JCS017137 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. 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.

• Supplemental Figure S2 - 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 DLG-1 along the lateral junction via LET-413. In addition, the SH3 domain is important during early elongation. The GUK domain is required to fully rescue DLG-1 function.

• Movie 1A
• Movie 1B -

  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 2A
• Movie 2B -

  Movies 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 3A
• Movie 3B -

  Movies 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 3A) or dlg-1(ok318) mutant embryos (B; compare with Movie 3A).

• Movie 4A
• Movie 4B -

  Movies 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 5A
• Movie 5B -

  Movies 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 3B). 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 3B). Thus, LET-413 and the SH3 domain likely function in a linear pathway.

• Movie 6 -

  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. 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.