DLG-1 is a MAGUK similar to SAP97 and is required for adherens junction formation

Abstract

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.

Figure 1

DLG-1 is a MAGUK protein most similar to SAP97. (A) Deduced amino acid sequence of DLG-1 with PDZ domains in blue, SH3 domain in red, and guanylate kinase domain in gray. (B) Alignment of the amino-terminal 99 amino acids of DLG-1 with SAP97. Identical residues are indicated in black, whereas conserved residues are indicated in gray. (C) Schematic diagrams of the DLG-1:: GFP chimeric proteins. Embryos expressing DLG-1:: GFP (green) in intestine (D) and hypodermis (E) are shown with DAPI-stained nuclei (blue). Adult worms express DLG-1:: GFP in the Pnp epithelial cells (F) and lateral seam cells (G). Scale bar represents 10 μm.

Figure 2

DLG-1 is localized to adherens junctions and required for AJM-1:: GFP localization. Immunostaining of embryos that express AJM-1:: GFP (green, A, D, G, J, M) with anti-PSD-95 antibodies (red, B, E, H) or with anti-PKC-3 antibodies (red, K, N). Nuclei are stained with DAPI (blue). Merged images (C, F, I, L, O) contain a 10 μm scale bar. AJM-1:: GFP and DLG-1 colocalize to adherens junction on the epidermis (A-C, lima bean stage) and in the intestine (D-F, tadpole stage) of wild-type embryos. (G-I, twofold stage) AJM-1:: GFP is found in small punctate structures between cells in dlg-1(RNAi) embryos No DLG-1 protein is detected in these embryos. (J-L, gastrulating embryo) AJM-1:: GFP is found at intestinal adherens junctions (green) whereas PKC-3 is localized to the apical surface of these cells (red) in wild-type embryos. (M-O, gastrulating embryo) AJM-1:: GFP is found in punctate structures, but PKC-3 is found at the apical surface of cells from dlg-1(RNAi) embryos.

Figure 3

Abnormal ultrastructure of adherens junctions in dlg-1(RNAi) embryos. (A) Electron microscopy of an adherens junction (arrow) in a wild-type embryo. (B) Electron-dense adherens junction material is often missing in dlg-1(RNAi) embryos. Arrowheads point to gaps that have formed between adjacent membranes. (C) When adherens junctions are visible in dlg-1(RNAi) embryos, they are often discontinuous (arrowheads). Scale bar is 200 nm.

Figure 4

DLG-1 is required for elongation and proper organization of actin filaments. (A-D) Nomarski interference microscopy of embryos. Wild-type (A) and dlg-1(RNAi) (B) embryos undergo proper ventral enclosure during gastrulation. However, unlike wild-type threefold embryos (C), dlg-1(RNAi) embryos fail to elongate and often contain large vacuoles (arrowhead). (E-I) Rhodamine-phalloidin staining of filamentous actin. Wild-type twofold embryos (E) develop an unelongated pharynx with radially-oriented actin filaments, which elongates into a complete pharynx (F) in threefold embryos. (G) The hypodermis of wild-type embryos contains circumferential filaments (cf) that wrap around the nematode, and adherens junction filaments (ajf) that have contracted circumferentially. The label “mf” indicates actin filaments of the longitudinal muscles. (H) In contrast, the pharynx of a dlg-1(RNAi) embryo fails to elongate and contains disorganized actin filaments. The labels “mc” and “tb” indicate metacorpus and terminal bulb, respectively. (I) The hypodermis of dlg-1(RNAi) embryos contains regions that lack circumferential actin bundles (black arrowhead). Adherens junction filaments are found at the adherens junctions of lateral hypodermis but have failed to contract circumferentially. Scale bar is 10 μm.

Figure 5

DLG-1:: GFP localization to adherens junctions does not require the cadherin-catenin system. Embryos expressing DLG-1:: GFP (A-H) and HMP-1:: GFP (I-J). Wild-type tadpole (A) and threefold stage (B) embryos localize DLG-1:: GFP to adherens junctions. (C) In contrast, let-413 mutant embryos localize DLG-1:: GFP to punctate structures between cells. (D) Tadpole stage hmr-1 embryos are disorganized but localize DLG-1:: GFP to adherens junctions. Tadpole (E) and terminal (threefold equivalent) stage (F) hmp-1 embryos localize DLG-1:: GFP to their disorganized adherens junctions. Tadpole (G) and terminal (threefold equivalent) stage (H) hmp-2 embryos localize DLG-1:: GFP to their disorganized adherens junctions. HMP-1:: GFP is found at adherens junctions of tadpole stage embryos from both wild-type (I) and dlg-1(RNAi) (J) embryos. Scale bar represents 10 μm.

Figure 6

DLG-1 is required to localize CPI-1. Embryos immunostained with anti-PSD-95 antibodies to detect DLG-1 (green, A, D, G) and anticypin antibodies to detect CPI-1 (red, B, E, H). DLG-1 (green) and CPI-1 (red) colocalize to the adherens junctions of wild-type intestine (A-C) and epidermis (D-F). However, in dlg-1(RNAi) embryos, CPI-1 is found in small clusters (arrowheads) along the adherens junctions of intestine (not shown) and epidermis (G-I). Merged images (C, F, I, L, O) contain a 10 μm scale bar.