The Hardin Lab - AJM-1 and DLG-1

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 5The 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.