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

Summary
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

Summary
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
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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)

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Supplemental AVIs
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.

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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.

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AVI movie 1B (0.4 Mb) Download


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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.

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AVI movie 2B (0.4 Mb) Download

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sm3_thumbSupplementary 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).

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AVI movie 3B (0.6 Mb) Download

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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).

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AVI movie 4B (0.3 Mb) Download

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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.

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AVI movie 5B (0.6 Mb) Download


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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.

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