Organizing a functional junctional complex requires specific domains of the Drosophila MAGUK Discs large - PubMed (original) (raw)

Organizing a functional junctional complex requires specific domains of the Drosophila MAGUK Discs large

C D Hough et al. Genes Dev. 1997.

Abstract

Discs large (Dlg) was the first identified member of an increasingly important class of proteins called membrane-associated guanylate kinase homologs (MAGUKs), which are often concentrated at cell junctions and contain distinct peptide domains named PDZ1-3, SH3, HOOK, and GUK. Dlg is localized at and required for the formation of both septate junctions in epithelial cells and synaptic junctions in neurons. In the absence of Dlg, epithelia lose their organization and overgrow. We tested the functions of each domain of Dlg in vivo by constructing transgenic flies expressing altered forms of the protein. In the first set of experiments each domain was examined for its ability to correctly target an epitope-tagged Dlg to pre-existing septate junctions. Based on these results the Hook domain is necessary for localization of the protein to the cell membrane and the PDZ2 is required for restricting the protein to the septate junction. In the second set of experiments each domain was tested for its role in growth regulation and organization of epithelial structure. These results show that PDZ1 and GUK are apparently dispensable for function, PDZ2 and PDZ3 are required for growth regulation but not for epithelial structure, and SH3 and HOOK are essential for both aspects of function. The results demonstrate the functional modularity of Dlg and clarify the functions of individual MAGUK domains in regulating the structure and growth of epithelial tissue.

PubMed Disclaimer

Figures

Figure 1

Summary of the Dlg–FLAG deletion analyses. (a) Cartoon of the domain-specific Dlg-deletion constructs. A V indicates a deleted domain and a straight line corresponds to the sequences present. The subcellular location of the deletion proteins in wing discs containing endogenous normal Dlg protein, and the ability of the deletion proteins to rescue the epithelial structure of wing discs from larvae hemizygous for a genetic null allele _dlg_m52 are listed in the columns to the right of each deletion tested. (b) Western blot analysis of the Dlg–FLAG proteins from larvae in which the Ptc-driven GAL4 transcriptional activator drives expression of the _dlg_–FLAG deletion constructs. (Lane P) Ptc-driver GAL4 line control; (lane t) Δ_t_40–FLAG; (lane 1) ΔPDZ1–FLAG; (lane 2) ΔPDZ2–FLAG (69B); (lane 3) ΔPDZ3–FLAG; (lane S) ΔSH3–FLAG; (lane H) ΔHOOK–FLAG; (lane G) ΔGUK–FLAG; (lane C) ΔC1/2–FLAG; (lane 2+) lane containing twice as much ΔPDZ2–FLAG as lane 2. Deficiency boundaries in base pairs for PDZ1, 109–399; PDZ2, 439–756; PDZ3, 1393–1695; SH3, 1810–2001; HOOK, 2002–2340; SH3–HOOK–GUK (C1/2), 1810–2805; and PDZ1,2,3–SH3 (N2/3), 109–2001.

Figure 2

Subcellular localization of Dlg-deletion proteins in wing discs expressing normal endogenous Dlg analyzed by confocal microscopy. All images are of folds within the epithelial layer that result in an apical surface facing another apical surface. Large, dividing nuclei in addition to the actin band at the adherens junctions denote the apical faces. Filamentous actin was detected with rhodamine–phalloidin (red) and the Dlg-deletion proteins were visualized using anti-FLAG M2 monoclonal antibodies (green), except in (a) where green staining corresponds to endogenous Dlg. (a) Wild-type (magnification, 800×) (A) apical surfaces; (B) basal end of the folds; (b) Δ_t_40–FLAG (1400×); (c) ΔPDZ1–FLAG (1200×); (d) ΔPDZ2–FLAG (1200×); (e) ΔHOOK–FLAG (1000×); (f) HOOK–FLAG (1400×); (g) PDZ2 + HOOK–FLAG (1400×). Note that the stripe of protein produced from the Ptc-driver is out of register in adjacent folds (b,d–f).

Figure 3

Ability of Dlg-deletion proteins to rescue the structural defects and the overgrowth phenotype of the dlg genetic null. Filamentous actin was visualized with rhodamine-phalloidin, except in (a) where the staining corresponds to the septate junction protein Cor, which is redistributed throughout the membrane in the dlg null allele that lacks septate junctions. (a) disc tissue from a larva hemizygous for the genetic null allele _dlg_m52 (magnification, 400×); (b) completely rescued wing disc from a _dlg_m52 larva expressing ΔGUK–FLAG (200×); (c) Δ_t_40–FLAG (200×); (d) ΔPDZ1–FLAG (200×); (e) ΔPDZ2–FLAG (200×); (f) ΔPDZ3–FLAG (200×); (g,h) the disc tissue from larvae expressing ΔSH3–FLAG and ΔHOOK–FLAG resemble the epithelium from the _dlg_m52 larva (a).

Figure 4

Ability of the Dlg-deletion protein DlgΔGUK–FLAG to restore apicobasal polarity in the dlg genetic null. The DlgΔGUK–FLAG construct was analyzed because of its ability to rescue the structural defects and overgrowth phenotype of the genetic null. The images show two apical surfaces facing each other within a fold of the wing disc epithelium. A wing disc stained with the septate junction marker Cor (a) detected with anti-Cor antibodies and with antibodies to the adherens junction protein DECad (b). Note the Cor staining is located just basal to the DECad at the apical end of the cells. A wing disc stained with antibodies to FasIII (c), which is enriched in the cells at the level of the septate junctions, just basal to the adherens junction band of filamentous actin detected with rhodamine-phalloidin (d). (A) Apical.

Figure 5

The ability of Dlg deletion proteins to rescue septate junction formation in the dlg genetic null. TEM of wing disc epithelia. (ap) Apical; (ba) basal. (a–c) Rescue of _dlg_m52 epithelial structure with (a) ΔGUK–FLAG, (b) ΔPDZ2–FLAG, and (c) ΔPDZ3–FLAG. Note the long, columnar epithelial cells from the ΔGUK–FLAG rescue (a). The epithelia from the ΔPDZ2–FLAG (b) and ΔPDZ3–FLAG (c) rescued discs have obvious apical and basal ends even though the cells are more cuboidal in shape. (d–h) Higher magnification of the rescued wing disc epithelia showing the apical junctions. (d) ΔGUK–FLAG; (e) Δ_t_40–FLAG; (f) ΔPDZ1–FLAG; (g) ΔPDZ2–FLAG; (h) ΔPDZ3–FLAG. Samples of septate junctions are marked with arrowheads and the apical adherens junctions with asterisks.

Figure 6

Model of Dlg function. (a) A summary of the function of each of the domains. (b) A three-step model of Dlg localization to the septate junctions and binding of the proteins important for structure and growth regulation. Step 1: Binding of the HOOK domain to a protein 4.1-like protein (green line) and membrane localization. Step 2: Transmembrane protein (dark blue rectangle) binding to PDZ1/2 and clustering at the septate junction. Step 3: Stabilization and binding of proteins involved in additional functions such as growth regulation. The relative order of these steps is arbitrary.

References

1. Aberle H, Schwartz H, Kemler R. Cadherin-catenin complex: Protein interactions and their implications for cadherin function. J Cell Biochem. 1996;61:514–523. - PubMed
1. Alley MR, Maddock JR, Shapiro L. Requirement of the carboxyl terminus of a bacterial chemoreceptor for its targeted proteolysis. Science. 1993;259:1754–1757. - PubMed
1. Bar-Sagi D, Rotin D, Batzer A, Mandiyan V, Schlessinger J. SH3 domains direct cellular localization of signalling molecules. Cell. 1993;74:83–91. - PubMed
1. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. - PubMed
1. Brower DL, Smith RJ, Wilcox M. A monoclonal antibody specific for diploid epithelial cells in Drosophila. Nature. 1980;285:403–405. - PubMed

Organizing a functional junctional complex requires specific domains of the Drosophila MAGUK Discs large - PubMed (original) (raw)