab-1(gk291) mutant showing wild-type embryonic morphogenesis and viability. (B) An apb-3(ok429) homozygous embryo, displaying characteristic `dumpy' (Dpy) morphology; arrow indicates presence of gut granules in the intestinal lumen. (C) An unc-101(sy108) embryo showing wild-type morphogenesis and viability. (D) An apb-1(ok429); dab-1(gk291) double-mutant embryo, which displays the same Dpy phenotype and gut granule defect (arrow) as ok429 single mutants. (E) An unc-101(sy108); dab-1(gk291) double-mutant embryo showing the synthetic body morphology defects seen in approximately 30% of unc-101; dab-1 double mutants. Arrowheads indicate vacuoles located at the approximate position of the excretory cell sometimes seen in the double-mutant embryos. Embryos are approximately 50 µm in length.

 

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© The Company of Biologists Ltd 2007