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First published online 14 April 2008
doi: 10.1242/jcs.017137

Journal of Cell Science 121, 1477-1487 (2008)
Published by The Company of Biologists 2008
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Dynamic analysis identifies novel roles for DLG-1 subdomains in AJM-1 recruitment and LET-413-dependent apical focusing

Christopher A. Lockwood1, Allison M. Lynch1 and Jeff Hardin1,2,*

1 Program in Genetics, University of Wisconsin-Madison, 1117 W. Johnson Street, Madison, WI 53706, USA
2 Department of Zoology, University of Wisconsin-Madison, 1117 W. Johnson Street, Madison, WI 53706, USA


Figure 1
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  		Fig. 1. The L27 domain of DLG-1 is necessary for the AJM-1–DLG-1 physical interaction and for DLG-1 multimerization. (A) Schematic showing the structure of the DLG-1 deletion proteins fused to the LexA DNA-binding domain. Constructs were co-transformed with a Gal4–ajm-1 (encoding amino acids 180-809) fusion. Deletions of the L27 domain abrogated the AJM-1–DLG-1 interaction. DLG-1 multimerization was also assayed by transformation of yeast with LexA–dlg-1 deletion constructs and a Gal4–dlg-1 fusion. Physical interaction was observed in constructs containing the N-terminus and the first two PDZ domains of DLG-1; this interaction was disrupted by removal of the L27 domain. (B) lacZ activity of yeast co-transformed with LexA–dlg-1 deletion constructs and Gal4–ajm-1. Numbers on the streaks correspond to the bait constructs from A. lacZ activity was detected in all streaks except 2 and 3. (C) lacZ activity of DLG-1 multimerization. Yeast were co-transformed with Gal4–dlg-1 and LexA–dlg-1 deletion constructs. lacZ activity was observed in streaks 1, 4, 5 and 6. Both streaks 2 and 3 lacked detectable activity.

 

Figure 2
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  		Fig. 2. dlg-1(ok318) is a strong loss-of-function allele, the phenotypes of which resemble dlg-1(RNAi). (A) Diagram displaying the locations of the dlg-1(ok318) deletions within the dlg-1 open reading frame. Deletion 1 is a 109-bp deletion spanning the third exon-intron boundary. Deletion 2 is a 1368-bp deletion removing most of exon 8 to exon 10. Deletion 1 is predicted to produce a frameshift mutation, truncating the protein after the first 153 amino acids. (B-D) Nomarski images of wild-type and dlg-1 loss-of-function embryos. (B) Wild-type embryo nearing the end of elongation. (E,H) dlg-1(ok318) embryos (E) resemble dlg-1(RNAi) embryos (H), arresting at the twofold stage of elongation and frequently forming large vacuoles (black arrowheads). (C,D,F,G,I,J) Wild-type and dlg-1 mutant embryos stained with anti-DLG-1 or anti-AJM-1 antibodies. In wild-type embryos, both DLG-1 (C) and AJM-1 (D) are well-distributed along the apical junctions of epithelia. DLG-1 protein is undetectable in both dlg-1(ok318) (F) and dlg-1(RNAi) (I) embryos. Similarly, AJM-1 is mislocalized into puncta along the junction in both dlg-1(ok318) (G) and dlg-1(RNAi) (J) embryos. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. The N-terminus and PDZ domain of DLG-1 are sufficient for membrane association in dlg-1(ok318) and dlg-1(ok318);ajm-1(RNAi) embryos. (A) Diagram displaying DLG-1::GFP deletion constructs and their localization in wild-type and dlg-1(ok318) embryos. (B,F,J) All constructs localized properly along the junctions of epidermal cells in wild-type embryos. (C,G,K) Localization of DLG::GFP deletions in dlg-1(ok318) homozygous mutants. (C) DLG-1(1-186)::GFP mislocalized into puncta along the junctional belt, with large regions of the junction lacking GFP. (G) DLG-1(1-468)::GFP localized along the lateral junction, although some gaps were present at the 1.5-fold stage of elongation. (K) DLG-1(1-710)::GFP localization was indistinguishable from wild type in dlg-1(ok318) embryos. (D,H,L) All constructs localized along the junctions of epidermal cells in ajm-1(RNAi) embryos. (E,I,M) Localization of DLG-1::GFP deletions in dlg-1(ok318);ajm-1(RNAi) double-mutant embryos. (E) DLG-1(1-186) was mislocalized away from the junction in the cytoplasm of epidermal cells in double mutants. (I) Localization of DLG-1(1-468)::GFP in dlg-1(ok318);ajm-1(RNAi) double mutants was similar to its localization in dlg-1(ok318) single-mutant embryos (G). (M) DLG-1(1-710)::GFP localization was indistinguishable from wild-type in dlg-1(ok318);ajm-1(RNAi) double mutants. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. The SH3 domain acts via a LET-413-dependent pathway to mediate lateral distribution of DLG-1 along the junction. (A) DLG-1(1-186)::GFP localization remains punctate throughout elongation in dlg-1(ok318) mutant embryos, demonstrating the importance of the PDZ domains for junctional localization of DLG-1. (B) DLG-1(1-468)::GFP localization is punctate at the beginning of elongation, but its localization becomes more continuous along the junction as elongation proceeds, but cannot restore proper rates of lateral distribution. (C) DLG-1(1-710) GFP is localized evenly along the junction prior to the beginning of elongation. (D) DLG-1({Delta}17-116)::GFP shows normal localization in a dlg-1(ok318) mutant. (E) DLG-1(1-710)::GFP in let-413 mutants displays delayed rates of lateral distribution, similar to DLG-1(1-468) in dlg-1(ok318) embryos (B). (F) DLG-1(1-468)::GFP shows similar rates of lateral distribution in dlg-1(ok318);let-413(RNAi) double mutants compared to dlg-1(ok318) alone (B). Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. DLG-1 constructs truncated after the PDZ domains can localize AJM-1. Imaging of anti-AJM-1 staining and direct imaging of DLG-1::GFP deletions in terminally arrested dlg-1(ok318) embryos at approximately the twofold stage of elongation. (A-C) DLG-1(1-186)::GFP;dlg-1(ok318) mutant embryo. (A) DLG-1(1-186)::GFP mislocalizes into punctate aggregates in dlg-1 mutant embryos. (B) The construct is unable to rescue AJM-1 localization, which is mislocalized into puncta. (C) AJM-1 and DLG-1(1-186)::GFP puncta co-localize in dlg-1(ok318) mutants. (D-F) DLG-1(1-468)::GFP;dlg-1(ok318) mutant embryo. (D) DLG-1(1-468)::GFP is distributed along the junction in arrested embryos. (E) The construct rescues AJM-1 localization, which is well-distributed in arrested DLG-1(1-468)::GFP;dlg-1(ok318) embryos. (F) AJM-1 and the GFP construct co-localize. (G-I) DLG-1(1-710)::GFP;dlg-1(ok318) mutant embryo. (G) DLG-1(1-710)::GFP localizes to the junction in dlg-1(ok318) mutants. (H) AJM-1 is properly localized in DLG-1(1-468)::GFP;dlg-1(ok318) mutant embryos. (I) GFP and AJM-1 co-localize. (J-L) DLG-1({Delta}17-116)::GFP;dlg-1(ok318) mutant stained. (J) DLG-1({Delta}17-116)::GFP localizes to the junction in a mutant embryo (K), but fails to localize AJM-1 along the entire junction (L). Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Constructs truncated after the SH3 domain can partially rescue the dlg-1(ok318) arrest phenotype. (A) Elongation in a wild-type embryo. From the beginning of elongation at the comma stage to approximately the twofold stage of elongation requires approximately 110 minutes. (B) DLG-1(1-710)::GFP restores elongation rates of dlg-1(ok318) embryos to near wild-type levels, with embryos reaching the twofold stage in approximately 115 minutes. (C) dlg-1(ok318) embryos display delayed rates of elongation, reaching the twofold stage of elongation after approximately 210 minutes. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. Summary and model of DLG-1 fragment localization. (A-D) DLG-1(1-186)::GFP localization. (A) In wild-type animals, DLG-1(1-186)::GFP (yellow) is able to localize to the junction via either endogenous DLG-1 (purple) or AJM-1 (teal). (B) In dlg-1 mutants, the L27 domain of DLG-1(1-186)::GFP binds mislocalized AJM-1 molecules, causing them to colocalize in puncta along the junctional belt. (C) In ajm-1 mutants, DLG-1(1-186)::GFP can localize to the junction via L27-domain-mediated multimerization with endogenous DLG-1. (D) In dlg-1 ajm-1 double mutants, neither of the L27 binding partners are expressed at the junction; thus, DLG-1(1-186)::GFP is mislocalized to the cytoplasm. (E-G) SH3-domain-mediated lateral distribution. (E) In wild-type animals, DLG-1(1-468)::GFP (blue) displays rapid lateral distribution along the junction through interaction with endogenous wild-type DLG-1 (purple). (F) In dlg-1 mutants, the PDZ domains of DLG-1(1-186)::GFP associate it with the junction, but the fragment is unable to restore lateral distribution, thus resulting in discontinuous junctional localization during the early stages of elongation. (G) The SH3 domain of DLG-1(1-710)::GFP (red) is sufficient to restore normal rates of lateral distribution in the dlg-1 mutants. (H-K) Model for the contribution of LET-413 to DLG-1 lateral distribution. (H) In dlg-1 mutants, basolateral LET-413 (green) can act via the SH3 domain of DLG-1(1-710)::GFP (red) to mediate its lateral distribution. (I) In let-413 mutants, DLG-1(1-710)::GFP is no longer able to distribute along the junction. (J) In dlg-1 mutants, DLG-1(1-468)::GFP shows slow rates of lateral distribution, because it lacks the SH3 domain. (K) DLG-1(1-186)::GFP localization is not significantly enhanced in dlg-1(ok318);let-413(RNAi) double mutants, suggesting that LET-413 acts via the SH3 domain to mediate DLG-1 lateral distribution.

 

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© The Company of Biologists Ltd 2008