Allison M. Lynch

In vitro and in vivo reconstitution of the cadherin–catenin–actin complex from Caenorhabditis elegans

The ternary complex of cadherin, β-catenin, and α-catenin regulates actin-dependent cell–cell adhesion. α-Catenin can bind β-catenin and F-actin, but in mammals α-catenin either binds β-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of α-catenin is evolutionarily conserved. The Caenorhabditis elegans α-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1–HMP-2(β-catenin)–HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of α-catenin and suggests that multiple mechanisms regulate α-catenin binding to F-actin.

The F-BAR domain of SRGP-1 facilitates cell–cell adhesion during C. elegans morphogenesis

SRGP-1 activity leads to outward bending and projections of membranes at cell–cell junctions, promoting robust adhesion between cells during embryonic closure events.

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2Colon, two colonsGFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.

Tropomodulin Protects α-Catenin-Dependent Junctional-Actin Networks under Stress during Epithelial Morphogenesis

α-catenin is central to recruitment of actin networks to the cadherin-catenin complex, but how such networks are subsequently stabilized against stress applied during morphogenesis is poorly understood. To identify proteins that functionally interact with α-catenin in this process, we performed enhancer screening using a weak allele of the C. elegans α-catenin, hmp-1, thereby identifying UNC-94/tropomodulin. Tropomodulins (Tmods) cap the minus ends of F-actin in sarcomeres. They also regulate lamellipodia, can promote actin nucleation, and are required for normal cardiovascular development and neuronal growth-cone morphology. Tmods regulate the morphology of cultured epithelial cells, but their role in epithelia in vivo remains unexplored. We find that UNC-94 is enriched within a HMP-1-dependent junctional-actin network at epidermal adherens junctions subject to stress during morphogenesis. Loss of UNC-94 leads to discontinuity of this network, and high-speed filming of hmp-1(fe4);unc-94(RNAi) embryos reveals large junctional displacements that depend on the Rho pathway. In vitro, UNC-94 acts in combination with HMP-1, leading to longer actin bundles than with HMP-1 alone. Our data suggest that Tmods protect actin filaments recruited by α-catenin from minus-end subunit loss, enabling them to withstand the stresses of morphogenesis.

A Conserved Phosphorylation Switch Controls the Interaction between Cadherin and β-Catenin In Vitro and In Vivo

In metazoan adherens junctions, β-catenin links the cytoplasmic tail of classical cadherins to the F-actin-binding protein α-catenin. Phosphorylation of a Ser/Thr-rich region in the cadherin tail dramatically enhances affinity for β-catenin and promotes cell-cell adhesion in cell culture systems, but its importance has not been demonstrated in vivo. Here, we identify a critical phosphorylated serine in the C. elegans cadherin HMR-1 required for strong binding to the β-catenin homolog HMP-2. Ablation of this phosphoserine interaction produces developmental defects that resemble full loss-of-function (Hammerhead and Humpback) phenotypes. Most metazoans possess a single gene for β-catenin, which is also a transcriptional coactivator in Wnt signaling. Nematodes and planaria, however, have a set of paralogous β-catenins; for example, C. elegans HMP-2 functions only in cell-cell adhesion, whereas SYS-1 mediates transcriptional activation through interactions with POP-1/Tcf. Our structural data define critical sequence differences responsible for the unique ligand specificities of these two proteins.

Specific conserved C-terminal amino acids of Caenorhabditis elegans HMP-1/α-catenin modulate F-actin binding independently of vinculin.

Background: α-Catenin is a crucial link between adherens junctions and F-actin. Results: C-terminal amino acids in HMP-1/α-catenin quantitatively modulate its ability to bind F-actin, but a putative vinculin-binding domain is not required in vivo. Conclusion: Key C-terminal residues in α-catenin modulate its ability to bind F-actin. Significance: This is the first genetic dissection of the ability of α-catenin to bind F-actin. Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687–742 and 826–927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315–494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan.

Dynamic analysis identifies novel roles for DLG-1 subdomains in AJM-1 recruitment and LET-413- dependent apical focusing

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 used structure-function experiments to dissect the role of domains of the Caenorhabditis elegans MAGUK DLG-1, a homolog of Drosophila Discs large and vertebrate SAP97. 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, using 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 found that inclusion of the SH3 domain can ameliorate dlg-1 mutant phenotypes, but full rescue of lethality required the complete C terminus, which includes the GUK and Hook domains, thereby demonstrating the importance of the C-terminus for DLG-1 function. Our results represent the first in vivo analysis of requirements for the L27 domain of a Discs-large/SAP97 protein, identify a crucial LET-413/Scribble regulatory motif and provide insight into how MAGUK subdomains function to maintain epithelial integrity during development.

Cadherins and their partners in the nematode worm Caenorhabditis elegans.

The extreme simplicity of Caenorhabditis elegans makes it an ideal system to study the basic principles of cadherin function at the level of single cells within the physiologically relevant context of a developing animal. The genetic tractability of C. elegans also means that components of cadherin complexes can be identified through genetic modifier screens, allowing a comprehensive in vivo characterization of the macromolecular assemblies involved in cadherin function during tissue formation and maintenance in C. elegans. This work shows that a single cadherin system, the classical cadherin-catenin complex, is essential for diverse morphogenetic events during embryogenesis through its interactions with a range of mostly conserved proteins that act to modulate its function. The role of other members of the cadherin family in C. elegans, including members of the Fat-like, Flamingo/CELSR and calsyntenin families is less well characterized, but they have clear roles in neuronal development and function.