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Dynamic analysis identifies novel roles for DLG-1 subdomains in AJM-1 recruitment and LET-413 dependent apical focusing
Christopher Lockwood1,3 , Allison Lynch1, and Jeff Hardin 2,4
1Program in Genetics and 2Department of Zoology, University of Wisconsin Madison, WI 53706 USA; 3Present address: Department of Biology, Washington University, 1 Brookings, Campus Box 1229, St. Louis, Mo. 63130 USA
4Corresponding author

Supplemental material for the following paper:
Lockwood, C.A., Lynch, A.M., and Hardin, J. (2008). Dynamic analysis identifies novel roles for DLG-1 subdomains in AJM-1 recruitment and LET-413 dependent apical focusing. J. Cell Sci. 121: 1477-1487. PubMed

Supplemental figure 1
Supplemental figure 2
Video sequence 1A & B
Video sequence 2A & B
Video sequence 3A & B
Video sequence 4A & B
Video sequence 5A & B
Video sequence 6

Cell-cell junctions are composed of a diverse array of specialized proteins that are necessary for the movement and integrity of epithelia. Scaffolding molecules, such as Membrane Associated GUanylate Kinases (MAGUKs) contain multiple protein-protein interaction domains that integrate these proteins into macromolecular complexes at junctions. We have utilized structure-function experiments to dissect the role of domains of the C. elegans MAGUK DLG-1, a homologue of Drosophila Discs large and vertebrate SAP-97. DLG-1 deletion constructs were analyzed in directed yeast two-hybrid tests as well as in vivo in a dlg-1 null mutant background. Our studies identify novel roles for several key domains. First, the L27 domain of DLG-1 mediates the physical interaction of DLG-1 with its binding partner AJM-1, as well as DLG-1 multimerization. Second, the PDZ domains of DLG-1 mediate its association with the junction. Third, through the use of dynamic in vivo imaging we demonstrate that the SH3 domain is required for rapid lateral distribution of DLG-1 via a LET-413/Scribble-dependent pathway. Finally, we find that inclusion of the SH3 domain can ameliorate dlg-1 mutant phenotypes, but full rescue of lethality required the complete C terminus, that includes the GUK and Hook domains, thereby demonstrating its importance for DLG-1 function. Our results represent the first in vivo analysis of requirements for the L27 domain of a Discs large/SAP-97 protein, identify a crucial LET-413/Scribble regulatory motif, and provide insight into how MAGUK subdomains function to maintain epithelial integrity during development.

Supplemental Figures
Supplemental Figure 1. Fragments possessing multiple PDZ domains show increased integration at the apical junction.
(A-C) Localization of DLG-1 deletion fragments containing one or more PDZ domains in dlg-1(ok318) homozygous embryos. Fragments truncated after PDZ1, PDZ2, and PDZ3 all displayed slowed rates of lateral distribution in dlg-1(ok318) mutants, but showed varying amounts of cytoplasmic staining and early puncta, depending on the number of PDZ domains. (A) DLG-1(1-302)::GFP displayed the most cytoplasmic localization and the most severe punctate localization. (B) DLG-1(1-468)::GFP displayed a moderate amount of cytoplasmic localization and punctate distribution. (C) DLG-1(1-710)::GFP displayed no detectable cytoplasmic localization and the least severe punctate localization. Scale bar, 10 µm.
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Supplemental Figure 2. Model of structure-function results.
Schematic of DLG-1 domain functions. The L27 domain of DLG-1 (1) mediates a physical interaction with the coiled-coil region of AJM-1 and (2) facilitates DLG-1 homotypic multimerization. The PDZ domains of DLG-1 are important for junctional integration; multiple PDZ domains increase the efficiency of junctional integration. The SH3 domain is required for rapid lateral distribution of AVI movie 1 (? Mb)
Supplemental Figure 2 - JPEG (140 Kb) Download

Supplemental Movies
Anterior is to the left, dorsal up in all movies. All movies are projections of 4-d data sets collected at 5 minute intervals. Movies are displayed at 7 frames per second.

Supplemental movies 1A and B. The N terminus of DLG-1 is insufficient for localization in dlg-1 mutants.
(A) DLG-1(1-186)::GFP expression in a wild-type embryo. DLG-1(1-186)::GFP displays wild type localization in the presence of endogenous DLG-1. (B) In dlg-1 mutants, DLG-1(1-186)::GFP is mislocalized into puncta along the junctional belt.
movie 1A (0.4 Mb) Download AVI | Download MP4
movie 1B (0.4 Mb) Download AVI | Download MP4

Supplementary Movie 2A and B. The PDZ domains of DLG-1 allow for junctional association, but deletion fragments display slower rates of lateral distribution.
(A) DLG-1(1-468)::GFP localizes normally in wild type animals. (B) DLG-1(1-468)::GFP shows slowed rates of lateral distribution in dlg-1(ok318) mutants. At the beginning of elongation DLG-1(1-468)::GFP expression is punctate, but as elongation progresses the GFP expression becomes more contiguous.
movie 2A (0.4 Mb) Download AVI | Download MP4
movie 2B (0.4 Mb) Download AVI | Download MP4
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Supplementary Movie 3A and B. Lateral distribution of DLG-1 does not require AJM-1.
Removal of AJM-1 via RNAi does not affect the localization of DLG-1(1-468)::GFP in wild type (A; compare with movie 2A) or dlg-1(ok318) mutant embryos (B; compare with movie 2A).
movie 3A (0.6 Mb) Download AVI | Download MP4
movie 3B (0.6 Mb) Download AVI | Download MP4

Supplementary Movie 4A and B. The SH3 domain can rescue lateral distribution defects.
DLG-1(1-710)::GFP expression restores wild type rates of lateral distribution displaying similar rates in wild type (A) and dlg-1 mutant embryos (B).
movie 4A (0.4 Mb) Download AVI | Download MP4
movie 4B (0.3 Mb) Download AVI | Download MP4
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Supplementary Movie 5A and B. SH3-mediated lateral distribution is dependent on LET-413 function.
(A) DLG-1(1-710)::GFP shows slowed rates of lateral distribution in let-413(RNAi) embryos similar to the rate observed for DLG-1(1-468)::GFP in dlg-1 mutants (A; compare with movie 2B). This result suggests that SH3 localization is dependent on LET-413. (B) DLG-1(1-468)::GFP localization rates are not significantly enhanced in let-413;dlg-1 double mutants compared to its expression in dlg-1 mutants alone (B; compare with movie 2B). Thus, LET-413 and the SH3 domain likely function in a linear pathway.
movie 5A (0.6 Mb) Download AVI | Download MP4
movie 5B (0.6 Mb) Download AVI | Download MP4

Supplementary Movie 6. A DLG-1 fragment lacking the L27 domain can localize apically.

DLG-1(Δ17-116)::GFP is apically localized in a dlg-1(ok318) mutant, although its distribution is slightly more punctate than in wild-type.
movie 6 (0.5 Mb) Download AVI | Download MP4

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