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Cooperative regulation of AJM-1 by Discs large and LET-413 controls junctional integrity of Caenorhabditis elegans epithelia

1Mathias Köppen, 1Jeffrey S. Simske, 1Paul A. Sims, 2Bonnie L. Firestein, 3David H. Hall, 4Anthony D. Radice, 5Christopher Rongo and 1*Jeffrey D. Hardin

1 Department of Zoology, University of Wisconsin-Madison, 1117 W. Johnson Street, Madison, Wisconsin 53706
2 Department of Cell Biology and Neuroscience, Department of Genetics, Rutgers University, Piscataway, NJ 08854
3 Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine,1410 Pelham Parkway, Bronx, New York 10461
4 Department of Stem Cell Biology, Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, New York 10021 5
The Waksman Institute, Rutgers University, Piscataway, NJ 08854

Supplemental material for the following paper:
Köppen, M., Simske, J.S., Sims, P.A., Firestein, B.L., Hall, D.H., Radice, A.D., Rongo, C., and Hardin, J.D. (2001). Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nat Cell Biol 3, 983-991. PubMed

Summary
The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighboring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR/HMP (cadherin/catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, while LET-413 mediates rapid accumulation of both DLG-1 and AJM-1 to the apical domain. Loss of both dlg-1 and let-413 function results in almost complete loss of AJM-1 from apical junctions in embryos, while HMP-1/a-catenin localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical subdomain of epithelial junctions in C. elegans.

Supplementary Data
Supplementary figure (AJM_POL.JPG): Markers of apicobasal polarity are correctly expressed in ajm-1 mutants
Description: Analysis of apical and basal markers in ajm-1(ok160) embryos. Analysis of the patterns of HMP-1/a-catenin, (G-spectrin, microfilaments and body wall muscle in an ajm-1(ok160) and wild-type embryo. a-d, ajm-1(ok160). HMP-1::GFP forms a continuous apical pattern in hypodermal cells (a), while anti-(G-spectrin immunostaining labels the lateral hypodermal junctions (b). anti-PAR-3 staining marks the apical junctions of the pharynx and intestine (c). Phalloidin staining reveals microfilaments that are circumferentially aligned and a body wall muscle quadrant that is positioned along the anterior-posterior body axix of the embryo. e-h, Wildtype. In each case, the pattern shows no significant difference compared to that of the ajm-1(ok160) embryo. Scale bar represents 10 um.

Movie #1 (AJM_LET.MOV): Dynamic analysis of AJM-1::GFP in a let-413(RNAi) and wild-type embryo
Description: A let-413(RNAi) embryo (on the left) and a similarly staged wild-type embryo (on the right) expressing ajm-1::gfp were analyzed simultaneously. Frames were acquired at 5 min intervals; ventral views, anterior is to the left. At the onset of ventral enclosure, the let-413 embryo shows a reduced and punctate junctional AJM-1::GFP pattern, while the pattern in the wild-type embryo is even and continuous. After enclosure, during the process of elongation, additional AJM-1::GFP accumulates in the junctions of the let-413 embryo, resulting in a significantly more continuous pattern. In the wild-type embryo, the AJM-1::GFP pattern remains even and continuous throughout development. This indicates a delay in junctional accumulation of AJM-1::GFP in the absence of let-413 function. The let-413 embryo arrests after the 1.5-fold stage, while the wild-type embryo elongates past the 3-fold stage.

Movie #2 (DLG_WT.MOV): Dynamic analysis of DLG-1::GFP in a wild-type embryo
Description: A wild-type embryo expressing dlg-1::gfp is shown during ventral enclosure and subsequent elongation past the 1.5-fold stage. Frames were acquired at 5 min intervals; ventral view, anterior is to the left. DLG-1::GFP forms an even, continuous pattern at all stages.

Movie #3 (DLG_LET.MOV): Dynamic analysis of DLG-1::GFP in a let-413(RNAi) embryo
Description: A let-413(RNAi) embryo expressing dlg-1::gfp was analyzed during ventral enclosure and subsequent elongation past the 1.5-fold stage. Frames were acquired at 5 min intervals; ventral view, anterior is to the left. At the onset of ventral enclosure, the let-413 embryo shows a reduced and punctate junctional DLG-1::GFP pattern. After enclosure, during the process of elongation, additional DLG-1-GFP accumulates in the junctions of the let-413 embryo resulting in a significantly more continuous pattern. This indicates a delay in junctional accumulation of DLG-1::GFP in the junctions in the absence of let-413 function.